A Rapid and Sensitive Method for Large-Scale Screening of CEBPA Mutations in AML Patients with Normal Karyotype.
Authors: Benthaus Tobias , Mellert Gudrun , Zellmeier Evelyn , Hiddemann Wolfgang , Spiekermann Karsten , Dufour Annika 
Published: October 2019

Abstract
In 45% of patients with de novo acute myeloid leukemia (AML) no cytogenetic abnormalities can be detected (normal karyotype AML, NK-AML). Recently, several molecular markers with prognostic significance have been described which define distinct subgroups of NK-AML patients. Mutations in the CEBPA gene have shown to occur at about 8% of NK-AML in Western countries and confer a favorable prognosis. The C/EBPα protein, a member of the family of basic region leucine zipper (bZIP) transcriptional regulators, is important for normal granulocytic differentiation and is frequently disrupted in AML. We retrospectively analyzed bone-marrow samples of 442 patients with de novo NK-AML for the presence of CEBPA mutations and established a fast and sensitive screening method. A multiplex-PCR-gene scanning assay for combined detection of CEBPA and NPM mutation has recently been described in which, however, the primers did not cover the whole CEBPA gene. CEBPA mutations have been found to be distributed over the entire CEBPA gene and their functional and clinical consequences are not yet clear. Therefore, we designed a rapid CEBPA specific multiplex PCR-gene scanning assay covering the entire coding region of the CEBPA gene. Four primer pairs were designed, fluorescently labeled and included in 2 multiplex PCR reactions. The PCR products were electrophoresed on a genetic analyzer and the amplicon sizes were compared to wildtype CEBPA of U937 cell line by fragment length analysis. In order to evaluate our method, we analyzed 120 patient samples in parallel by both multiplex PCR and sequencing analysis. Using sequencing analysis as a gold standard, all of the CEBPA mutations could be detected by multiplex PCR and fragment length analysis. Thus, our multiplex PCR assay reached a sensitivity of 100%. The specificity was 89% due to the false positive detection of a 6 basepair duplication polymorphism in 2.5% of patients (Wouters BJ et al, Blood, 2007). 322 patient samples were subsequently screened for CEBPA mutations by the multiplex PCR assay. In case of alterations in the fragment length analysis, the relevant CEBPA region was sequenced to identify the exact type of mutation. Among 442 patients with NK-AML, 32 patients (7%) showed CEBPA mutations. Taken together, we identified 47 mutations in 32 patients, of which 17 patients had a single CEBPA mutation and 15 patients had more than one CEBPA mutation. We identified 30 out of frame insertion/deletion nonsense mutations resulting in an N-terminal stopcodon and 14 in frame insertion/deletion mutations as well as 3 out of frame insertion/deletion mutations in the C-terminal bZIP region. The only limitation of this method might be that single basepair substitutions, which do not affect the length of the amplicon, cannot be detected. Substitutions in the CEBPA gene are, however, rare events and often silent. In 120 sequenced AML patients we did not find any non-silent substitution. We established a fast and sensitive screening method suitable for large-scale detection of CEBPA mutation and applicable for inclusion in routine AML diagnostics. This is so far the largest reported analysis of CEBPA mutations in patients with NK-AML and might provide further insights into the functional and clinical relevance of the different types of CEBPA mutations and their correlation to other molecular markers in NK-AML.