scholarly journals Targeted next-generation sequencing of familial platelet disorder with predisposition to acute myeloid leukaemia

2015 ◽  
Vol 175 (1) ◽  
pp. 161-163 ◽  
Author(s):  
Karl Haslam ◽  
Stephen E. Langabeer ◽  
Amjad Hayat ◽  
Eibhlin Conneally ◽  
Elisabeth Vandenberghe
Keyword(s):  
Next Generation Sequencing ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Platelet Disorder ◽  
Familial Platelet Disorder ◽  
Generation Sequencing ◽  
Acute Myeloid

scholarly journals Longitudinal targeted next‐generation sequencing in a patient with acute myeloid leukaemia

2019 ◽  
Vol 186 (6) ◽  
pp. 801-801
Author(s):  
Priya D. Velu ◽  
Alexander E. Perl ◽  
Selina M. Luger ◽  
Adam Bagg ◽  
Jennifer J. D. Morrissette
Keyword(s):  
Next Generation Sequencing ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Generation Sequencing ◽  
Acute Myeloid

CEBPA mutational analysis in acute myeloid leukaemia by a laboratory-developed next-generation sequencing assay

2017 ◽  
Vol 71 (6) ◽  
pp. 522-531 ◽  
Author(s):  
Christopher Wai Siong Ng ◽  
Bustamin Kosmo ◽  
Peak-Ling Lee ◽  
Chun Kiat Lee ◽  
Jingxue Guo ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Mutational Analysis ◽  
Gc Content ◽  
Amplification Efficiency ◽  
Transactivation Domain ◽  
Next Generation ◽  
Generation Sequencing ◽  
Acute Myeloid

AimThe presence of biallelic CEBPA mutations is a favourable prognostic feature in acute myeloid leukaemia (AML). CEBPA mutations are currently identified through conventional capillary sequencing (CCS). With the increasing adoption of next-generation sequencing (NGS) platforms, challenges with regard to amplification efficiency of CEBPA due to the high GC content may be encountered, potentially resulting in suboptimal coverage. Here, the performance of an amplicon-based NGS method using a laboratory-developed CEBPA-specific Nextera XT (CEBNX) was evaluated.MethodsMutational analyses of the CEBPA gene of 137 AML bone marrow or peripheral blood retrospective specimens were performed by the amplification of the CEBPA gene using the Expand Long Range dNTPack and the amplicons processed by CCS and NGS. CEBPA-specific libraries were then constructed using the Nextera XT V.2 kit. All FASTQ files were then processed with the MiSeq Reporter V.2.6.2.3 using the PCR Amplicon workflow via the customised CEBPA-specific manifest file. The variant calling format files were analysed using the Illumina Variant Studio V.2.2.ResultsA coverage per base of 3631X to 28184X was achieved. 22 samples (16.1%) were found to contain CEBPA mutations, with variant allele frequencies (VAF) ranging from 3.8% to 58.2%. Taking CCS as the ‘gold standard’, sensitivity and specificity of 97% and 97% was achieved. For the transactivation domain 2 polymorphism (c.584_589dupACCCGC/p.His195_Pro196dup), the CEBNX achieved 100% sensitivity and 100% specificity relative to CCS.ConclusionsOur laboratory-developed CEBNX workflow shows high coverage and thus overcomes the challenges associated with amplification efficiency and low coverage of CEBPA. Therefore, our assay is suitable for deployment in the clinical laboratory.

scholarly journals Next-generation sequencing-based molecular genetic profiling in adults with acute myeloid leukaemia

2020 ◽  
Vol 65 (4) ◽  
pp. 444-459
Author(s):  
A. I. Kashlakova ◽  
E. N. Parovichnikova ◽  
B. V. Biderman ◽  
Y. V. Sidorova ◽  
Y. A. Chabaeva ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Molecular Genetic ◽  
Driver Mutations ◽  
Next Generation ◽  
Old Patients ◽  
Generation Sequencing ◽  
Acute Myeloid

Introduction. Acute myeloid leukaemia (AML) is associated with multiple driver mutations, which prognostic value remains understudied.Aim. Assessment of the frequency of mutations in various genes and their impact on acute myeloid leukaemia outcome in adults.Materials and methods. The study included 90 adult patients with newly diagnosed AML; 76 were aged under 60, 14 were 60 and more years old. Patients under 60 had chemotherapy (CT) “7+3” as induction, the elder cohort had variant low-dose CT with hypomethylating agents. The molecular genetic status of patients was determined using next-generation sequencing; the in-house gene panel included ASXL1, BCOR, DNMT3, FLT3, IDH1, IDH2, PIGA, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53 and U2AF2.Results. Nucleotide substitutions were identified in genes DNMT3, TET2, TP53, SETBP1, BCOR, RUNX1, IDH2, IDH1, FLT3, U2AF2, SF3B1 in 57.8 % of the patients (n = 52), with 17.8 % (n = 16) having compound mutations in two or three genes. Treatment efficacy and long-term outcomes were assessed against age, ELN-2017 risk groups and mutations in genes TP53, RUNX1, IDH1, IDH2 and DNMT3. In the long term, a reliable variation was revealed in the overall survival (OS) rate with respect to mutations in genes TP53 and RUNX1. Patients with mutant TP53 had 30 % OS, those with the intact gene — 53.4 % (p = 0.0037). Similar results were obtained with RUNX1: mutations marked 20 % OS, intact patients had 54% OS (p = 0.0466).Conclusion. Mutations in genes FLT3-ITD, NPM1 and CEBPA are proxy to AML. However, a more accurate prognosis and optimal choice of therapy require detailed molecular profiling due to genetic heterogeneity of AML patients.

Single Nucleotide Polymorphism Array (SNP-A) Karyotype Is the Best Predictor of Prognosis In Normal Cytogenetics Acute Myeloid Leukaemia (AML)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 411-411
Author(s):  
Azim M Mohamedali ◽  
Robert K Hills ◽  
Erick E Nasser ◽  
Atiyeh Abdallah ◽  
Sneha Shinde ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Prognostic Significance ◽  
Next Generation ◽  
Chromosome 13 ◽  
Median Size ◽  
Genomic Aberrations ◽  
Generation Sequencing ◽  
Acute Myeloid

Abstract Abstract 411 The herald of next generation sequencing has ushered an era for the discovery of frequent novel mutations such as DNMT3a, IDH1/2 and ASXL1 in acute myeloid leukaemia with a normal karyotype (AML-NK). The frequency of these mutations has been extensively documented; however, there is inconclusive evidence for their prognostic significance. Furthermore, whilst having a ‘normal' cytogenetic karyotype, AML-NK has been characterized by SNP-A karyotyping revealing multiple cryptic genomic aberrations. To predict prognosis, mutations of FLT3, NPM1 and CEBPA as well as gene expression profiles have been used as biomarkers. Using 454 next generation sequencing (NGS), we sought to determine the prognostic significance of mutations in ASXL1, DNMT3a, FLT3, IDH1/2, NPM1 and TP53 as well as detect cryptic genomic aberrations using 250K SNP microarrays in 92 AML-NK patients (pts) uniformly treated from the UK MRC/NCRI AML clinical trials, excluding pts with induction deaths. We demonstrate the presence of 54 cryptic genomic aberrations in 37 pts consisting of 11 deletions (del) (8 pts), 10 gains (10 pts) and 33 regions of UPD (29 pts). Aberrations were excluded as CNV's if there was >50% overlap with variants from the DGV and an internal cohort of 91 healthy subjects. The median size of del was 1.6Mb(0.18Mb-3.4Mb) with chromosome (chr) 4(q24) and 17(q11.2 and q22.1) deleted in two pts. One pt had micro del on chr12p13.2-p12.3 and 21q22.11-q22.12 affecting the ETV6 and AML1 genes, respectively, that was confirmed by FISH. Gains (median 0.37Mb(0.26–0.71Mb)) were identified on chr8 (4 pts). The median size of UPD's was 83.3Mb(20.5Mb-158Mb) with interstitial UPD <20Mb excluded from the analysis. Chromosome 13 (q12.11-q34) and 6(p25.3-p22.1) were the most frequently affected with UPD observed in 15 and 5 pts, respectively. Of the 15 pts with UPD of chr13, 11 pts had this occurrence in small clones. Additional regions affected by UPD included chromosomes 1, 2, 4, 5, 7 and 11. NPM1, DNMT3a, FLT3-ITD, IDH2, FLT3-TKD, IDH1, TP53 and ASXL1 mutations occurred in 64%, 49%, 22%, 5%, 3%, 2% and 1% of patients, respectively with 13 cases having no detectable mutations. The R882 mutation hotspot of DNMT3a occurred in 33/45 cases with clone size ranging from 34–65% and occurred concurrently with mutations in NPM1 (31 pts), FLT3-ITD/TKD (30 pts) and IDH2 (9 pts). IDH1 (R130H) and IDH2 (R140Q and R172K) mutations were mutually exclusive and occurred in 23 pts, with 4 pts having concurrent FLT3, NPM1 and DNMT3a mutations. Only 1 patient had an ASXL1 mutation with a concurrent IDH2 mutation and 2 cases had mutation of TP53. FLT3-TKD/ITD mutations were exclusive of each other. Not all pts with FLT3-ITD mutation had UPD of chromosome 13. There was no correlation between genomic aberrations and mutations of any other assayed genes. The median duration of follow-up from time of diagnosis of the study group was 44 months (range 2–160). There were no significant correlations between the presence of genomic aberrations and age, sex, WBC, secondary disease or mutations in IDH1/2, FLT3-ITD or DNMT3a. Patients with FLT3-TKD were likely to have gains (2/5) compared to those with wild type (8/85, p=0.04). Patients with genomic aberrations had worse overall survival (OS) compared to pts without aberrations, (5 year OS 12% vs 40%, hazard ratio (HR) adjusted for other prognostic variables 2.56 (1.43–4.60), p=0.001); this was also seen when considering UPD on OS ((8% for those with UPD compared to 37% for those without UPD (adjusted HR 2.52(1.40–4.54) p=0.002) and cumulative incidence of relapse (CIR) (78% in pts with UPD compared to 54% for those without UPD (adjusted HR 4.04(1.84–8.90) p=0.0003). Adjusted analysis showed a dose effect for the number of UPD's on OS (p=0.007) and relapse (p=0.001). In a model building analysis, adjusted for age, WBC, sex, secondary disease, performance status and mutations of IDH1, IDH2, FLT3-ITD/TKD, DNMT3a and NPM1, only UPD proved independently prognostic for relapse and survival. We conclude that cryptic genomic aberrations in uniformly treated AML-NK pts provide better prognostic value and outcome prediction than metaphase cytogenetics and mutation detection, thereby, helping to identify patient groups with poor prognosis and requiring specific therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.

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