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

Ng, Christopher; Kosmo, Bustamin; Lee, Peak-Ling; Lee, Chun Kiat; Chiu, Lily; Lee, Hong Kai; Ho, S.; Zhou, Jianbiao; Lin, Mingxuan; Tan, Karen; Ban, Kenneth H K; Tan, Tin Wee; Chng, Wee Joo; Yan, Benedict

doi:10.1136/jclinpath-2017-204825

Cited by 16 publications

(13 citation statements)

References 13 publications

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“…However, some genes frequently mutated in myeloid malignancies are particularly difficult to sequence with NGS, especially with amplicon-based or RNA bait capture-based panels. For example, technical difficulties hinder the ability to capture targets with high GC content, such as CEBPA, which is associated with poor prognosis of AML, or repetitive genomic regions, such as FLT3-ITD, which is associated with poor prognosis of AML in the absence of NPM1 mutation [10,12,[21][22][23][24][25][26][27]. Aguilera-Diaz and colleagues evaluate the performance of four different targeted NGS gene panels, three commercial panels and one in-house developed panel, and noted that CEBPA, CALR and FLT3 genes remain challenging the use of NGS for diagnosis of myeloid neoplasms in compliance with current guidelines [21].…”

Section: Introductionmentioning

confidence: 99%

Analytical validation and performance characteristics of a 48-gene next-generation sequencing panel for detecting potentially actionable genomic alterations in myeloid neoplasms

Rosenthal

Герасимова

et al. 2021

PLoS ONE

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Identification of genomic mutations by molecular testing plays an important role in diagnosis, prognosis, and treatment of myeloid neoplasms. Next-generation sequencing (NGS) is an efficient method for simultaneous detection of clinically significant genomic mutations with high sensitivity. Various NGS based in-house developed and commercial myeloid neoplasm panels have been integrated into routine clinical practice. However, some genes frequently mutated in myeloid malignancies are particularly difficult to sequence with NGS panels (e.g., CEBPA, CARL, and FLT3). We report development and validation of a 48-gene NGS panel that includes genes that are technically challenging for molecular profiling of myeloid neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). Target regions were captured by hybridization with complementary biotinylated DNA baits, and NGS was performed on an Illumina NextSeq500 instrument. A bioinformatics pipeline that was developed in-house was used to detect single nucleotide variations (SNVs), insertions/deletions (indels), and FLT3 internal tandem duplications (FLT3-ITD). An analytical validation study was performed on 184 unique specimens for variants with allele frequencies ≥5%. Variants identified by the 48-gene panel were compared to those identified by a 35-gene hematologic neoplasms panel using an additional 137 unique specimens. The developed assay was applied to a large cohort (n = 2,053) of patients with suspected myeloid neoplasms. Analytical validation yielded 99.6% sensitivity (95% CI: 98.9–99.9%) and 100% specificity (95% CI: 100%). Concordance of variants detected by the 2 tested panels was 100%. Among patients with suspected myeloid neoplasms (n = 2,053), 54.5% patients harbored at least one clinically significant mutation: 77% in AML patients, 48% in MDS, and 45% in MPN. Together, these findings demonstrate that the assay can identify mutations associated with diagnosis, prognosis, and treatment options of myeloid neoplasms even in technically challenging genes.

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Section: Introductionmentioning

confidence: 99%

Analytical validation and performance characteristics of a 48-gene next-generation sequencing panel for detecting potentially actionable genomic alterations in myeloid neoplasms

Rosenthal

Герасимова

et al. 2021

PLoS ONE

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“…63 Next-generation sequencing (NGS) is another molecular technique employed in the evaluation of CEBPA. 36,37,66 NGS allows for the simultaneous evaluation of multiple genes with high sensitivity. 36 The major obstacle to the NGS evaluation of CEBPA mutations is the high GC content in the coding region of the gene, causing poor amplicon coverage.…”

Section: Abor Atory E Valuati On Of Ceb Pamentioning

confidence: 99%

Laboratory evaluation and prognostication among adults and children with CEBPA‐mutant acute myeloid leukemia

Mendoza

Podoltsev

Siddon

2021

Int J Lab Hematology

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CEBPA‐mutant acute myeloid leukemia (AML) encompasses clinically and biologically distinct subtypes of AML in both adults and children. CEBPA‐mutant AML may occur with monoallelic (moCEBPA) or biallelic (biCEBPA) mutations, which can be somatic or germline, with each entity impacting prognosis in unique ways. BiCEBPA AML is broadly associated with a favorable prognosis, but differences in the type and location of CEBPA mutations as well as the presence of additional leukemogenic mutations can lead to heterogeneity in survival. Concurrent FLT3‐ITD mutations have a well‐documented negative effect on survival in adult biCEBPA AML, whereas support for a negative prognostic effect of mutations in TET2, DNMT3A, WT1, CSF3R, ASXL1, and KIT is mixed. NPM1 and GATA2 mutations may have a positive prognostic impact. MoCEBPA AML has similar survival outcomes compared to AML with wild‐type CEBPA, and risk stratification is determined by other cytogenetic and molecular findings. Germline CEBPA mutations may lead to familial biCEBPA AML after acquisition of second somatic CEBPA mutation, with variable penetrance and age. BiCEBPA AML in children is likely a favorable‐risk diagnosis as it is in adults, but the role of a single CEBPA mutation and the impact of concurrent leukemogenic mutations are not clear in this population. Laboratory evaluation of the CEBPA gene includes PCR‐based fragment‐length analysis, Sanger sequencing, and next‐generation sequencing. Phenotypic analysis using multiparameter flow cytometry can also provide additional data in evaluating CEBPA, helping to assess for the likelihood of mutation presence.

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“…The intron is low and rich in GC (approximately 75% containing GC) and encodes a protein that belongs to basic leucine zipper family of transcription factor. It is expressed in the cells of the myelomonocytic cell and specifically up regulated during granulocyte differentiation [54]. The finding that the improved prognosis associated with AML with mutated CEBPA, which is seen in 5% to14% of AML, is associated with biallelic, but not single, mutations of the gene which resulted in a change in that disease definition to require biallelic mutations (Table 4).…”

Section: Aml With Biallelic Mutations Of Cebpamentioning

confidence: 99%

Expanding the Classification of Leukemia by the World Health Organization over Time

Tamaddon¹

2019

HIJ

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The classification has always been the basis for many treatments of the disease. The French-American-British has performed leukemia classification base on morphological finding, but the World Health Organization apply gene or chromosomal mutations in its classification. This approach has revolutionized the diagnosis, choose strategy of treatment and follow-up ever since. Although WHO has classified leukemia on the basis of FAB; but genetic mutations has made the wider and more specific outcome. Obviously, identifying these damages provide an extensive filed for researchers and physicians to effective assistance for patients. Therefore, the purpose of this review is comparing revisions of WHO classification in the years 2008 and 2016, because these categories are highlighted unknown cases that give researchers incentives to further study.

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

Abstract: Our laboratory-developed CEBNX workflow shows high coverage and thus overcomes the challenges associated with amplification efficiency and low coverage of Therefore, our assay is suitable for deployment in the clinical laboratory.

Cited by 16 publications

References 13 publications

Analytical validation and performance characteristics of a 48-gene next-generation sequencing panel for detecting potentially actionable genomic alterations in myeloid neoplasms

Analytical validation and performance characteristics of a 48-gene next-generation sequencing panel for detecting potentially actionable genomic alterations in myeloid neoplasms

Laboratory evaluation and prognostication among adults and children with CEBPA‐mutant acute myeloid leukemia

Expanding the Classification of Leukemia by the World Health Organization over Time

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