NEXT-GENERATION SEQUENCING AS A TOOL FOR ASSESSING MINIMAL RESIDUAL DISEASE IN AML PATIENTS WITH CEBPA MUTATIONS

Mutations in the CCAAT/enhancer binding protein alpha (CEBPA) gene occur approximately in 5 – 10 % of acute myeloid leukemia (AML) patients and can be used as molecular markers for monitoring of minimal residual disease (MRD). However, detection of MRD using quantitative real-time PCR (qPCR) is complicated due to GC-rich regions and thus validation of leukemia-specific and sensitive MRD assay can be technically difficult. 

The goal of our work was the application of next-generation amplicon-based deep sequencing (NGS) as a quantitative detection method for MRD monitoring.

Since 2010, we have performed mutational analysis of the CEBPA gene coding region in 411 AML patients at the time of diagnosis using Sanger sequencing. In patients with the CEBPA mutation/s as the only detected mutation/s, i.e. the examined patients did not have other available MRD markers after standard molecular analysis of fusion transcripts and mutations in hematological prognostic genes, we designed leukemia-specific assays using qPCR. However, in 5 patients the assay did not provide sufficiently sensitive and specific detection of residual leukemic cells. In these cases MRD was monitored by NGS technology (GS Junior System, Roche). For MRD analysis starting at first diagnosis and following the course of the treatment we analyzed bone marrow (n = 36) or peripheral blood (n = 2) samples. The result of the MRD assessment was evaluated as a percentage of the mutated reads from all reads (reads with mutation/all reads). 20 000 reads per sample was considered as a minimum for valid MRD analysis. 

From January 2013 to February 2015 we examined 38 samples from 5 AML patients. CEBPA mutations found in these patients and used as a marker for MRD analysis were short insertions or deletions. The median of reads per sample was 58 376 (mean 58 076, range 27 829 - 110 564). The assay detection sensitivity achieved the threshold of 10^-4 to 10^-5 (1 leukemic cell in 10 000 cells to 1 leukemic cell in 100 000 cells). The MRD levels of residual leukemic cells correlated with clinical outcome. In patients with relapse (three out of five patients), the occurrence of the CEBPA mutation/s was also confirmed by conventional Sanger sequencing.  

Quantitative assessment of CEBPA mutations using next-generation amplicon-based deep sequencing enabled MRD monitoring in AML patients, where the design of quantitative real-time PCR assay didn not achieve sensitivity of at least four orders of magnitude. NGS technology represents another approach for MRD assessment of the mutated CEBPA gene.