RT Journal Article
SR Electronic
T1 <em>CEBPA</em> mutational analysis in acute myeloid leukaemia by a laboratory-developed next-generation sequencing assay
JF Journal of Clinical Pathology
JO J Clin Pathol
FD BMJ Publishing Group Ltd and Association of Clinical Pathologists
SP 522
OP 531
DO 10.1136/jclinpath-2017-204825
VO 71
IS 6
A1 Ng, Christopher Wai Siong
A1 Kosmo, Bustamin
A1 Lee, Peak-Ling
A1 Lee, Chun Kiat
A1 Guo, Jingxue
A1 Chen, Zhaojin
A1 Chiu, Lily
A1 Lee, Hong Kai
A1 Ho, Sherry
A1 Zhou, Jianbiao
A1 Lin, Mingxuan
A1 Tan, Karen M L
A1 Ban, Kenneth H K
A1 Tan, Tin Wee
A1 Chng, Wee Joo
A1 Yan, Benedict
YR 2018
UL http://jcp.bmj.com/content/71/6/522.abstract
AB Aim The 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.Methods Mutational 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.Results A 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.Conclusions Our 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.