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    The establishment of a prognostic scoring model based on the new tumor immune microenvironment classification in acute myeloid leukemia. Zeng Tiansheng,Cui Longzhen,Huang Wenhui,Liu Yan,Si Chaozeng,Qian Tingting,Deng Cong,Fu Lin BMC medicine BACKGROUND:The high degree of heterogeneity brought great challenges to the diagnosis and treatment of acute myeloid leukemia (AML). Although several different AML prognostic scoring models have been proposed to assess the prognosis of patients, the accuracy still needs to be improved. As important components of the tumor microenvironment, immune cells played important roles in the physiological functions of tumors and had certain research value. Therefore, whether the tumor immune microenvironment (TIME) can be used to assess the prognosis of AML aroused our great interest. METHODS:The patients' gene expression profile from 7 GEO databases was normalized after removing the batch effect. TIME cell components were explored through Xcell tools and then hierarchically clustered to establish TIME classification. Subsequently, a prognostic model was established by Lasso-Cox. Multiple GEO databases and the Cancer Genome Atlas dataset were employed to validate the prognostic performance of the model. Receiver operating characteristic (ROC) and the concordance index (C-index) were utilized to assess the prognostic efficacy. RESULTS:After analyzing the composition of TIME cells in AML, we found infiltration of ten types of cells with prognostic significance. Then using hierarchical clustering methods, we established a TIME classification system, which clustered all patients into three groups with distinct prognostic characteristics. Using the differential genes between the first and third groups in the TIME classification, we constructed a 121-gene prognostic model. The model successfully divided 1229 patients into the low and high groups which had obvious differences in prognosis. The high group with shorter overall survival had more patients older than 60 years and more poor-risk patients (both P< 0.001). Besides, the model can perform well in multiple datasets and could further stratify the cytogenetically normal AML patients and intermediate-risk AML population. Compared with the European Leukemia Net Risk Stratification System and other AML prognostic models, our model had the highest C-index and the largest AUC of the ROC curve, which demonstrated that our model had the best prognostic efficacy. CONCLUSION:A prognostic model for AML based on the TIME classification was constructed in our study, which may provide a new strategy for precision treatment in AML. 10.1186/s12916-021-02047-9
    A novel scoring system integrating molecular abnormalities with IPSS-R can improve the risk stratification in patients with MDS. Gu Siyu,Xia Jingya,Tian Yulu,Zi Jie,Ge Zheng BMC cancer BACKGROUND:The treatment strategies for Myelodysplastic Syndromes (MDS) are usually based on the risk stratification system. However, few risk signatures which integrate the revised international prognostic scoring system (IPSS-R) with gene mutations can be easily applied in the real world. METHODS:The training cohort of 63 MDS patients was conducted at Zhongda Hospital of Southeast University from January 2013 to April 2020. The validation cohort of 141 MDS patients was obtained from GSE129828. The mutation scoring system was based on the number of mutations and a unique favorable prognostic factor, which is SF3B1 mutation. Univariate Cox, multivariate Cox, and LASSO regression analyses were used to determine the significant factors that influenced the overall survival. The receiver operating characteristic curve (ROC) was used to evaluate the efficiency of the prognostic model. RESULTS:A novel risk scoring system we named "mutation combined with revised international prognostic scoring system (MIPSS-R)" was developed based on the results derived from multivariate analysis which assigned points to the IPSS-R and the mutation scores according to their relative statistical weight. Based on the quintile of the new scores, patients were divided into five risk levels. The Kaplan-Meier curves showed the superiority of MIPSS-R in separating patients from different groups, comparing with IPSS-R both in the training cohort (p = 1.71e-08 vs. p = 1.363e-04) and validation cohort (p = 1.788e-04 vs. p = 2.757e-03). The area under the ROC of MIPSS-R was 0.79 in the training cohort and 0.62 in the validation cohort. The retrospective analysis of our house patients showed that the risk levels of 57.41% of patients would adjust according to MIPSS-R. After changing risk levels, 38.71% of patients would benefit from treatment strategies that MIPSS-R recommends. CONCLUSION:A mutation scoring system was conducted based on the number of mutations and a unique favorable prognostic factor. MIPSS-R, the novel integral risk stratification system was developed by integrating IPSS-R and the mutation scores, which is more effective on prognosis and treatment guidance for MDS patients. 10.1186/s12885-021-07864-y
    Evaluation of different scoring systems and gene mutations for the prognosis of myelodysplastic syndrome (MDS) in Chinese population. Du Meng-Yi,Xu Min,Deng Jun,Liu Lin,Guo Tao,Xia Ling-Hui,Hu Yu,Mei Heng Journal of Cancer MDS is a heterogeneous disease with diverse clinical manifestations, and an effective prognostic evaluation tool for MDS patients is needed. To achieve more accurate prognosis assessment for Chinese MDS patients, here we examined several scoring systems and explored the implications of gene mutations. The prognostic conditions were stratified against three different score systems (International Prognostic Scoring System (IPSS), WHO Prognostic Scoring System (WPSS), and Revised International Prognostic Scoring System (IPSS-R)) were retrospectively applied to 110 de novo MDS patients in study cohort in our hospital and the prognostic conditions were stratified respectively. IPSS-R out-performed the others, since it had less overlaps in survival curve, especially in the relatively low-risk group. Furthermore, genetic mutations were identified in 84 out of 110 patients and their association with overall survival (OS) were determined. Among them, sixty-three percent patients had at least one-point mutation, including thirty-five patients with normal karyotypes. The presence of TP53 mutations, but not TET2, DNMT3A or ASXL1 mutations was significantly correlated with shorter OS. A new model incorporating IPSS-R and TP53 mutations into survival analysis was proposed, and the prognostic value of this model was validated to be predominant in a 190-primary MDS patient independent cohort. Our data suggested that IPSS-R was more suitable for Chinese population. Attentions should be paid to the unfavourable mutations that might exert impact on the survival, especially in patients with relatively low risk. 10.7150/jca.30363
    Does mutational burden add to other prognostic factors in MDS? Nazha Aziz Best practice & research. Clinical haematology Myelodysplastic syndromes (MDS) are clonal bone marrow disorders characterized by complex genomic abnormalities that define disease phenotype, prognosis, and progression. The overall outcomes of MDS patients are very heterogeneous and can be measured in months in some patients and years in others. Several scoring systems have been developed in MDS, with the International Prognostic Scoring System (IPSS) and its revised version (IPSS-R) the most widely accepted risk stratification tools in clinical practice and trial eligibility. Recently, somatic mutations have been shown to impact overall survival and the risk of progression to acute myeloid leukemia. Attempts to add this information to current models or develop newer models are underway, but the optimal approach remains controversial. Newer methods to develop a personalized prediction model that provides outcomes specific for a patient were developed and could change the prognostic paradigm for MDS patients in the near future. 10.1016/j.beha.2019.101098
    Comparison of clinical outcomes and prognostic utility of risk stratification tools in patients with therapy-related vs de novo myelodysplastic syndromes: a report on behalf of the MDS Clinical Research Consortium. Zeidan A M,Al Ali N,Barnard J,Padron E,Lancet J E,Sekeres M A,Steensma D P,DeZern A,Roboz G,Jabbour E,Garcia-Manero G,List A,Komrokji R Leukemia While therapy-related (t)-myelodysplastic syndromes (MDS) have worse outcomes than de novo MDS (d-MDS), some t-MDS patients have an indolent course. Most MDS prognostic models excluded t-MDS patients during development. The performances of the International Prognostic Scoring System (IPSS), revised IPSS (IPSS-R), MD Anderson Global Prognostic System (MPSS), WHO Prognostic Scoring System (WPSS) and t-MDS Prognostic System (TPSS) were compared among patients with t-MDS. Akaike information criteria (AIC) assessed the relative goodness of fit of the models. We identified 370 t-MDS patients (19%) among 1950 MDS patients. Prior therapy included chemotherapy alone (48%), chemoradiation (31%), and radiation alone in 21%. Median survival for t-MDS patients was significantly shorter than for d-MDS (19 vs 46 months, P<0.005). All models discriminated survival in t-MDS (P<0.005 for each model). Patients with t-MDS had a significantly higher hazard of death relative to d-MDS in every risk model, and had inferior survival compared to patients with d-MDS within all risk group categories. AIC Scores (lower is better) were 2316 (MPSS), 2343 (TPSS), 2343 (IPSS-R), 2361 (WPSS) and 2364 (IPSS). In conclusion, subsets of t-MDS patients with varying clinical outcomes can be identified using conventional risk stratification models. The MPSS, TPSS and IPSS-R provide the best predictive power. 10.1038/leu.2017.33
    The utility of a myeloid mutation panel for the diagnosis of myelodysplastic syndrome and myelodysplastic/myeloproliferative neoplasm. Ibrar Warda,Zhang Weiwei,Cox Jesse Lee,Cushman-Vokoun Allison,Fu Kai,Greiner Timothy C,Yuan Ji International journal of laboratory hematology INTRODUCTION:The diagnosis of myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) is based on morphology and cytogenetics/FISH findings per 2017 WHO classification. With rare exceptions, somatic mutations have not been incorporated as the diagnostic criteria. METHODS:We analyzed the utility of mutational analysis with a targeted 54-gene or 40-gene next-generation sequencing (NGS) panel in the diagnosis of MDS and MDS/MPN. RESULTS:We retrospectively collected 92 patients who presented with unexplained cytopenia with or without cytosis, including 32 low-grade MDS (MDS-L), 18 high-grade MDS (MDS-H), 5 therapy-related MDS (MDS-TR), 19 MDS/MPN, and 18 negative cases. Of 92 patients, 197 somatic mutations involving 38 genes were detected and had variant allele frequency (VAF) ranging from 3% to 99%. The most common mutated genes were TET2, ASXL1, RUNX1, TP53, SRSF2, and SF3B1. MDS-L, MDS-H, MDS-TR, and MDS/MPN showed an average number of somatic mutations with a mean VAF of 1.9/33%, 2.6/30%, 2/36%, and 4/41%, respectively. SF3B1 mutations were exclusively observed in MDS-L and MDS/MPN. TP53 gene mutations were more frequently seen in MDS-H and MDS-TR. Among 34 patients with a diagnosis of MDS or MDS/MPN with normal cytogenetics, 31 patients (91%) had at least 1 mutation and 24 patients (71%) had ≥2 mutations with ≥10% VAF. CONCLUSION:A myeloid mutational panel provides additional evidence of clonality besides cytogenetics/FISH studies in the diagnosis of cytopenia with or without cytosis. Two or more mutations with ≥10% VAF highly predicts MDS and MDS/MPN with a positive predictive value of 100%. 10.1111/ijlh.13659
    Design, implementation and clinical utility of next generation sequencing in myeloid malignancies: acute myeloid leukaemia and myelodysplastic syndrome. Hughes Charlotte F M,Gallipoli Paolo,Agarwal Rishu Pathology Next generation sequencing (NGS) based technology has contributed enormously to our understanding of the biology of myeloid malignancies including acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS). Assessment of clinically important mutations by NGS is a powerful tool to define diagnosis, determine prognostic risk, monitor measurable residual disease and uncover predictive mutational markers/therapeutic targets, and is now a routine component in the workup and monitoring of haematological disorders. There are many technical challenges in the design, implementation, analysis and reporting of NGS based results, and expert interpretation is essential. It is vital to distinguish relevant somatic disease associated mutations from those that are known polymorphisms, rare germline variants and clonal haematopoiesis of indeterminate potential (CHIP) associated variants. This review highlights and addresses the technical and biological challenges that should be considered before the implementation of NGS based testing in diagnostic laboratories and seeks to outline the essential and expanding role NGS plays in myeloid malignancies. Broad aspects of NGS panel design and reporting including inherent technological, biological and economic considerations are covered, following which the utility of NGS based testing in AML and MDS are discussed. In current practice, patient care is now strongly shaped by the results of NGS assessment and is considered a vital piece of the puzzle for clinicians as they manage these complex haematological disorders. 10.1016/j.pathol.2021.01.005
    Impact of gene variants on iron overload, overall survival and leukemia-free survival in myelodysplastic syndromes. Schneeweiss-Gleixner Mathias,Greiner Georg,Herndlhofer Susanne,Schellnegger Julia,Krauth Maria-Theresa,Gleixner Karoline V,Wimazal Friedrich,Steinhauser Corinna,Kundi Michael,Thalhammer Renate,Schwarzinger Ilse,Hoermann Gregor,Esterbauer Harald,Födinger Manuela,Valent Peter,Sperr Wolfgang R American journal of cancer research Although iron overload is a clinical challenge, little is known about the clinical impact of -variants in myelodysplastic syndromes (MDS) to date. We analyzed the status in 167 MDS patients and 494 healthy controls. One or more of the 3 -variants (H63D, C282Y, S65C) were found in 65/167 (38.9%) MDS patients and in 164/494 (33.2%) controls. At diagnosis, the median serum ferritin levels were higher in MDS patients with -variants (409 µg/L; range: 23-7415) compared to those without -variants (346.5 µg/L; range: 10-5450) (P=0.62). Moreover, '-mutated' patients had a slightly faster increase in serum ferritin in follow up examinations. The percentage of patients with -variants was higher in refractory anemia (RA) (22/53=41.5%) or RA with ring sideroblasts (RARS) (17/39=43.6%) compared to RA with excess of blasts (RAEB) (16/46=34.8%) or RAEB in transformation (RAEB-T) (5/17=29.4%). Differences were also detectable when comparing low- and high-risk MDS variants defined by the World Health Organization classification. There was no significant correlation between -variants and MDS-related somatic mutations. Progression-free survival was substantially longer in patients with -variants compared to those without -variants H63D and C282Y (P=0.089). Together, the -variants H63D and C282Y are frequently detected in Austrian MDS patients. These patients have substantially higher ferritin levels at diagnosis, accumulate iron slightly faster and have a better progression-free survival than non-mutated patients.
    Getting personal with myelodysplastic syndromes: is now the right time? Chokr Nora,Pine Alexander B,Bewersdorf Jan Philipp,Shallis Rory M,Stahl Maximilian,Zeidan Amer M Expert review of hematology INTRODUCTION:Commonly used scoring systems rely on blood counts, histological and cytological examination of bone marrow and peripheral blood as well as cytogenetic assessments to estimate prognosis of patients with myelodysplastic syndromes (MDS) and guide therapy decisions. Next-generation sequencing (NGS) has identified recurrent genetic abnormalities in up to 90% of patients with MDS and may provide important information regarding the pathogenesis of the disease, diagnostic and prognostic evaluation, and therapy selection. Areas covered: Herein, the authors review the role of NGS in diagnosis, treatment, and prognosis of MDS at various disease stages, and discuss advantages and caveats of incorporating molecular genetics in routine management of MDS. While a vast majority of patients harbor recurrent mutations implicated in MDS pathogenesis, similar mutations can be detected in otherwise healthy individuals with other hematologic malignancies. Besides establishing a diagnosis, NGS may be used to monitor minimal residual disease following treatment. Expert opinion: As more targeted therapies become available, assessment of genetic mutations will become central to individualized therapy selection and may improve diagnostic accuracy and further guide management for each patient. However, multiple challenges remain before NGS can be incorporated into routine clinical practice. 10.1080/17474086.2019.1592673
    Pathogenic Mutations and Atypical Flow Cytometric Findings Characterize the Majority of Unclassifiable Myelodysplastic/Myeloproliferative Neoplasms. Li Yanchun,Beck Rose C,Moore Erika M American journal of clinical pathology OBJECTIVES:Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are a group of rare and heterogeneous hematopoietic disorders that frequently present a diagnostic challenge. Here we present our institutional experience with next-generation sequencing (NGS), together with morphologic, flow cytometric, and cytogenetic evaluation, in the diagnosis of MDS/MPN, with particular emphasis on MDS/MPN unclassifiable (MPN-U). METHODS:We evaluated the morphologic, flow cytometric, cytogenetic, and molecular characteristics of all MDS/MPN cases that underwent NGS at our institution between April 2016 and February 2019. RESULTS:Thirty-seven cases of MDS/MPN were identified, including 14 cases of MDS/MPN-U. Ninety-seven percent harbored mutations and immunophenotypic aberrancies (36/37), while only 38% had cytogenetic abnormalities (12/32). The MDS/MPN-U group had the highest rate of myeloblast phenotypic abnormalities and had a high mutation rate of approximately 2.7 mutated genes per case, most commonly in JAK2, SRSF2, and ASXL1. CONCLUSIONS:No single ancillary study was abnormal in every case, but all cases had at least one abnormal finding, demonstrating the usefulness of a multiparameter approach to the diagnosis of MDS/MPN. Although a few specific mutations were found exclusively in MDS/MPN-U and JAK2 mutations were most prevalent, larger studies are needed to determine whether MDS/MPN-U has a mutational "fingerprint," which may aid in diagnosis and targeted therapy. 10.1093/ajcp/aqaa281
    Clinical molecular testing for ASXL1 c.1934dupG p.Gly646fs mutation in hematologic neoplasms in the NGS era. Montes-Moreno Santiago,Routbort Mark J,Lohman Elijah J,Barkoh Bedia A,Kanagal-Shamanna Rashmi,Bueso-Ramos Carlos E,Singh Rajesh R,Medeiros L Jeffrey,Luthra Raja,Patel Keyur P PloS one ASXL1 (additional sex combs like 1) is a gene that is mutated in a number of hematological neoplasms. The most common genetic alteration is c.1934dupG p.Gly646fs. Previous publications have shown that ASXL1 mutations have a negative prognostic impact in patients with MDS and AML, however, controversy exists regarding the molecular testing of ASXL1 c.1934dupG as polymerase splippage over the adjacent homopolymer could lead to a false-positive result. Here, we report the first study to systematically test different targeted next generation sequencing (NGS) approaches for this mutation in patients with hematologic neoplasms. In addition, we investigated the impact of proofreading capabilities of different DNA polymerases on ASXL1 c.1934dupG somatic mutation using conventional Sanger sequencing, another common method for ASXL1 genotyping. Our results confirm that ASXL1 c.1934dupG can be detected as a technical artifact, which can be overcome by the use of appropriate enzymes and library preparation methods. A systematic study of serial samples from 30 patients show that ASXL1 c.1934dupG is a somatic mutation in haematological neoplasms including MDS, AML, MPN and MDS/MPN and often is associated with somatic mutations of TET2, EZH2, IDH2, RUNX1, NRAS and DNMT3A. The pattern of clonal evolution suggests that this ASXL1 mutation might be an early mutational event that occurs in the principal clonal population and can serve as a clonal marker for persistent/relapsing disease. 10.1371/journal.pone.0204218
    Clinical Utility of Targeted Next-Generation Sequencing Assay to Detect Copy Number Variants Associated with Myelodysplastic Syndrome in Myeloid Malignancies. Jiang Liqun,Pallavajjala Aparna,Huang Jialing,Haley Lisa,Morsberger Laura,Stinnett Victoria,Hardy Melanie,Park Rebecca,Ament Candice,Finch Alexandra,Shane Alison,Parish Rebecca,Nozari Azin,Long Patty,Adams Emily,Smith Kirstin,Parimi Vamsi,Dougaparsad Sam,Long Lori,Gocke Christopher D,Zou Ying S The Journal of molecular diagnostics : JMD Copy number variants (CNVs) and gene mutations are important for diagnosis and treatment of myeloid malignancies. In a routine clinical setting, somatic gene mutations are detected by targeted next-generation sequencing (NGS) assay, but CNVs are commonly detected by conventional chromosome analysis and fluorescence in situ hybridization (FISH). The aim of this proof-of-principle study was to investigate the feasibility of using targeted NGS to simultaneously detect both somatic mutations and CNVs. Herein, we sequenced 406 consecutive patients with myeloid malignancies by targeted NGS and performed a head-to-head comparison with the results from a myelodysplastic syndrome (MDS) FISH and conventional chromosome analysis to detect CNVs. Among 91 patients with abnormal MDS FISH results, the targeted NGS revealed all 120 CNVs detected by MDS FISH (including -5/5q-, -7/7q-, +8, and 20q-) and 193 extra CNVs detected by conventional chromosome analysis. The targeted NGS achieved 100% concordance with the MDS FISH. The lower limit of detection of MDS CNVs by the targeted NGS was generally 5% variant allele fraction for DNA, based on the lowest percentages of abnormal cells detected by MDS FISH in this study. This proof-of-principle study demonstrated that the targeted NGS assay can simultaneously detect both MDS CNVs and somatic mutations, which can provide a more comprehensive genetic profiling for patients with myeloid malignancies using a single assay in a clinical setting. 10.1016/j.jmoldx.2021.01.011
    Challenges in the introduction of next-generation sequencing (NGS) for diagnostics of myeloid malignancies into clinical routine use. Bacher Ulrike,Shumilov Evgenii,Flach Johanna,Porret Naomi,Joncourt Raphael,Wiedemann Gertrud,Fiedler Martin,Novak Urban,Amstutz Ursula,Pabst Thomas Blood cancer journal Given the vast phenotypic and genetic heterogeneity of acute and chronic myeloid malignancies, hematologists have eagerly awaited the introduction of next-generation sequencing (NGS) into the routine diagnostic armamentarium to enable a more differentiated disease classification, risk stratification, and improved therapeutic decisions. At present, an increasing number of hematologic laboratories are in the process of integrating NGS procedures into the diagnostic algorithms of patients with acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPNs). Inevitably accompanying such developments, physicians and molecular biologists are facing unexpected challenges regarding the interpretation and implementation of molecular genetic results derived from NGS in myeloid malignancies. This article summarizes typical challenges that may arise in the context of NGS-based analyses at diagnosis and during follow-up of myeloid malignancies. 10.1038/s41408-018-0148-6
    Clinical value of next-generation sequencing compared to cytogenetics in patients with suspected myelodysplastic syndrome. Kawata Eri,Lazo-Langner Alejandro,Xenocostas Anargyros,Hsia Cyrus C,Howson-Jan Kang,Deotare Uday,Saini Lalit,Yang Ping,Broadbent Robert,Levy Michael,Howlett Christopher,Stuart Alan,Kerkhof Jennifer,Santos Stephanie,Lin Hanxin,Sadikovic Bekim,Chin-Yee Ian British journal of haematology Next-generation sequencing (NGS) increasingly influences diagnosis, prognosis and management of myelodysplastic syndrome (MDS). In addition to marrow morphology and flow cytometry, our institution performs cytogenetics (CG) and NGS-based testing routinely in patients with suspected MDS. We evaluated the relative value of NGS in the assessment of patients with suspected MDS. We initially compared the diagnostic and prognostic information derived from CG and NGS in 134 patients. NGS enhanced the diagnostic yield compared to CG for clonal myeloid disorders (sensitivity 77% vs. 42·2%; specificity 90·2% vs. 78%; positive predictive value 92·8% vs. 76%; and negative predictive value 70·8% vs. 45·5%). The identification of poor prognosis mutations by NGS altered risk category in 27/39 (69·2%) patients with MDS with good/intermediate risk CG. Subsequently, we prospectively evaluated 70 patients with suspected MDS using an 'NGS-first approach' with CG restricted to samples with morphological abnormalities. We rarely identified mutations or CG abnormalities in patients without dysplastic features. NGS has a superior diagnostic performance compared to CG in patients with suspected MDS. We estimate that by using an 'NGS-first approach' we could reduce karyotyping by approximately 30%. 10.1111/bjh.16891
    AMLVaran: a software approach to implement variant analysis of targeted NGS sequencing data in an oncological care setting. Wünsch Christian,Banck Henrik,Müller-Tidow Carsten,Dugas Martin BMC medical genomics BACKGROUND:Next-Generation Sequencing (NGS) enables large-scale and cost-effective sequencing of genetic samples in order to detect genetic variants. After successful use in research-oriented projects, NGS is now entering clinical practice. Consequently, variant analysis is increasingly important to facilitate a better understanding of disease entities and prognoses. Furthermore, variant calling allows to adapt and optimize specific treatments of individual patients, and thus is an integral part of personalized medicine.However, the analysis of NGS data typically requires a number of complex bioinformatics processing steps. A flexible and reliable software that combines the variant analysis process with a simple, user-friendly interface is therefore highly desirable, but still lacking. RESULTS:With AMLVaran (AML Variant Analyzer), we present a web-based software, that covers the complete variant analysis workflow of targeted NGS samples. The software provides a generic pipeline that allows free choice of variant calling tools and a flexible language (SSDL) for filtering variant lists. AMLVaran's interactive website presents comprehensive annotation data and includes curated information on relevant hotspot regions and driver mutations. A concise clinical report with rule-based diagnostic recommendations is generated.An AMLVaran configuration with eight variant calling tools and a complex scoring scheme, based on the somatic variant calling pipeline appreci8, was used to analyze three datasets from AML and MDS studies with 402 samples in total. Maximum sensitivity and positive predictive values were 1.0 and 0.96, respectively. The tool's usability was found to be satisfactory by medical professionals. CONCLUSION:Coverage analysis, reproducible variant filtering and software usability are important for clinical assessment of variants. AMLVaran performs reliable NGS variant analyses and generates reports fulfilling the requirements of a clinical setting. Due to its generic design, the software can easily be adapted for use with different targeted panels for other tumor entities, or even for whole-exome data. AMLVaran has been deployed to a public web server and is distributed with Docker scripts for local use. 10.1186/s12920-020-0668-3
    [Application of Next Generation Sequencing for AML/MDS Diagnosis and Treatment]. Cheng Huan-Chen,Liu Sheng-Wei,Liu Yu,Zhao Xue-Fei,Li Wei,Qiu Lin,Ma Jun Zhongguo shi yan xue ye xue za zhi OBJECTIVE:To detect the mutations of AML/MDS- related genes by using next generation sequencing (NGS), to analyze the mutation levels of each genes in the AML/MDS and the sensitivity of NGS, and to evaluate the feasibility of gene mutations for monitoring the MRD and predicating the progression of diseases. METHODS:The specimens were collected from primary AML (68 cases) and MDS (57 cases) patients from August 2015 to June 2016 in the Harbin Institute of Hematology and Oncology. The mutations of 22 related genes were detected by using AML/MDS-NGS chips. RESULTS:TET2 gene showed the highest mutation rate in AML (55.9%) and MDS (56.1%). The gene mutations were as follows: CEBPA (11.8%), DNMT3A (7.4%), C-KIT (7.4%) and FLT3-ITD (7.4%) in AML, and U2AF1 (10.5%) and SRSF2 (10.5%) in MDS. All the genes had specific mutation sites except TP53 and CEBPA. The mutations of FLT3, C-KIT and CEBPA became negative in the 5 AML patients in remission when compared with those at primary attack, but the mutation rate of TET2 gene was not obviously changed, whereas the mutation rate of the 5 MDS patients was not significantly changed. The new gene mutations appeared in 3 MDS patients with disease progression, but the mutation rate was not changed significantly in the disease progression. The gene mutation rate still has not been changed significantly even after remission. CONCLUSION:Both AML and MDS have their own specific mutated genes and sites. Some gene mutations, such as CEBPA, can be used as an effective indicator to monitoring MRD in AML patients, but those only used for the evaluation of the disease progression and prognosis in MDS patients. 10.7534/j.issn.1009-2137.2017.06.009
    Gene mutational analysis by NGS and its clinical significance in patients with myelodysplastic syndrome and acute myeloid leukemia. Yu Jifeng,Li Yingmei,Li Tao,Li Yafei,Xing Haizhou,Sun Hui,Sun Ling,Wan Dingming,Liu Yanfang,Xie Xinsheng,Jiang Zhongxing Experimental hematology & oncology Background:In this study, we retrospectively summarized the differences of molecular gene mutations between MDS and AML patients, as well as the young and older age groups of MDS and AML patients. We also analyzed the response of newly diagnosed AML patients to standard DA or IA induction chemotherapy and the relationship between the chemotherapy outcome and the frequency of different gene mutation abnormalities. Methods:NGS assay covering 43 genes was studied in 93 de novo MDS and 325 non-M3 AML patients. Bone marrow samples from all patients underwent gene mutational analysis by NGS. Results:At least one non-synonymous gene mutation was detected in 279 AML patients (85.8%) and 85 MDS patients (91.4%). Contrary to 59 years and younger AML patients, there was a significantly higher incidence of gene mutation in 60 years and older AML patients (2.37 vs 1.94, p = 0.034). Gene mutation incidence in 60 years and older MDS patients increased, but no statistical significance was present (1.95 vs 1.64, p = 0.216). AML patients had a significantly higher gene mutation incidence compared with MDS-MLD patients (2.02 vs 1.63, p = 0.046). Gene mutation incidence was higher in patients with MDS-EB1/EB2 compared with patients with MDS-MLD but there was no statistical significance present (2.14 vs 1.63, p = 0.081). AML patients had significantly higher incidences of CEBPA, FLT3-ITD, DNMT3A, NPM1 and IDH1/2 gene mutations (p = 0.0043, 0.000, 0.030962, 0.002752, and 0.000628, respectively) and a lower incidence of TET2 and U2AF1 gene mutations (p = 0.000004 and 0.000, respectively) compared with MDS patients. Among the individual genes in different age groups, there were significantly higher incidences of RUNX1, IDH2, TP53 and SF3B1 gene mutations (p = 0.0478, 0.0028, 0.0024 and 0.005, respectively) as well as a trend of higher ASXL gene mutation (p = 0.057) in 60 years and older AML patients compared to 59 years and younger patients. There was no statistically significant difference in MDS patients with the different age groups and among the individual genes. Between AML patients and MDS patients among the different gene functional groups, AML patients had a significantly higher incidence of transcriptional deregulation (27.4% vs 15.1%, p = 0.014963), activated signalling (36.3% vs 10.8%, p = 0.000002) related gene mutations as well as a significantly lower incidence of RNA spliceosome (6.15% vs 60.1%, p = 0.000) related gene mutations. Furthermore, among the patients who received either IA or DA regimen for induction chemotherapy, patients with IA regimen had a significantly better CR rate than those with DA regimen (76.6% vs 57.1%, p = 0.0228). Conclusions:Different gene mutations had been found in majority of MDS and AML patients. MDS and AML patients had different gene mutation patterns. AML patients with fewer or no gene mutations had a better chance of achieving CR when treated with IA and DA regimen induction chemotherapy. 10.1186/s40164-019-0158-5
    Prognostic significance of serial molecular annotation in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). Yun Seongseok,Geyer Susan M,Komrokji Rami S,Al Ali Najla H,Song Jinming,Hussaini Mohammad,Sweet Kendra L,Lancet Jeffrey E,List Alan F,Padron Eric,Sallman David A Leukemia The implementation of next-generation sequencing (NGS) has influenced diagnostic, prognostic, and therapeutic decisions in myeloid malignancies. However, the clinical relevance of serial molecular annotation in patients with myelodysplastic syndrome (MDS) undergoing active treatment is unknown. MDS or secondary acute myeloid leukemia (sAML) patients who had at least two NGS assessments were identified. Outcomes according to mutation clearance (NGS-) on serial assessment were investigated. Univariate and multivariate Cox regression models were used to evaluate the prognostic impact of NGS trajectory on overall survival (OS). A total of 157 patients (MDS [n = 95]; sAML [n = 52]; CMML [n = 10]) were identified, with 93% of patients receiving treatment between NGS assessments. Magnitude of VAF delta from baseline was significantly associated with quality of response to treatment. Patients achieving NGS- had significantly improved OS compared to patients with mutation persistence (median OS not reached vs. 18.5 months; P = 0.002), which was confirmed in multivariate analysis (HR,0.14; 95%CI = 0.03-0.56; P = 0.0064). Serial TP53 VAF evaluation predicts outcomes with TP53 clearance representing an independent covariate for superior OS (HR,0.22; 95%CI = 0.05-0.99; P = 0.048). Collectively, our study highlights the clinical value of serial NGS during treatment and warrants prospective validation of NGS negativity as a biomarker for treatment outcome. 10.1038/s41375-020-0997-4
    Clinical, biological, and prognostic implications of SF3B1 co-occurrence mutations in very low/low- and intermediate-risk MDS patients. Janusz Kamila,Izquierdo Marta Martín,Cadenas Félix López,Ramos Fernando,Sánchez Jesús María Hernández,Lumbreras Eva,Robledo Cristina,Del Real Javier Sánchez,Caballero Juan Carlos,Collado Rosa,Bernal Teresa,Pedro Carme,Insunza Andrés,de Paz Raquel,Xicoy Blanca,Salido Eduardo,García Joaquín Sánchez,Mínguez Sandra Santos,García Cristina Miguel,Muñoz Ana María Simón,Barba Mercedes Sánchez,Rivas Jesús María Hernández,Abáigar María,Campelo María Díez Annals of hematology SF3B1 is a highly mutated gene in myelodysplastic syndrome (MDS) patients, related to a specific subtype and parameters of good prognosis in MDS without excess blasts. More than 40% of MDS patients carry at least two myeloid-related gene mutations but little is known about the impact of concurrent mutations on the outcome of MDS patients. In applying next-generation sequencing (NGS) with a 117 myeloid gene custom panel, we analyzed the co-occurrence of SF3B1 with other mutations to reveal their clinical, biological, and prognostic implications in very low/low- and intermediate-risk MDS patients. Mutations in addition to those of SF3B1 were present in 80.4% of patients (median of 2 additional mutations/patient, range 0-5). The most frequently mutated genes were as follows: TET2 (39.2%), DNMT3A (25.5%), SRSF2 (10.8%), CDH23 (5.9%), and ASXL1, CUX1, and KMT2D (4.9% each). The presence of at least two mutations concomitant with that of SF3B1 had an adverse impact on survival compared with those with the SF3B1 mutation and fewer than two additional mutations (median of 54 vs. 87 months, respectively: p = 0.007). The co-occurrence of SF3B1 mutations with specific genes is also linked to a dismal prognosis: SRSF2 mutations were associated with shorter overall survival (OS) than SRSF2wt (median, 27 vs. 75 months, respectively; p = 0.001), concomitant IDH2 mutations (median OS, 11 [mut] vs. 75 [wt] months; p = 0.001), BCOR mutations (median OS, 11 [mut] vs. 71 [wt] months; p = 0.036), and NUP98 and STAG2 mutations (median OS, 27 and 11 vs. 71 months, respectively; p = 0.008 and p = 0.002). Mutations in CHIP genes (TET2, DNMT3A) did not significantly affect the clinical features or outcome. Our results suggest that a more comprehensive NGS study in low-risk MDS SF3B1 patients is essential for a better prognostic evaluation. 10.1007/s00277-020-04360-4
    A Single-Run Next-Generation Sequencing (NGS) Assay for the Simultaneous Detection of Both Gene Mutations and Large Chromosomal Abnormalities in Patients with Myelodysplastic Syndromes (MDS) and Related Myeloid Neoplasms. Liquori Alessandro,Lesende Iván,Palomo Laura,Avetisyan Gayane,Ibáñez Mariam,González-Romero Elisa,Boluda-Navarro Mireia,Morote-Faubel Mireya,Garcia-Ruiz Cristian,Martinez-Valiente Cristina,Santiago-Balsera Marta,Gomez-Seguí Inés,Sanjuan-Pla Alejandra,Sanz Miguel A,Sanz Guillermo,Solé Francesc,Such Esperanza,Cervera José Cancers Myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms are clonal disorders that share most of their cytogenetic and molecular alterations. Despite the increased knowledge of the prognostic importance of genetics in these malignancies, next-generation sequencing (NGS) has not been incorporated into clinical practice in a validated manner, and the conventional karyotype remains mandatory in the evaluation of suspected cases. However, non-informative cytogenetics might lead to an inadequate estimation of the prognostic risk. Here, we present a novel targeted NGS-based assay for the simultaneous detection of all the clinically relevant genetic alterations associated with these disorders. We validated this platform in a large cohort of patients by performing a one-to-one comparison with the lesions from karyotype and single-nucleotide polymorphism (SNP) arrays. Our strategy demonstrated an approximately 97% concordance with standard clinical assays, showing sensitivity at least equivalent to that of SNP arrays and higher than that of conventional cytogenetics. In addition, this NGS assay was able to identify both copy-neutral loss of heterozygosity events distributed genome-wide and copy number alterations, as well as somatic mutations within significant driver genes. In summary, we show a novel NGS platform that represents a significant improvement to current strategies in defining diagnosis and risk stratification of patients with MDS and myeloid-related disorders. 10.3390/cancers13081947