Next Generation Sequencing and Functional Analysis of Mirna Expression in Acute Myeloid Leukemia Patients with Different FLT3 Mutations: Block of MiR-155 in FLT3-ITD Driven AML Leads to Downregulation of Myeloid Blasts in Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2438-2438 ◽  
Author(s):  
Dennis Gerloff ◽  
Alexander Arthur Wurm ◽  
Jens-Uwe Hartmann ◽  
Nadja Hilger ◽  
Anne-Marie Müller ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Next Generation Sequencing ◽  
Mirna Expression ◽  
Leukemic Cells ◽  
Next Generation ◽  
Mirna Expression Pattern ◽  
Differentially Expressed Mirnas ◽  
Flt3 Mutations ◽  
Generation Sequencing

Abstract Up to 30% of all acute myeloid leukemias (AMLs) are associated with an activating mutation in the FMS-like tyrosine kinase 3 receptor (FLT3). Two distinct groups of FLT3 mutations are found: (1) the most common are internal tandem duplications (ITDs) of the FLT3 juxtamembrane region, and (2) point mutations within the tyrosine kinase domains (TKDs). While FLT3-TKD mutations seem to have no prognostic relevance in AML, patients bearing an FLT3-ITD mutation have a significantly worse outcome compared with AML patients with wild-type FLT3 (FLT3-WT). MicroRNAs (miRNAs) are small (~22 bp) noncoding RNAs, which regulate protein expression posttranscriptionally by recruitment of the RNA-induced silencing complex (RISC) to the 3′-untranslated region (3′-UTR) of target mRNAs. We and others have shown that miRNAs are crucial regulators in myeloid differentiation and in leukemogenesis. Furthermore, it was shown that several miRNAs have a prognostic impact. Hence, we hypothesized that the different FLT3 mutations lead to altered miRNA expression. To find different expression patterns of miRNAs, we performed next generation sequencing of normal karyotype bone marrow patient samples with FLT3-WT (n=5), FLT3-TKD (n=3) and FLT3-ITD (n=3). Sequencing was performed with an Illumina HighScan-SQ sequencer using version 3 chemistry and flowcell according to the instructions of the manufacturer. For normalization the method of trimmed mean of M values (TMM) was used. Data analyses were performed using the Qlucore Omics Explorer 3.1. In a multi group analyses of miRNA expression pattern, we found 17 significant differentially expressed miRNAs (p ≤ 0.05). The expression of 6 miRNAs (miR-10a-5p, miR-10a-3p, miR-18a-5p, let-7b-3p, miR-155-5p and miR-576-5p) was increased only in the FLT3-ITD associated patient samples. In the FLT3-WT samples we found 8 miRNAs (miR-141-3p, 342-3p, 181a-2-3p, 374b-5p, 30b-5p, 29c-3p, 23b-3p and 125a-3p) with an increased expression. The miR-92a-3p showed an enhanced expression in FLT3-WT and FLT3-ITD patient samples. The multi group analyses showed only 2 miRNAs (miR-3615 and miR-193b-3p) induced in FLT3-TKD patient samples. The two FLT3-ITD induced miRNAs, miR-10a-5p and miR-155-5p were the most abundant and most differentially expressed miRNAs in the screen. From our data we hypothesize that miR-155 and miR-10a could play an important role in disease progression and clinical outcome of FLT3-ITD induced AMLs To analyze a block of miR-155 in FLT3-ITD driven AML in vivo, we transfected 32D cells, stably expressing human FLT3-ITD, with unspecific scramble or miR-155 specific locked nucleic acids (LNAs (Exiqon)). 24h after transfection, we injected 1x106 cells into C3H mice (scr. n=5; LNA-155 n=5). All animals rapidly developed a leukemia like disease with hepatosplenomegaly. The animals died 17 - 21 days after 32DFLT3-ITD cell injection. We could not observe a difference in survival. In flow cytometry analysis of the peripheral blood we found a strong increase of human FLT3 (huCD135) expressing cells (32DFLT3-ITD) 1 to 2 days before the mice died. At death of the animals we analyzed the accumulation of leukemic cells (32DFLT3-ITD) in bone marrow, spleen and liver by flow cytometry for the human FLT3 (huCD135). Here we could observe a significantly (p≤ 0.05) reduced number of leukemic cells in the bone marrow of the mice with the LNA-155 transfected 32DFLT3-ITD cells in comparison to the group with the scramble transfected 32D cells. In spleen we could not observe a difference in accumulation of leukemic cells, but in the liver we could show a tendentially reduced accumulation of 32DFLT3-ITD cells transfected with LNA-155. The next generation sequencing screen gives insight into the altered miRNA expression pattern of FLT3-WT, FLT3-TKD and FLT3-ITD related AMLs. The miR-10a-5p and miR-155-5p are highly expressed in FLT3-ITD associated AMLs. The block of the FLT3-ITD induced miR-155 in vivo significantly reduces the accumulation of leukemic cells in the bone marrow of transplanted mice. The results give the evidence that miR-155 could be a novel therapeutic target in FLT3-ITD associated AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mapping DNA Topoisomerase Binding and Cleavage Genome Wide Using Next-Generation Sequencing Techniques

Genes ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 92 ◽  
Author(s):  
Shannon J. McKie ◽  
Anthony Maxwell ◽  
Keir C. Neuman
Keyword(s):  
Next Generation Sequencing ◽  
Dna Cleavage ◽  
Next Generation ◽  
Dna Breaks ◽  
Genome Wide ◽  
Extension Activity ◽  
Nucleotide Resolution ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Next-generation sequencing (NGS) platforms have been adapted to generate genome-wide maps and sequence context of binding and cleavage of DNA topoisomerases (topos). Continuous refinements of these techniques have resulted in the acquisition of data with unprecedented depth and resolution, which has shed new light on in vivo topo behavior. Topos regulate DNA topology through the formation of reversible single- or double-stranded DNA breaks. Topo activity is critical for DNA metabolism in general, and in particular to support transcription and replication. However, the binding and activity of topos over the genome in vivo was difficult to study until the advent of NGS. Over and above traditional chromatin immunoprecipitation (ChIP)-seq approaches that probe protein binding, the unique formation of covalent protein–DNA linkages associated with DNA cleavage by topos affords the ability to probe cleavage and, by extension, activity over the genome. NGS platforms have facilitated genome-wide studies mapping the behavior of topos in vivo, how the behavior varies among species and how inhibitors affect cleavage. Many NGS approaches achieve nucleotide resolution of topo binding and cleavage sites, imparting an extent of information not previously attainable. We review the development of NGS approaches to probe topo interactions over the genome in vivo and highlight general conclusions and quandaries that have arisen from this rapidly advancing field of topoisomerase research.

scholarly journals A rare atypical chronic myeloid leukemia BCR-ABL1 negative with concomitant JAK2 V617F and SETBP1 mutations: a case report and literature review

2020 ◽  
Vol 11 ◽  
pp. 204062072092710
Author(s):  
Tianqi Gao ◽  
Changhui Yu ◽  
Si Xia ◽  
Ting Liang ◽  
Xuekui Gu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Targeted Therapy ◽  
Next Generation Sequencing ◽  
Myeloid Leukemia ◽  
Jak2 V617f ◽  
Next Generation ◽  
Bone Marrow Fibrosis ◽  
Atypical Chronic Myeloid Leukemia ◽  
Generation Sequencing ◽  
Marrow Fibrosis

Atypical chronic myeloid leukemia (aCML) BCR-ABL1 negative is a rare myelodysplastic syndromes/myeloproliferative neoplasm (MDS/MPN) for which no standard treatment currently exists. The advent of next-generation sequencing has allowed our understanding of the molecular pathogenesis of aCML to be expanded and has made it possible for clinicians to more accurately differentiate aCML from similar MDS/MPN overlap syndrome and MPN counterparts, as MPN-associated driver mutations in JAK2, CALR, or MPL are typically absent in aCML. A 55-year old male with main complaints of weight loss and fatigue for more than half a year and night sweats for more than 2 months was admitted to our hospital. Further examination revealed increased white blood cells, splenomegaly, and grade 1 bone marrow fibrosis with JAK2 V617F, which supported a preliminary diagnosis of pre-primary marrow fibrosis. However, in addition to JAK2 V617F (51.00%), next-generation sequencing also detected SETBP1 D868N (46.00%), ASXL1 G645fs (36.09%), and SRSF2 P95_R102del (33.56%) mutations. According to the 2016 World Health Organization diagnostic criteria, the patient was ultimately diagnosed with rare aCML with concomitant JAK2 V617F and SETBP1 mutations. The patient received targeted therapy of ruxolitinib for 5 months and subsequently an additional four courses of combined hypomethylating therapy. The patient exhibited an optimal response, with decreased spleen volume by approximately 35% after therapy and improved symptom scores after therapy. In diagnosing primary bone marrow fibrosis, attention should be paid to the identification of MDS/MPN. In addition to basic cell morphology, mutational analysis using next-generation sequencing plays an increasingly important role in the differential diagnosis. aCML with concomitant JAK2 V617F and SETBP1 mutations has been rarely reported, and targeted therapy for mutated JAK2 may benefit patients, especially those not suitable recipients of hematopoietic stem cell transplants.

scholarly journals Next-Generation Sequencing in a Direct Model of HIV Infection Reveals Important Parallels to and Differences from In Vivo Reservoir Dynamics

2020 ◽  
Vol 94 (9) ◽  
Author(s):  
Marilia Rita Pinzone ◽  
Maria Paola Bertuccio ◽  
D. Jake VanBelzen ◽  
Ryan Zurakowski ◽  
Una O’Doherty
Keyword(s):  
T Cells ◽  
Next Generation Sequencing ◽  
Cd4 T Cells ◽  
Next Generation ◽  
Activated T Cells ◽  
Selection Pressures ◽  
Hiv Dna ◽  
Generation Sequencing

ABSTRACT Next-generation sequencing (NGS) represents a powerful tool to unravel the genetic make-up of the HIV reservoir, but limited data exist on its use in vitro. Moreover, most NGS studies do not separate integrated from unintegrated DNA, even though selection pressures on these two forms should be distinct. We reasoned we could use NGS to compare the infection of resting and activated CD4 T cells in vitro to address how the metabolic state affects reservoir formation and dynamics. To address these questions, we obtained HIV sequences 2, 4, and 8 days after NL4-3 infection of metabolically activated and quiescent CD4 T cells (cultured with 2 ng/ml interleukin-7). We compared the composition of integrated and total HIV DNA by isolating integrated HIV DNA using pulsed-field electrophoresis before performing sequencing. After a single-round infection, the majority of integrated HIV DNA was intact in both resting and activated T cells. The decay of integrated intact proviruses was rapid and similar in both quiescent and activated T cells. Defective forms accumulated relative to intact ones analogously to what is observed in vivo. Massively deleted viral sequences formed more frequently in resting cells, likely due to lower deoxynucleoside triphosphate (dNTP) levels and the presence of multiple restriction factors. To our surprise, the majority of these deleted sequences did not integrate into the human genome. The use of NGS to study reservoir dynamics in vitro provides a model that recapitulates important aspects of reservoir dynamics. Moreover, separating integrated from unintegrated HIV DNA is important in some clinical settings to properly study selection pressures. IMPORTANCE The major implication of our work is that the decay of intact proviruses in vitro is extremely rapid, perhaps as a result of enhanced expression. Gaining a better understanding of why intact proviruses decay faster in vitro might help the field identify strategies to purge the reservoir in vivo. When used wisely, in vitro models are a powerful tool to study the selective pressures shaping the viral landscape. Our finding that massively deleted sequences rarely succeed in integrating has several ramifications. It demonstrates that the total HIV DNA can differ substantially in character from the integrated HIV DNA under certain circumstances. The presence of unintegrated HIV DNA has the potential to obscure selection pressures and confound the interpretation of clinical studies, especially in the case of trials involving treatment interruptions.

scholarly journals Next-Generation Sequencing Analysis of MiRNA Expression in Control and FSHD Myogenesis

PLoS ONE ◽  
2014 ◽  
Vol 9 (10) ◽  
pp. e108411 ◽  
Author(s):  
Veronica Colangelo ◽  
Stéphanie François ◽  
Giulia Soldà ◽  
Raffaella Picco ◽  
Francesca Roma ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Mirna Expression ◽  
Sequencing Analysis ◽  
Next Generation ◽  
Next Generation Sequencing Analysis ◽  
Generation Sequencing

Mutation Analysis in 200 Cases of Newly Diagnosed Myelodysplastic Syndrome By Next Generation Sequencing: Association with Established Prognostic Variables and IPSS-R

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1703-1703
Author(s):  
Kankana Ghosh ◽  
Parsa Hodjat ◽  
Priyanka Priyanka ◽  
Beenu Thakral ◽  
Keyur P. Patel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Next Generation Sequencing ◽  
Myelodysplastic Syndrome ◽  
Mutation Analysis ◽  
Conflicts Of Interest ◽  
International Prognostic Scoring System ◽  
P Value ◽  
Newly Diagnosed ◽  
Next Generation ◽  
Generation Sequencing

Abstract INTRODUCTION Myelodysplastic syndrome (MDS) is known to have numerous genomic aberrations that predict response to treatment and overall survival. We aimed to assess various mutations in newly diagnosed MDS cases by next generation sequencing (NGS) and their association with various well-established clinicopathologic parameters and the Revised International Prognostic Scoring System (IPSS-R). MATERIALS AND METHODS We performed molecular studies on DNA extracted from bone marrow aspirate specimens in 200 newly diagnosed treatment naïve MDS patients presenting at a single institution from 08/2013 to 03/2015 as part of routine clinical work up in a CLIA certified molecular diagnostics laboratory. Cases met criteria for MDS per WHO 2008 criteria. The entire coding sequences of 28 genes (ABL1, ASXL1, BRAF, DNMT3A, EGFR, EZH2, FLT3, GATA1, GATA2, HRAS, IDH1, IDH2, IKZF2, JAK2, KIT, KRAS, MDM2, MLL, MPL, MYD88, NOTCH1, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53, WT1) were sequenced using a NGS-based custom-designed assay using TruSeq chemistry on Illumina MiSeq platform. FLT3 internal tandem duplications (ITD) and codon 835/836 point mutation were detected by PCR followed by capillary electrophoresis. CEBPA mutation analysis was performed by PCR followed by Sanger sequencing on 186 patients. RESULTS Median age was 67 years. Patients included 139 males (69.5%) and 61 females (30.5%). Hematologic parameters are as follows [median (range)]: Hb 9.6 g/dL (5-16.7), platelets 75 K/μ L (5-652), WBC: 2.8 K/μ L (0.4-20.8), ANC 1.3 K/μ L (0.0 -12.0), AMC 0.2 K/μ L (0.0-3). Bone marrow (BM) blasts [median (range)] were 4% (0-19). Of 192 patients with cytogenetic analysis performed, 65 (33.85%) had diploid karyotype, 53 (27.6%) had one, 21 (10.93%) had two, 13 (6.77%) had three, 40 (20.83%) had > three abnormalities. IPSS-R risk categorization of the 200 cases is as follows: very low (17 cases, 8.5%), low (46, 23%) intermediate (42, 21%), high (47, 23.5%), very high (48, 24%). Mutations identified by NGS are as detailed in Table 1. Of the 4 patients with FLT mutations detected, the breakdown is as follows: FLT3 ITD (3, 75%), FLT3 D835 (1, 25%), FLT3, ITD + D835 (0, 0%). CEBPA mutation was detected in 12 of 186 (6.45%) cases assessed. CEBPA was detected in 12 (6.45%). Sixty three (31.5%) cases had no mutations detected in the genes analyzed by NGS or PCR, 80 (40%) had mutations in one, 42 (21%) had mutations in two, 8 (4%) in three and 7 (3.5%) in > three genes. We found positive associations between mutated genes and various parameters as detailed in Table 2. No association was found between frequency of any particular mutation and the IPSS-R score. CONCLUSIONS: MDS is a heterogeneous group of myeloid neoplasms at the genetic level. Multiple genetic mutations in a large subset of cases likely indicate clonal evolution. A subset of mutations has significant association with well-established clinico-pathologic parameters like WBC and BM blast percentage. With longer follow-up, we could use this data to refine IPSS-R. Table 1. Number of cases % cases TP53 46 23 TET2 33 16.5 RUNX1 27 13.5 ASXL1 25 12.5 DNMT3A 17 8.5 EZH2 12 6 IDH2 8 4 IDH1 7 3.5 NRAS 7 3.5 JAK2 5 2.5 FLT3 4 2 PTPN11 3 1.5 EGFR 2 1 MPL 2 1 WT1 2 1 GATA2 1 0.5 KIT 1 0.5 KRAS 1 0.5 MYD88 1 0.5 NPM1 1 0.5 BRAF 1 0.5 Table 2. Mutated genes p value WBC ASXL1 <0.042 AEC TET2 <0.016 BM blast % RUNX1, CEBPA <0.008, p<0.02 BM myelocyte % TP53, TET2, RUNX1, DNMT3A <0.014, <0.014, <0.015, <0.038 AEC: absolute eosinophil count, BM: bone marrow Disclosures No relevant conflicts of interest to declare.

miRNA profiling in resectable NSCLC by multiplex next-generation sequencing.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 7060-7060
Author(s):  
Sandra Gallach ◽  
Eloisa Jantus-Lewintre ◽  
David Montaner ◽  
Marta Usó ◽  
Elena Sanmartin ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
High Throughput ◽  
Mirna Expression ◽  
Early Stage ◽  
Normal Lung ◽  
Next Generation ◽  
Early Stage Nsclc ◽  
Nsclc Patients ◽  
Generation Sequencing ◽  
Independent Cohort

7060 Background: Early-stage NSCLC has a relapse rate around 40% within 5 years. With the advent of microRNA (miRNA), which seems to regulate many genes critical for tumorigenesis, there is a growing interest in the characterization of miRNAome in NSCLC specimens and to correlate them with prognosis. Next generation sequencing is a useful tool to study the miRNA content of solid tumors. Here, we applied high-throughput SOLiD transcriptome sequencing to study miRNAs expression in a cohort of early-stage NSCLC patients (tumor vs normal lung). Methods: RNA was isolated from frozen lung specimens (tumor and normal lung) from resectable NSCLC patients (n=35). Samples with a RIN ≥ 7 were analyzed and enriched in the miRNA fraction. miRNAs were sequencing using a bar-code multiplex SOLiD protocol. Data normalization was carried out by rescaling all data according to their counts. Readings were mapped against mature and no-mature miRNAs using miRBase. Statistical analysis was performed with CLCbio software and considered significant when p-adj<0.005. Results: Using the SOLiD high throughput sequencing, we performed a systemic miRNA expression profiling analysis of paired samples (tumor vs normal lung). A total of 1268 miRNAs (mature and no-mature) have been detected in at least one sample. The differential expression between normal and tumor samples shown that 6 miRNAs (miR-193b, miR-182, miR-96, miR-148a, miR-299, miR-590) were upregulated and 7 (miR-145, miR-133a, miR-218, miR-125a, miR-30a, miR-126 and miR-139) were down-regulated significantly in tumor samples compared with normal lung tissues. We are performing studies using qRT-PCR in an independent cohort to further validate these findings. Conclusions: The deep sequencing technology used for differential miRNA expression is useful and novel. The use of barcoding allows multiplexing and lowers cost per sample. Several miRNAs were differentially expressed between tumor and normal tissue, but this point needs to be further validated in an independent cohort. Supported by grants TRA09-0132 (MICINN) and RD06/0020/1024 (ISCIII).

Genome study of PDGFRA D842V mutant GIST using next generation sequencing approach.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 10540-10540
Author(s):  
Maria A. Pantaleo ◽  
Annalisa Astolfi ◽  
Milena Urbini ◽  
Valentina Indio ◽  
Margherita Nannini ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Tyrosine Kinases ◽  
Therapeutic Targets ◽  
Mapk Signaling ◽  
Gastric Gist ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Generation Sequencing

10540 Background: Mutations of the receptors KIT and PDGFRA in GIST are the oncogenetic events of disease as well as the targets of molecular therapies. Within the PDGFRA mutations, the D842V mutation in exon 18 confers in vitro and in vivo resistance to imatinib. Next generation sequencing techniques may completely dissect all the possible somatic mutations and genomic rearrangements in order to identify novel therapeutic targets in this patients population. Methods: Five patients with gastric GIST were analyased (3 M, 2 F; mean age 65,5 years, range 51-77). The tumor dimension ranged between 3 and 15 cm, MI < 2 and 8 /50 HPF. No metastases were present in all cases. The KIT and PDGFRA anlysis showed a D842V mutation in exon 18 of PDGFRA in all cases. Whole transcriptome sequencing was performed with Illumina HiScanSQ platform with a paired-end strategy (75x2). After performing quality controls, the short reads were mapped with Tophat-Botie pipeline against the human reference genome (HG19). The variations, such as Single nucleotide variants (SNVs) and InDels, were called by SNVMix2 (a software suited for SNVs discovery in tumor samples) implementing an accurate filtering procedures developed in our laboratory. Two predictors of mutations effect at protein level (SNPs&GO and Provean) were employed in order to prioritize the emerging variation. Results: An average of 206 coding non-synonymous variants were highlighted in the five GIST samples, of which ~ 30% were predicted as deleterious with at least one predictor. In addition to PDGFRA D842V mutation, in all five patients were found mutations on different receptor tyrosine kinases, such as FGFR4 and DDR2. Moreover three out of five patients harboured mutations on members of the MDR/TAP subfamily that are involved in multidrug resistance, in particular on ABCB1, ABCB4 and on ABCB6 genes. Other mutations were found on the hedgehog and MAPK signaling pathway and on SNF/SWI chromatin remodeling complex. Conclusions: New molecular events have been identified in PDGFRA D842V mutant GIST. These data should be validated in larger series and the role of these mutations as therapeutic targets should be further investigated.

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