ASXL1 Research Articles

A genomics-informed computational biology platform prospectively predicts treatment responses in AML and MDS patients

AbstractPatients with myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) are generally older and have more comorbidities. Therefore, identifying personalized treatment options for each patient early and accurately is essential. To address this, we developed a computational biology modeling (CBM) and digital drug simulation platform that relies on somatic gene mutations and gene CNVs found in malignant cells of individual patients. Drug treatment simulations based on unique patient-specific disease networks were used to generate treatment predictions. To evaluate the accuracy of the genomics-informed computational platform, we conducted a pilot prospective clinical study (NCT02435550) enrolling confirmed MDS and AML patients. Blinded to the empirically prescribed treatment regimen for each patient, genomic data from 50 evaluable patients were analyzed by CBM to predict patient-specific treatment responses. CBM accurately predicted treatment responses in 55 of 61 (90%) simulations, with 33 of 61 true positives, 22 of 61 true negatives, 3 of 61 false positives, and 3 of 61 false negatives, resulting in a sensitivity of 94%, a specificity of 88%, and an accuracy of 90%. Laboratory validation further confirmed the accuracy of CBM-predicted activated protein networks in 17 of 19 (89%) samples from 11 patients. Somatic mutations in the TET2, IDH1/2, ASXL1, and EZH2 genes were discovered to be highly informative of MDS response to hypomethylating agents. In sum, analyses of patient cancer genomics using the CBM platform can be used to predict precision treatment responses in MDS and AML patients.

Mutational profile of myeloproliferative neoplasms detected by a customized NGS-based gene panel

Objective By a sequencing panel consisting of 50 targeted genes, aiming at depicting the molecular landscape of ET, PV, and PMF, which are three major subtypes of MPN, to provide valuable information in the diagnosis and prognosis of MPN. Methods A retrospective study was conducted of 53 patients from Huashan hospital and Changhai hospital. All patients were diagnosed in accordance with the 2016 WHO diagnostic criteria for MPN, including 31 cases of ET(11 males, 20 females, median age 55 years), 17 cases of PV(12 males, 5 females, median age 65 years), and 5 cases of PMF(4 males, 1 females, median age 67 years), and underwent next-generation of DNA sequencing of their bone marrow or blood samples. The genetic analyses were performed on bone marrow or peripheral blood. Referring to COSMIC, dbSNP, Clinvar and other public databases, we analyzed the sequencing data, and elucidated the mutation profile of MPN patients, combining with their clinic information. Results In addition to the typical JAK2, CALR, and MPL mutations, pathogenic mutations in other 11 genes were detected, as well as 4 SNPs that confer individual susceptibility to MPNs (rs4858647, rs9376092, rs58270997, rs621940). The average rate of mutated genes was 2.3 genes per patient. In all patients (53 cases), the mutated genes detected were TET2, EZH2, ASXL1, MIR662, SF3B1, BARD1, DNMT3A, KIT, RUNX1, TP53, NRAS according to their mutational frequency. Conclusions Applying next-generation sequencing technology, multi-gene sequencing of a bunch of typical BCR-ABL-negative MPN patients can be performed at one time within 2 working days, and pathogenic mutations other than JAK2, CALR, MPL can be found, which has a bright prospection in clinic. Key words: Myeloproliferative disorders; Mutation; High-throughput nucleotide sequencing

PS1434 GENOMIC LANDSCAPE AND MOLECULAR RISK IN PATIENTS WITH DIPSS LOW‐ AND INTERMEDIATE‐1‐RISK PRIMARY MYELOFIBROSIS: A STUDY OF THE GERMAN STUDY GROUP FOR MYELOPROLIFERATIVE NEOPLASMS (GSG‐MPN)

Background:The ́Dynamic International Prognostic Scoring Systeḿ (DIPSS) incorporates 5 clinical variables to estimate prognosis in primary myelofibrosis (PMF) by stratification of patients into the low‐, intermediate‐1‐ (int1), intermediate‐2‐ (int2), or high‐risk category (Passamonti et al.; Blood 2010). Independent of the DIPSS, PMF patients carrying mutations (mut) in the ́High Molecular Risḱ (HMR) genes ASXL1, SRSF2, EZH2, IDH1 and/or IDH2 have a higher risk of death or leukemic transformation (Vannucchi et al., Leukemia 2013). Therefore, analysis of the genomic landscape in low‐ and int1‐risk PMF patients may refine prognostic scoring and identify those who benefit from earlier therapeutic intervention.Aims:To assess the genomic landscape in DIPSS low‐ and int1‐ risk PMF and to explore the risk of disease progression in patients with an adverse molecular profile.Methods:Using a next generation sequencing approach (NGS) [HaloPlex HS (Agilent); Miseq (Illumina)], we performed a retrospective analysis of the complete coding regions of 42 genes associated with myeloid malignancies, including the HMR genes. 78 patients with low‐ and int1‐ risk PMF enrolled in our German Study Group‐ (GSG‐) MPN Bioregistry (NCT 03125707) were analyzed.Results:Overall, 182 mut were identified in 74/78 patients (95%). Of the three classical MPN driver mut, 50/78 (64%) patients carried JAK2 V617F, 4/78 (5%) had MPL W515 (n = 3) and Y591 (n = 1), and 20/78 (26%) were CALR mutated; co‐existing mut in JAK2 and MPL occurred in two cases; 6/78 (8%) patients were triple‐negative. Of note, 59% of the patients carried non‐driver mut, most commonly TET2 (13%), or DNMT3A (6%) as well as FLT3, KMT2A, KRAS, CBL, CEBPA, and ZRSR2 (2% each).HMR mut (HMRmut) were identified in 20/78 of the patients (26%) and were more frequently present in the int1‐ (39%) than low‐risk (14%) category (p = .019); mut in ASXL1 occurred in 19% (15/78), SRSF2 in 9% (7/78), EZH2 and IDH2 in 4% each (3/78), and IDH1 in 1% (1/78). 11/78 patients (14%) harbored two HMRmut, of these 10/11 co‐occurred with ASXL1 (5/10 in combination with SRSF2 mut); no patient had more than 2 HMRmut.HMRmut patients harbored more co‐occurring mut than HMR wildtype (HMRwt) patients (median 4 vs 2; p < .0001). In addition, HMRmut were associated with the presence of JAK2 V617F (p = .03). However, no unique mutational pattern beside JAK2 V617F was identified for HMRmut and HMRwt patients.We were able to perform an explorative analysis of clinical outcomes in 47/78 patients. In this subcohort, the median follow‐up (FU)‐time was 3.5 years for HMRmut vs 6.5 years for HMRwt patients. Despite the shorter FU more HMRmut patients 5/13 (38%) than HMRwt patients 4/34 (12%) shifted to a higher DIPSS category (n = 8) or experienced leukemic transformation (n = 1) [p = .09]. All affected HMRmut patients carried either ASXL1 (n = 2) or SRSF2 (n = 2) or both mut ASXL1/SRSF2 (n = 1).Summary/Conclusion:Using a targeted sequencing approach we identified mutations in 95% of 78 DIPSS low‐ and int‐1‐ risk PMF patients. 26% of these harbored HMR mutations, underlining the clinical importance of HMR marker screening for the identification of patients potentially at higher risk. However, prospective studies with longer observation periods are needed to demonstrate the clinical impact of prognostically adverse HMR mutations. Such studies can provide the rationale for evaluating the impact of earlier therapeutic intervention. NGS in larger patient cohorts will allow the identification of distinct mutational patterns as well as gene‐gene interactions to further refine risk categorization in low/int‐1 PMF.

PF544 THE DIFFERENT MUTATIONAL LANDSCAPES OF A PANEL GENES BY NEXT GENERATION SEQUENCING IN 967 CASES OF MDS AND AML

Background: Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are myeloid disease with dysplasia characterized by multiple gene mutations in most cases. With advances in Next-generation sequencing techniques, the molecular landscapes in MDS or AML have been reported with further understanding the disease pathogenesis. As well known, MDS may end up in evolving into AML in most cases, especially the subtypes of MDS with excessive blasts. The driver mutation in MDS developing to AML is still open to study. The comparison of mutational landscapes between MDS and AML will provide evidence to better understand the underlying molecular mechanisms of the myeloid dysplasia evolution. Aims: To analyze the different mutational status of 22 genes commonly observed in MDS and AML. Methods: The bone marrow samples were collected from patients with AML (749 cases) and MDS (218 cases) with written consent from June 2017 to January 2019. Next generation sequencing of a panel of 22 genes was used to evaluate the mutations in MDS/AML patients. Results: 719/ 967(74.4%) cases with MDS/AML were detected at least one gene mutation. There were 274 gene mutations in 142/ 218 (65.1%) cases with MDS and 1224 gene mutations in 577/749(77.0%) cases with AML. Both the median mutations and the frequency of gene mutation between MDS and AML were significantly difference (p < 0.05). Compared to MDS, the mutation rate of CEBPA (11.10% vs 1.40%, p < 0.01), NRAS (14.00% vs 6.40%, p < 0.01), KIT (7.30% vs 0.00%,p < 0.01), NPM1 (17.80% vs 5.00%, p < 0.01), FLT3-ITD (15.40% vs 0.90%, p < 0.01), DNMT3A (10.90% vs 4.10%, p < 0.01), IDH2 (9.5% vs 2.8%, p < 0.01), and IDH1 (6.00% vs 1.40%, p < 0.01) genes were significantly higher in AML. While the mutation rate of ASXL1 (5.30% vs 14.7%, p < 0.01), U2AF1 (5.90% vs 22.00%, p < 0.01), RUNX1 (6.30% vs 10.60%, p < 0.05), SRSF2 (2.30% vs 6.00%, p < 0.05), EZH2 (0.50% vs 3.20%, p < 0.01), SETBP1 (0.70% vs 5.00%, p < 0.01), SF3B1 (2.50% vs 7.80%, p < 0.01), and CBL (0.80% vs 3.20%, p < 0.05) genes were significantly higher in MDS. There were no significant difference of the incidence in TET2, FLT3-TKD, JAK2, ETV6, TP53, PHF6 and ZRSR2 gene mutations. Considering the gene mutation increasing with age, the cases were divided into the young (age < 60 years old) and the old (age≥60 years old). In the young cases, the gene mutation profile was similar to the cohort with incidences of CEBPA, NRAS, KIT, NPM1, FLT3-ITD, DNMT3A, IDH1, plus TET2 gene mutations were significantly higher in AML (p < 0.05). The incidences of ASXL1, U2AF1, TP53, EZH2, SETBP1, and CBL gene mutations were significantly higher in MDS (p < 0.05), and the incidences of FLT3-TKD, JAK2, RUNX1, IDH2, ETV6, PHF6, SRSF2, SF3B1 and ZRSR2 gene mutations were not significantly different between AML and MDS cases (p>0.05). The old cohort showed a different gene mutation profile as compared to the young cohort with the incidences of CEBPA, NPM1, FLT3-ITD, and IDH2 gene mutations were significantly higher in AML (p < 0.01), and the incidences of U2AF1 and SF3B1 gene mutations were significantly higher in MDS. There were no significant differences of all the other gene mutations between AML and MDS cases in the old cohort. Summary/Conclusion: There are typical gene mutation patterns in MDS and AML cases. Some genes mutate in MDS and AML cases without differences and others mutate mainly in AML or MDS. The roles and significances of these gene mutation profiles warranted further investigations to understand the pathogenesis underlying MDS/AML.

PB2193 GENE EXPRESSION PROFILE IN PRIMARY MYELOFIBROSIS: ROLE OF ASXL1

Background:Primary myelofibrosis (PMF) is a BCR‐ABL1‐negative myeloproliferative neoplasm characterized by aberrant differentiation of all myeloid lineages and profound disruption of the bone marrow niche. The presence of JAK2, CALR or MPL [R1] mutation supports the diagnosis. In approximately 10% of cases classified as PMF, the genetic cause remains unknown and they are known as triple negative. Recent prognostic scores like GIPSS or MIPSS recommend the analysis of other mutations in non‐driver genes, such as ASXL1, SRSF2, EZH2, IDH1/2 and U2AF1Q157, since they influence the risk stratification.Aims:To analyze the mRNA expression profiles of EZH2, ASXL1, RB1 and TET2 genes in a series of PMF patients with known mutational status of JAK2V617F, CALR and MPL MethodsOur series consisted of 21 PMF patients, 6 females and 15 males, with a mean age of 70 years (range 41–87), diagnosed and treated between 1996 and 2018 at the Hospital Universitario de Gran Canaria Dr. Negrín, Spain. Four patients of our series were triple negative, 13 JAK2V167mut, 3 CALRmut and 1 MPLmut. mRNA expression levels were determined by real time PCR in a LightCycler 480 Instrument II (Roche) using ABL as a control gene. Results were normalized with a cDNA pool from the peripheral blood of 10 healthy donors, which was introduced as an internal control in each experiment. SPPS V.23 software was used for statistical analyses.Results:We found that ASXL1 expression levels were significantly higher in patients with JAK2V617F mutated compared with JAK2V617F non‐mutated (mean 0.47 vs. 0.25, p = 0.037, Mann–Whitney U test). In the study of progression free survival, we detected a 36% decrease in the probability of progression to AML or death per unit of ASXL1 expression (p = 0.007, Cox regression test). No significant associations were found between expression levels of the other genes studied (RB1, TET2 and EZH2) and triple negative status.Summary/Conclusion:ASXL1 mutations are common in myeloid diseases and, in these cases, ASXL1 mutations are associated with worse overall survival and leukemia‐free survival. While the impact of ASXL1 mutations has been well‐studied, the impact of ASXL1 expression levels on patient outcome is less studied. The positive association between higher ASXL1 expression plus JAK2V617Fmut and better progression free survival is in agreement with the fact that the opposite, that is, inactivating ASXL1 mutations plus triple negative status, contribute to poor prognosis. These preliminary results should be confirmed in further studies with a larger series.

A new case of Bainbridge–Ropers syndrome (BRPS): delineating the phenotype and review of literature

Background: Bainbridge-Ropers syndrome (BRPS) is characterized by failure to thrive, global developmental delay, feeding problems, hypotonia, profound speech delays, and intellectual disability and dysmorphic features. It is an autosomal dominant condition caused by heterozygous mutations in the ASXL3 gene (OMIM #615115) on chromosome 18q12. As per the literature available, only 39 cases including the current patient were reported with BRPS across the globe. Case presentation: A 4-year-old girl with confirmed BRPS. She had the characteristic features of the disease including psychomotor delay, hypotonia, profound speech impairment, neonatal feeding difficulties, postnatal, and dysmorphic features. The array CGH and whole exome sequencing were negative, but the whole genome sequencing detected a novel heterozygous de novo insertion in the ASXL3 gene c.3592_3593insGAT; p.Leu1198X. Conclusion: With the advent of whole exome/genome sequencing we would expect to diagnose more cases of BRPS from different ethnic populations. Further clinical and functional studies are needed to delineate the long-term course of the disease and to elaborate on the exact role of the ASXL3 gene in brain development.

Clinical features and curative effect analysis of patients with myeloid neoplasms and RUNX1 mutations

目的研究伴RUNX1突变髓系肿瘤的临床特征和异基因造血干细胞移植（allo-HSCT）的疗效。方法回顾性分析2014年7月至2018年4月在苏州大学附属第一医院行二代测序检出RUNX1基因突变的42例髓系肿瘤患者的临床资料。结果全部42例伴RUNX1突变髓系肿瘤患者中，男27例，女15例，中位年龄43.5（16~68）岁，急性髓系白血病（AML）30例，骨髓增生异常综合征（MDS）12例。共突变基因中频率最高的是FLT3（26.2%，11/42），携带FLT3共突变基因的均为AML患者（P=0.014）。而MDS患者中最常见的共突变为ASXL1（25%，3/12）。allo-HSCT组（31例）1年总生存（OS）、无病生存（DFS）率分别为（70.6±9.0）%、（61.0±9.4）%，化疗组（11例）1年OS、DFS率分别为（34.4±16.7）%、（22.4±15.3）%，两组OS、DFS率差异有统计学意义（χ2=4.843，P=0.036；χ2=4.320，P=0.047）。单因素分析提示移植年龄>45岁为影响患者OS及DFS的预后不良因素[HR=4.819（95%CI 1.145~20.283），P=0.032；HR=5.945（95%CI 1.715~20.604），P=0.005]，染色体核型复杂异常为影响OS的预后不良因素[HR=5.572（95%CI 1.104~28.113），P=0.038]。结论allo-HSCT可以改善伴RUNX1突变髓系肿瘤患者预后，移植年龄>45岁、染色体核型复杂异常是影响allo-HSCT疗效的不良预后因素。

Next-generation sequencing-based genetic landscape and its clinical implications for Chinese acute myeloid leukemia patients

BackgroundAcute myeloid leukemia (AML) is a clinically and biologically heterogeneous disease. The survival of older patients is generally poor. In the current study, we sought to investigate the differences in molecular gene mutations between younger and older AML patients, and to identify those newly diagnosed AML patients who are more likely to respond to standard cytarabine and daunorubicin induction chemotherapy.MethodsWe retrospectively evaluated 179 patients who were newly diagnosed with non-M3 AML. A next-generation sequencing assay covering 34 genes was used to investigate recurrently mutated genes. The mutational status of fusion genes was determined by real time PCR.ResultsThe median age at diagnosis was 53 years (range 18–88 years). Sixty-eight patients were 60 years or older with a median age of 67 years (range 60–88 years). Eighteen patients (10.1%) carried t(8;21)(q22;q22.1) or RUNX1–RUNX1T1 gene fusion, and there was a significantly higher incidence in younger patients (p = 0.019). At least one non-synonymous gene mutation was detected in 159 patients (88.8%). The median number of gene mutations was two (range 0–6). The mean number of molecular gene mutations at diagnosis was higher in older patients than younger patients (2.5 vs 1.83, p = 0.003). Older patients had significantly higher incidences of ASXL1 (22.1% vs 13.5%, p = 0.025) and TP53 mutations (13.2% vs 3.6%, p = 0.034). In total, 78 patients received DA60 (daunorubicin 60 mg/m2 per day on days 1–3 and cytarabine 100 mg/m2 twice per day on days 1–7) as the induction therapy, and information was available on their response to induction treatment. Patients with RUNX1–RUNX1T1 gene fusion were significantly more likely to achieve complete remission (CR) after DA60 induction therapy (p = 0.026), as were patients without the ASXL1 mutation (p = 0.007).ConclusionOlder AML patients had a lower incidence of favorable cytogenetics and higher frequencies and burdens of molecular mutations that are associated with poor prognosis compared to younger patients. Patients with RUNX1–RUNX1T1 gene fusion or without the ASXL1 gene mutation had a better chance of achieving CR when treated with cytarabine and daunorubicin induction chemotherapy.

A brief, but comprehensive, guide to clonal evolution in aplastic anemia.

Acquired aplastic anemia (AA) is an immune-mediated bone marrow aplasia that is strongly associated with clonal hematopoiesis upon marrow recovery. More than 70% of AA patients develop somatic mutations in their hematopoietic cells. In contrast to other conditions linked to clonal hematopoiesis, such as myelodysplastic syndrome (MDS) or clonal hematopoiesis of indeterminate potential in the elderly, the top alterations in AA are closely related to its immune pathogenesis. Nearly 40% of AA patients carry somatic mutations in the PIGA gene manifested as clonal populations of cells with the paroxysmal nocturnal hemoglobinuria phenotype, and 17% of AA patients have loss of HLA class I alleles. It is estimated that between 20% and 35% of AA patients have somatic mutations associated with hematologic malignancies, most characteristically in the ASXL1, BCOR, and BCORL1 genes. Risk factors for evolution to MDS in AA include the duration of disease, acquisition of high-risk somatic mutations, and age at AA onset. Emerging data suggest that several HLA class I alleles not only predispose to the development of AA but may also predispose to clonal evolution in AA patients. Long-term prospective studies are needed to determine the true prognostic implications of clonal hematopoiesis in AA. This article provides a brief, but comprehensive, review of our current understanding of clonal evolution in AA and concludes with 3 cases that illustrate a practical approach for integrating results of next-generation molecular studies into the clinical care of AA patients in 2018.

A Novel Cell Line, Tnet-2, Derived from “Triple-Negative” Essential Thrombocythemia

【Introduction】 Myeloproliferative neoplasm (MPN) is a heterogeneous group of disorders, including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). It is known that mutations of JAK2, CALR, or MPL gene are causative alternations of MPNs. However, 10-15% of MPNs have no mutations in any of these three genes, so-called “triple negative” (TN) MPN. It remains unclear that what genetic alternations cause TN-MPN. Cell lines derived from clonal hematopoietic disorders are useful tools to characterize diseases as well as to identify effective reagents for such diseases. To our knowledge, there is no cell line derived from TN-MPN although there are several lines with JAK2 V617F or CALR mutation. We have encountered an aggressive ET case with TN phenotype. Three years after initial diagnosis, blastic transformation occurred in this patient. Both conventional chemotherapy and allogeneic transplantation failed to cure the patient. After relapse, various therapies including ruxolitinib (JAK inhibitor) failed to control the disease. We have successfully established a cell line from the leukemic cells of this patient. We have also characterized the cell line by whole genome sequencing (WGS), RNA sequencing (RNA-Seq), as well as in vitro assays.【Material and Methods】 We have established a novel cell line, TNET-2, from the leukemic cells. WGS and RNA-Seq were performed at Beijing Genome Institute (BGI) Japan. Proliferation assays were performed in the presence or absence of ruxolitinib, AC220 (FLT3 inhibitor), giltertinib (FLT3/AXL inhibitor), etoposide, cytarabine or hydroxycarbamide.【Results】 The patient's leukemic cells have been cultured in RPMI1640 culture media with 10% fetal bovine serum and antibiotics. The cells were exponentially growing more than one year and the cell line, TNET-2, has been established. WGS has found 116 missense mutations in 110 genes encoding proteins. Also, five nonsense mutations causing early termination codons were found. InDel mutations causing frame-shifts were found in 28 genes. In-frame deletions were found in seven genes and in-frame insertions were found in nine genes. No mutations were found in JAK2, MPL, or CALR gene. A nonsense mutation was found in ASXL1 gene. Multiple genes encoding proteins associated with myeloid proliferation/differentiation, including CBL, A-RAF, HOXB9 and BCL11B, were mutated. Genes involved in DNA repair, for example ERCC6, were also mutated. Genes involved in apoptosis including CASP7, TRAK1 and SOCS3 were mutated. RNA-Seq revealed high expression of IRAK1 mRNA as well as low expression of TET2. Cell proliferation assays showed TNET-2 cells were resistant to ruxolitinib and conventional chemotherapeutic reagents including etoposide, cytarabine and hydroxycarbamide. However, both AC220 and giltertinib induced cell death.【Discussion】 It has been reported that alternations of ASXL1 and/or TET2 genes are common in TN-MPNs. Consistent with the reports, a nonsense mutation of ASXL1 gene and low expression of TET2 were detected in TNET-2 cells. Besides these well-known genetic changes, we have found more than 150 mutated genes. Some of them seem “actionable” / “targetable” mutations. There are no optimal treatments targeting an altered signaling pathway for TN-MPNs. Our novel cell line is a useful tool to characterize this subtype of MPN and find novel treatment options for this rare type of disease. In summary, we have, for the first time, established a cell line derived from TN-MPN and found novel altered signaling pathways by WGS, RNA-Seq and in vitro assays. DisclosuresNo relevant conflicts of interest to declare.

Clinical Utility of Routine Targeted Next-Generation Sequencing of Peripheral Blood in the Evaluation of Patients with Cytopenias

BackgroundWith the increasing accessibility of next-generation sequencing (NGS), many institutions are incorporating routine mutational profiling to assist in the evaluation of patients with a variety of hematologic disorders, including cytopenias. Such testing can be helpful in distinguishing neoplastic causes of cytopenias from others. Mutation profiling of peripheral blood is a particularly attractive option because of its potential application as a minimally invasive screen for hematologic malignancies, particularly myeloid neoplasms. However, it is not entirely clear how such molecular data can inform hematology practice as there is limited data on the clinical value of routine NGS testing in cytopenic patients. Here we report the clinical utility of peripheral blood screening by targeted NGS testing in a large institutional cohort of patients with cytopenias.MethodsAfter institutional review board approval, we identified all patients presenting with peripheral blood cytopenias over a 30-month period (January 2015 - June 2017) to the Adult Hematology Clinic at Dana-Farber/Brigham and Women's Cancer Center (n=1491). Of these, 244 patients (median age: 66, range: 22-93) underwent testing of a peripheral blood specimen using a custom 95-gene, amplicon-based sequencing panel (Rapid Heme Panel, Kluk et al., 2016) surveying genes recurrently mutated in hematologic malignancies (Table 1). In addition, these patients also received a complete hematologic work-up to determine the cause for the cytopenia(s). All patients with a known history of a hematologic malignancy were excluded.ResultsOverall, 60 (25%) patients had a pathogenic somatic mutation in at least one of the 95 genes studied (Figure 1). An underlying hematologic malignancy was identified in 26 (44%) of these patients at the time of presentation, most commonly a myeloid neoplasm (19/26; 73%); 11 (18%) had a non-neoplastic etiology of the cytopenia(s); and 23 (38%) had unexplained cytopenia(s) following complete work-up. The most frequently mutated genes in patients with unexplained cytopenias were TET2, SF3B1 and SRSF2. Of the 184 (75%) patients without a pathogenic somatic mutation, 15 (8%) of these patients were found to have an underlying hematologic malignancy at the time of presentation, most commonly a lymphoid neoplasm (11/15; 73%); 84 (46%) had a non-neoplastic etiology of the cytopenia(s); and 85 (46%) had unexplained cytopenia(s) following complete work-up. With a median follow-up of 22 months, two patients with non-clonal unexplained cytopenias went on to develop MDS and splenic marginal zone lymphoma, respectively. Overall, the presence of a pathogenic mutation was strongly associated with the diagnosis of a myeloid neoplasm (RR 11.6, 95% CI 4.5-29.8). Conversely, if no pathogenic mutation were identified, the likelihood of developing a myeloid neoplasm in 2 years was low (5/184; 2.7%).To further investigate the diagnostic utility of peripheral blood mutational profiling as a screening test in predicting the presence of a myeloid neoplasm on bone marrow biopsy, we studied its diagnostic characteristics using bone marrow biopsy diagnosis within a 6-month interval as the gold standard (n=57). The absence of a pathogenic mutation in peripheral blood was highly predictive of absence of a myeloid neoplasm on bone marrow biopsy (NPV 94%, 95% CI 84-99%). The presence of specific mutations in spliceosome genes (SF3B1, SRSF2, U2AF1) and co-mutation patterns involving DNMT3A, TET2 and ASXL1 genes and other genes were predictive of a myeloid neoplasm with positive predictive values of 75% (95% CI 41-93%) and 70% (95% CI 41-88%) respectively. Finally, larger clone sizes (≥20%) and higher numbers of mutations (≥2) were also predictive of a myeloid neoplasm (PPV: 80%, 95% CI 60-90% and 75%, 95% CI 49-90%, respectively).ConclusionsIn conclusion, mutation profiling of peripheral blood is a valuable minimally invasive test in the diagnostic work-up of the cytopenic patient. After a complete hematologic work-up and in the absence of a pathogenic somatic mutation, the likelihood of developing a myeloid neoplasm in 2 years is very low. Also, the lack of a pathogenic mutation in peripheral blood is highly predictive of the absence of a myeloid neoplasm on a bone marrow biopsy. Finally, certain molecular features (number and type of mutations, clone size) are predictive of a myeloid neoplasm. [Display omitted] DisclosuresKim:LabCorp, Inc.: Consultancy; Papgene, Inc: Consultancy; Aushon Biosciences: Consultancy.

Diagnostic Utility of Flow Cytometric Immunophenotyping in Chronic Myelomonocytic Leukemia

INTRODUCTIONThe diagnosis of chronic myelomonocytic leukemia (CMML) according to WHO 2017 requires the presence of ≥1x109/L and ≥10% of monocytes in peripheral blood (PB). Establish an accurate diagnostic is difficult since many clinical situations present persistent monocytosis. The presence of dysplasia, mainly dysgranulopoiesis, is frequent but not always present in CMML. Cytogenetic aberrations are infrequent in this disease (20-25% of cases). Although 85-90% of CMML patients present at least one mutation in TET2, SRSF2 or ASXL1 genes, the use of NGS panels is not widespread. Furthermore, mutations in these genes are among the most frequently observed in age-related clonal hematopoiesis. Therefore, complementary techniques are required to support the diagnosis of this entity. The study of the peripheral monocyte subsets by flow cytometry (FC) has gained special interest due to a high sensitivity and specificity for the diagnosis of CMML (S = 90.6%, E = 95.1%, Selimoglu-Buet et al., Blood, 2015). An increase in the fraction of classical monocytes (Mo1) to >94% of total monocytes is an event frequently observed in CMML. There are no specific bone marrow (BM) FC panels for the diagnosis of CMML and very few have been validated for the diagnosis of MDS. “Ogata score”, the only multicenter validated score in MDS, has not been applied in CMML. The aim of our study was to evaluate the usefulness of FC in PB and BM for the diagnosis of CMML.METHODSTwenty-two CMML were prospectively studied from 02/2016 to 04/2018. Patients' characteristics are summarized in Table 1. Diagnostic procedure consisted of morphological, cytochemical (Perls, myeloperoxidase, nonspecific esterase), cytogenetic and FC studies in BM, and morphological and FC studies in PB. “Ogata Score” was applied in BM samples (Table 2). Aberrant coexpression of CD2, CD7 and CD56 in BM monocytes was assessed. Immunophenotypic maturation profile of the monocytic elements in BM distinguishes: promonocytes (CD34-/CD117-/CD64++/CD14- or dim/CD45+/HLA-DR+++), mature monocytes (CD34-/CD117-/CD64++/CD14++/CD45++/HLA-DR++) and mature monocytes in terminal stage (CD300e+). In PB, the monocytes FC subsets (Mo1, Mo2 and Mo3) were studied, as well as the aberrant coexpression of CD2, CD7 and CD56 (Table 3).RESULTSThe presence of ≥2 aberrations in Ogata Score predicted properly the diagnosis of CMML in all patients analyzed (100% sensitivity). Due to the study design, we could not obtain results about specificity.An increase in Mo1 (classical monocytes) > 94% was detected in 18/20 patients (Table 3). This method predicted the diagnosis of CMML with a sensitivity of 91%, a result almost identical to the original study (Selimoglu-Buet et al., Blood, 2015).A good positive correlation was established between the percentage of BM promonocytes detected by morphology and by FC (Rho Spearman 0.61, P = 0.003).A negative correlation was found between the percentage of promonocytes by FC in MO and the expression of CD56 (Rho Spearman -0.612, P = 0.002). Similarly, CD56+ CMML presented a percentage of promonocytes by FC significantly lower than the CD56- CMML group (median: 24.5% (14-40) vs. 41% (23-71), P = 0.005). The expression of CD56 seems to be related to a more mature immunophenotypic profile of the monocytic population. On the other hand, the correlation between the percentage of CD56+ monocytes in BM and PB was almost perfect (Rho Spearman 0.928, P <0.001).CONCLUSIONOur findings support the usefulness of flow cytometry in bone marrow and peripheral blood for the diagnosis of CMML. DisclosuresNo relevant conflicts of interest to declare.

Combined Impact of Aberrant Immunophenotypes, Somatic Mutations and Cytogenetic Aberrations on the Probability of Developing Myelodysplasia in Patients with Cytopenias of Undetermined Significance

Background: Myelodysplastic syndromes (MDS) are diagnosed according to WHO based on cytomorphology and cytogenetics. Various conditions without dysplasia have been described which are associated with an increased although low risk of developing myeloid malignancy (Steensma et al. Blood 2015): non-clonal idiopathic cytopenias of undetermined significance (ICUS, cytopenias and no mutations), clonal hematopoiesis of indeterminate potential (CHIP, mutations and no cytopenias) and clonal cytopenias of undetermined significance (CCUS, both cytopenias and mutations). The spectrum of encountered mutations resembles the one of MDS. MDS-typical immunophenotypes have been identified and are recommended by ELN in the diagnostic work-up of patients (pts) with suspected MDS (Malcovati et al., Blood 2013). Similar to mutations described in both MDS and CHIP/CCUS, MDS-typical immunophenotypes are found in cytopenic non-MDS pts. Our study aims at the determination of the diagnostic potential of both of these findings.Aims: To determine the impact of aberrant immunophenotypes, somatic mutations and cytogenetic aberrations in the absence of dysplasia on the later diagnosis of MDS.Patients and methods: Between 2005 and 2018 a total of 2279 bone marrow samples of pts with suspected MDS were analyzed by a combination of cytomorphology and cytogenetics as well as NGS and flow cytometry. NGS targeted the following genes in all pts: ASXL1, CBL, DNMT3A, EZH2, JAK2, RUNX1, SF3B1, SRSF2, TET2, TP53 and U2AF1. Further genes analyzed in subsets of pts only were BCOR, CSF3R, CSNK1A1, ETV6, GATA1, GATA2, IDH1, IDH2, KIT, KRAS, NPM1, NRAS, PTPN11, SETBP1, WT1 and ZRSR2. Flow cytometry was performed according to ELN standards (Westers et al., Leukemia 2012). For 368 pts at least one follow-up bone marrow analysis was available, these pts are the subject of the present study. Patients ages ranged from 20 to 90 years (median 70 years), male:female ratio was 1.9:1.Results: 277/368 (75%) pts were diagnosed with MDS by cytomorphology. In 263/368 (72%) of pts MDS-typical immunophenotypes were present. Findings concordant between cytomorphology and flow cytometry were present in 304/368 (83%) pts reflecting published large series. Focus of further analyses were the 91 pts without diagnosis of MDS by cytomorphology. In 34 of these 91 pts MDS was diagnosed at follow-up, median follow-up time was 16.3 months, median time to MDS was 38.5 months. At initial evaluation, in 25 of these 91 (27%) pts MDS-typical immunophenotypes were present and resulted in a shorter time to MDS as compared to those without MDS-typical immunophenotypes (median 15.9 vs. 39.9 months, p=0.012). Frequencies of mutations with impact on time to MDS were as follows: ASXL1, present in 9/91 (10%), median time to MDS ASXL1 mutated vs non-mutated 14.2 vs. 39.9 months, p=0.010; EZH2, 3/91 (3%), 7.4 vs. 39.9 months, p=0.020; JAK2, 3/91 (3%), 4.9 vs. 39.9 months, p=0.003; RUNX1, 5/91 (5%), 7.4 vs. 39.9 months, n.s.; SRSF2, 7/91 (8%), 11.9 vs. 39.9 months, p=0.008; TET2, 14/91 (15%), 14.2 vs. 57.1 months, p=0.029; TP53, 4/91 (4%), 6.5 vs. 44.1 months, p=0.007. Other genes were less frequently found mutated without an impact on the time to MDS. An aberrant karyotype was present in 9 of 89 evaluable pts and was associated with a shorter time to MDS (median 4.9 vs. 57.1 months, p<0.001). Also age was associated with a shorter time to MDS (HR 1.47 per decade, p=0.029), peripheral blood cell counts were not. Patients with two out of the three parameters 1) aberrant immunophenotype, 2) at least one mutation in the genes ASXL1, EZH2, JAK2, RUNX1, SRSF2, TET2, TP53, 3) aberrant karyotype had a significantly shorter duration to MDS as compared to those without: 1) and 2), 7.4 vs. 44.1 months, p<0.001; 1) and 3), 4.9 vs. 39.9 months, p=0.004; 2) and 3), 16.0 vs. 44.1 months, p=0.041. Patients presenting with any two out of the parameters 1), 2) and 3) had a highly significantly shorter time to MDS as compared to those presenting with only one or none: 6.5 vs. 57.1 months, p<0.0001.Conclusions: In the absence of cytomorphologic signs of dysplasia, findings commonly present in MDS patients, i.e. aberrant immunophenotypes, somatic mutations and cytogenetic aberrations, occur in patients with cytopenias of undetermined significance. The presence of two of these three findings may be considered as a pre-diagnostic state of MDS. Further studies should clarify the diagnostic potential of this approach. DisclosuresKern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Incorporation of Clinical Information to Decipher Driver Mutations in Myeloproliferative Neoplasms

Introduction: Mutations in the epigenetic regulators are commonly found in myeloid disorders including Myeloproliferative Neoplasm (MPN). Primary myelofibrosis, dysplastic changes and severity of the disease were associated with the mutation load. Most of the studies had a limited number of targeted genes and included a mixture of JAK2 positive and negative disease. The objective of this study is to assess the impact of mutations in the epigenetic regulators on the presentation of patients with JAK2 V617F positive MPN.Methods: We retrieved the clinical and laboratory information on 61 consecutive eligible patients. Mutation analysis of the entire coding regions of ASXL1, ASXL2, CBL, CEBPA, CSF3R, DNMT3A, EZH2, IDH2, TET1 and TET2 genes was performed using next generation sequencing (NGS; Ion PGM Sequencer; Thermo Fisher ScientificÒ). The library was constructed and the templates were prepared using the PGM tool. The variants were annotated using the ClinVar database and the prediction from the Scale-Invariant Feature Transform (SIFT) and or Polymorphism Phenotyping (Polyphen) algorithms. Alignment, variant filtering and annotation were performed using Ion Torrent Suite. Standard descriptive and analytical statistics were used as appropriate to describe and compare different groups. The MPN subtype, bleeding, thrombosis, hemoglobin, platelet count, White Cell Count (WBC), Lactate Dehydrogenase (LDH) and erythropoietin level were compared for each candidate variant. An alpha threshold of 0.05 was used with no adjustment for multiple comparisons as the analyses were considered exploratory. All statistics were performed using R program. Variants were selected for further experimentation based on their frequency and association with the clinical information at diagnosis.Results: Sixty-one patients were included (Polycythemia Vera: 29, Essential Thrombocythemia: 21, Primary Myelofibrosis: 9, MPN unclassifiable: 2) with a median age of 62 years (Interquartile Range [IQR]: 44 - 70). Male to female ratio was 35:26. The median hemoglobin, WBC, platelet count, LDH and erythropoietin were 14.6 g/dL (IQR: 12.8 - 16.8), 11.5 *109/L (IQR: 11.5 - 14.4), 507 *109/L (IQR: 391 - 779), 265 mU/mL (IQR: 231 - 409) and 1.2 mU/mL (IQR: 1.0 - 4.8) respectively. At presentation, 54% had splenomegaly, 23% had an arterial or venous thrombosis, and 5% had bleeding. Sixty-three variants were found in the samples tested. The median mutation load was 13 variants (Range: 11-14). Patients with higher than the median mutation load had higher mean erythropoietin (7.8 vs. 0.9 g/dL; p = 0.02016). ASXL1 p.Leu815Pro variant was found in all patients. Only three variants were found in the ClinVar database. Seven variants were predicted to be pathogenic (ASXL1: 1, EZH2: 1, IHD1: 1, TET1: 1 and TET2: 3). Patients with TET2: p.Leu1721Trp variant had 6.4 higher odds of bleeding (p = 0.04345). Patients with TET2: p.His1778Arg variant had a lower WBC (9.1 vs. 13.9 *109/L; p = 0.01699) and LDH (213 vs. 348 mU/mL; p = 0.0006528) while those with TET1: p.Lle1123Met variant had a higher WBC (13.5 vs. 8.3 *109/L; p = 0.02756). None of the remaining comparisons were statistically significant.Conclusions: Incorporation of clinical information facilitates the prioritization of variants from DNA sequencing experiments. In MPN, we recommend ASXL1: p.Leu815Pro (expressed in all patients), TET2: p.Leu1721Trp (associated with bleeding), TET2: p.His1778Arg (correlated with the WBC and LDH) and TET1: p.Lle1123Met (correlated with LDH) for further functional experimentation. DisclosuresNo relevant conflicts of interest to declare.

Prospective Evaluation of Prognostic Relevance of KIT Mutations in Core-Binding Factor Acute Myeloid Leukemia: Results from the JALSG CBF-AML209-KIT Study

Prospective Evaluation of Prognostic Relevance of KIT Mutations in Core-Binding Factor Acute Myeloid Leukemia: Results from the JALSG CBF-AML209-KIT Study

Clinical Significance of Germ Line and Acquired Somatic Mutations of 30 High Frequency Genes in Hematologic Malignancies

Objective: To analyse germ line and acquired somatic mutations of 30 high frequency genes and explore their clinical significance in hematologic malignancies. Methods: A total of 254 patients with hematologic diseases (145 males and 109 females) from Jan 2017 to Apr 2018 in the Southwest China were included and the samples from bone marrow and oral mucosa were collected, and the gene mutations of germ line and acquired somatic cells were examined by the next-generation sequencing technology or PCR. The frequency and locus of germ line and acquired somatic mutations of 30 high frequency genes, as well as their clinical significance to the diagnosis and classification in hematologic diseases were analysed. Results: 1.The frequency and locus of germ line and acquired somatic mutations, and their clinical significance: no mutation was detected in 24 cases (9.4%), and a total of 659 genetic locus mutations were detected in 230 cases (90.6%), including 520 somatic cell mutations (78.9%) and 139 germ line mutations (21.1%). The results of gene mutations are listed in order: the number of acquired somatic mutation: the number of germ line mutation: common mutation locus: the type of hematological disease for the patients diagnosed in the West China hospital, as described in Table 1. 2.Characteristics of acquired somatic and germ line mutations: the genes of WT1, U2AF1, TP53, TET2, STAG2, RUNX1, PIGA, NRAS, KIT, IDH2, EZH2, DNMT3A, CBL and BCOR were dominated by acquired somatic mutations, while the genes of ZRSR2, JAK2 and BCORL1 were dominated by germ line mutations, the genes of SRSF2, SF3B1, SETBP1, PTPN11, NPM1, PHF6, KRAS, IDH1, FLT3-ITD, ETV6 and CEBPA were detected only in acquired somatic mutations, BCOR and ASXL1 have a similar proportion of acquired somatic and germ line mutation; only 42-4C>A of ZRSR2 mutation was existed in both somatic and germ line, however, the mutation sites of the acquired somatic and germ line of other genes have not been found to be existed at the same time. 3.The clinical significance of the acquired somatic and germ line mutation: It was found that the genes of TP53, SF3B1, EZH2 and DNMT3A tend to appear in MDS, the genes of ZRSR2, WT1, U2AF1, TET2, RUNX1, NRAS, NPM1, KRAS, KIT, IDH1/2, FLT3-ITD, CEBPA and BCOR tend to appear in AML, and the similar probability of the STAG2, PTPN11, PHF6, ETV6, CBL, BCORL1, ASXL1 and NF1 genes appear in AML and MDS, while the genes of PIGA tend to appear in PNH, and BCOR in AA. Conclusion: The acquired somatic and germ line mutations are significantly different in the frequency, mutation site and the type of hematological disease, and the further research will clarify their potential roles in the pathogenesis of hematological tumors, and have important clinical significance for the classification and prognosis of the hematological diseases.Key words: gene mutation, somatic, germ line, hematologic malignancies DisclosuresNo relevant conflicts of interest to declare.

A 17-Gene Leukemia Stem Cell (LSC) Score in Adult Patients (Pts) with Acute Myeloid Leukemia (AML) Reveals a Distinct Mutational Landscape and Refines Current European Leukemianet (ELN) Genetic Risk Stratification

Introduction: Prognosis of AML pts is still poor mainly because of refractoriness to or relapse after intensive chemotherapy. High rates of relapse are also attributed to LSCs, which are a small subset of cells with acquired abnormal self-renewal capacity and increased resistance to chemotherapy. A better understanding of LSCs is critical to improve outcomes of pts with AML. Ng et al. (Nature 2016;540:433) defined a 17 stemness-associated gene score that was highly prognostic.Aims: The aim of this study was to validate the prognostic relevance of the 17-gene LSC score and explore its utility in the context of the ELN classification. We also examined gene mutations associated with the 17-gene LSC score.Methods: We analyzed a total of 934 pts [729 aged <60 years (y) and 205 aged ≥60 y] with de novo AML. We used whole transcriptome expression data (RNAseq) to calculate the aforementioned 17-gene LSC score for each pt in our cohort. Similar to Ng et al., we used the median of the whole cohort to discriminate between pts with LSChigh and LSClow scores. The mutational status of 80 cancer- and leukemia-associated genes (Eisfeld et al. Leukemia 2017;31:2211) were determined using a targeted next-generation sequencing panel, CEBPA mutations using Sanger sequencing, and an internal tandem duplication (ITD) of the FLT3 gene using fragment analysis in pretreatment bone marrow or blood samples. All pts were treated on frontline Cancer and Leukemia Group B/Alliance protocols.Results: A comparison of pretreatment clinical and genetic features revealed that LSChigh pts were older (P<.001; median age, 53 vs 46 y) and had higher platelet counts (P<.001; median, 63 vs 50x109/L) than LSClow pts. Pts with a LSChigh score more frequently had FLT3-ITD (P<.001) and mutations in the ASXL1 (P=.001), DNMT3A (P<.001), RUNX1 (P=.002), SRSF2 (P=.02), STAG2 (P=.009), TET2 (P=.008) and TP53 (P<.001) genes. Conversely, these pts had a lower frequency of biallelic CEBPA (P<.001), GATA2 (P=.008) and KIT (P<.001) mutations.Because of differences in treatment intensity, we analyzed outcomes of younger and older pts separately. Younger pts with a LSChigh score had a lower complete remission (CR) rate (P<.001; 63% vs 87%), shorter disease-free survival (DFS; P<.001; 3-y rates, 26% vs 48%; Figure 1A) and overall survival (OS; P<.001; 3-y rates, 27% vs 59%; Figure 1B) compared to those of LSClow pts. In multivariable analyses including clinical and genetic factors that impact on outcome, a LSChigh score associated with lower remission rates (P<.001; HR: 0.36), shorter DFS (P<.001; HR: 1.67) and OS (P<.001; HR: 1.88) after adjusting for other co-variates. We also analyzed the prognostic impact of the LSC score with respect to the 2017 ELN classification. We found that LSC score associated with different ELN groups (P<.001), with LSChigh pts being more often classified in the Adverse or Intermediate group and less often in the Favorable group. Within the ELN Favorable and Adverse groups, LSChigh score retained its prognostic impact and identified pts with a lower CR rate and shorter DFS and OS (Table1).In older pts, a LSChigh score also associated with lower CR rate (P=.004; 50% vs 72%), shorter DFS (P=.04; 3-y rates, 6% vs 17%; Figure 1C) and OS (P<.001; 3-y rates, 9% vs 27%; Figure 1D). In multivariable analyses, LSC score remained significant only for OS (P<.003; HR: 1.70) after adjusting for other co-variates. Regarding the ELN classification, pts with LSChigh score in the Favorable group had shorter OS (P=.05; 3-y rates, 17% vs 50%) and, by trend, shorter DFS (P=.09; 3-y rates, 17% vs 39%); no significant differences were found in Intermediate or Adverse groups.Conclusions: We used RNAseq expression data and applied the previously established 17-gene LSC signature to score 934 de novo AML pts. We detected distinct mutational differences between LSChigh and LSClow pts, with LSChigh pts more often carrying gene mutations associated with age-related clonal hematopoiesis (i.e., ASXL1, DNMT3A, TET2, SRSF2 and TP53 mutations). Moreover, this score, derived from the expression of stemness-associated genes, has not only a prognostic impact on its own but also in the context of the current 2017 ELN classification. [Display omitted] DisclosuresKolitz:Magellan Health: Consultancy, Honoraria. Powell:Rafael Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Targeted Next Generation Sequencing for Myeloid Neoplasm in Pakistani Patients

Introduction:Myeloid malignancies are heterogenous diseases caused by excessive accumulation of apparently myeloid clone of cells. Genomic studies on myeloid malignancies in recent years have identified new genetic alterations with biological and clinical significance. In addition to cytogenetics and morphological examination these genetic mutation play an important role in diagnosis, prognosis and treatment of the patient. We assessed the frequency and clinicopathologic significance of 54 genes in myeloid neoplasm patients by using targeted next-generation sequencing.Methods:About 50 samples were collected from OPD at National Institute of Blood Diseases (NIBD), that consisting of 17 MDS, 18 AML and other myeloid neoplasms. They comprises of 33 males and 17 females with median age of 33 years (range: 5-69 years). The myeloid sequencing panel of 54 genes (complete coding exons of 15 genes and exonic hotspots of 39 genes) was sequenced. The panel total coverage was 141 kb in genomic sequence. TruSight myeloid sequencing (Illumina, CA) libraries were prepared and runs were performed on a MiSeq (Illumina) genome sequencer. The generated data were analyzed by on-instrument software or TruSeq Amplicon® and BaseSpace Apps®.Results:Overall 3092 variants were identified, after excluding intronic and synonymous variants, 380 missense variants were found in 50 patients. Around 38 mutations in 22 genes were identified in 23 out of 50 samples (46 %). The recurrent mutations found in RUNX1, ASXL1, GATA2 and CEPBA genes in our cohort.Conclusion:Most of the myeloid neoplasms are not easily manageable with limited treatment options. Therefore, targeted gene panel by next generation sequencing was an appropriate method for precise identification of mutations in myeloid neoplasms at our institution. Based on the obtained findings we will be able to design patient management plan with respect to individualize genetic mutations in the clinical setting. DisclosuresNo relevant conflicts of interest to declare.

Monosomy 7 As the Initial Hit Followed By Sequential Acquisition of SETBP1 and ASXL1 Driver Mutations in Childhood Myelodysplastic Syndromes

Childhood myelodysplastic syndromes (MDS) account for less than 5% of pediatric hematologic malignancies and differ from their adult counterpart in terms of biology, genetics, and cure rates. Complete (-7) or partial loss (del7q) of chromosome 7 constitutes the most common cytogenetic abnormality and is associated with more advanced disease typically requiring timely hematopoietic stem cell transplantation (HSCT). Previously, we and others established a link between -7 and germline GATA2 mutations in pediatric MDS (37% of MDS/-7 cases are GATA2-deficient) as well as constitutional SAMD9/9L disorders where -7 is utilized as an escape mechanism from the growth-restrictive effect of SAMD9/9L mutations. To date, comprehensive sequencing studies have been performed in 96 children with primary MDS, as reported by Pastor et al, Leukemia 2017 and Schwartz et al, Nature Comm 2017. This work established mutations in SETBP1, ASXL1, PTPN11, RUNX1 and RAS pathway genes as common somatic drivers. However, little is known about the clonal development of -7 and the role of additional somatic mutations. The knowledge about clonal hierarchies is essential for the understanding of disease progression on molecular level and for mapping potential drug targets. The rationale for the current study was to i) define the most common somatic drivers in a large cohort of patients with childhood MDS, ii) identify clonal/subclonal mutations, iii) infer clonal architecture of monosomy 7 and track the changes over time.We studied a cohort of 576 children and adolescents with primary MDS diagnosed between 1998 and 2016 in Germany, consisting of 482 (83%) patients with refractory cytopenia of childhood (RCC) and 94 (17%) MDS with excess blasts (EB). All patients underwent deep sequencing for 30 genes relevant to pediatric MDS and additional WES was performed in 150/576 patients. Using 20 computational predictors (including CADD and REVEL), population databases and germline testing, we identified the most likely pathogenic mutations.First, we excluded germline predisposing mutations in GATA2, SAMD9/SAMD9L and RUNX1 detected in 7% (38/576), 8% (43 of 548 evaluable) and 0.7% (4/576) of patients, respectively. Then we focused on the exploration of somatic aberrations. Most common karyotype abnormalities were monosomy 7 (13%, 77/576) and trisomy 8 (3%, 17/576). A total of 104 patients carried somatic mutations, expectedly more prevalent in the MDS-EB group as compared to RCC (56%, 53/94 vs 10.6%, 51/482; p<0.0001). The most recurrent somatic hits (≥ 1% frequency within 576 cases) were in SETBP1 (4.2%), ASXL1 (3.8%), RUNX1 (3.3%), NRAS (2.9%), KRAS (1.6%), PTPN11 (1.4%) and STAG2 (1%). We next focused on the -7 karyotype as a common denominator for the mutated group. Mutations were found in 54% (43/79), and the mutational load was significantly higher in -7 vs. non-7 (1.1 vs. 0.1 mutations per patient; p<0.001). In 11 patients with -7 and concomitant SETBP1/ASXL1 driver mutations, SETBP1 surpassed ASXL1 hits (median allelic frequency: 38% vs. 24%, p<0.05), while mutations in other genes were subclonal. Notably, these clonal patterns were independent of the underlying hereditary predisposition (4/11 GATA2; 3/11 SAMD9L). To explore the clonal hierarchy in MDS/-7 we performed targeted sequencing of several hundreds of single bone marrow derived colony forming cells (CFC) in 7 patients with MDS/-7. In all cases, the -7 clone was the founding clone followed by stepwise acquisition of mutations (i.e. -7>SETBP1>ASXL1; -7>SETBP1>ASXL1>PTPN11; -7>SETBP1>ASXL1>CBL, -7>EZH2>PTPN11). Finally, we tracked clonal evolution over time in 12 cases with 2-12 available serial samples using deep sequencing complemented by serial CFC-analysis. This confirmed that SETBP1 clones are rapidly expanding, while ASXL1 subclones exhibit an unstable pattern with clonal sweeping, while additional minor clones are acquired as late events. In 2 of 11 transplanted patients who experienced relapse, the original clonal architecture reappeared after HSCT.In summary, the hierarchy of clonal evolution in pediatric MDS with -7 follows a defined pattern with -7 aberrations arising as ancestral event followed by the acquisition of somatic hits. SETBP1 mutations are the dominant driver while co-dominant ASXL1 mutations are unstable. The functional interdependence and potential pharmacologic targetability of such somatic lesions warrants further studies. DisclosuresNiemeyer:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Correlations between Pre-Transplant Treatment and Bone Marrow Response with Post-Transplant Outcomes in Patients with Myelodysplastic Syndrome Undergoing Allogeneic Stem Cell Transplantation

Correlations between Pre-Transplant Treatment and Bone Marrow Response with Post-Transplant Outcomes in Patients with Myelodysplastic Syndrome Undergoing Allogeneic Stem Cell Transplantation