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CEBPA mutational analysis in acute myeloid leukaemia by a laboratory-developed next-generation sequencing assay
  1. Christopher Wai Siong Ng1,
  2. Bustamin Kosmo1,
  3. Peak-Ling Lee1,
  4. Chun Kiat Lee1,
  5. Jingxue Guo2,
  6. Zhaojin Chen3,
  7. Lily Chiu1,
  8. Hong Kai Lee1,
  9. Sherry Ho1,
  10. Jianbiao Zhou4,5,
  11. Mingxuan Lin1,
  12. Karen M L Tan1,
  13. Kenneth H K Ban2,
  14. Tin Wee Tan6,
  15. Wee Joo Chng4,5,7,
  16. Benedict Yan1,8
  1. 1Department of Laboratory Medicine, Molecular Diagnosis Centre, National University Health System, Singapore, Singapore
  2. 2Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
  3. 3Investigational Medicine Unit, National University Health System, Singapore, Singapore
  4. 4Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
  5. 5Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore
  6. 6National Supercomputing Centre Singapore, Singapore, Singapore
  7. 7Department of Haematology-Oncology, National University Cancer Institute, National University Health System, Singapore, Singapore
  8. 8Translational Centre for Development and Research, National University Health System, Singapore, Singapore
  1. Correspondence to Christopher Wai Siong Ng; christopher.ws.ng{at}gmail.com and Dr Benedict Yan, Department of Laboratory Medicine, Molecular Diagnosis Centre, National University Health System, Singapore, 119074, Singapore; tranceblues{at}gmail.com

Abstract

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.

  • leukaemia
  • haemato-oncology
  • molecular pathology

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Footnotes

  • CWSN and BK contributed equally.

  • Handling editor Runjan Chetty

  • Funding This study was funded by Centre for Personalized and Precision Health (CPPH) of National University Hospital (NUH), Singapore (CPPH/FY2016/ProjectBudget/003).

  • Competing interests None declared.

  • Patient consent Obtained.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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