Development of a quantitative real‐time polymerase chain reaction method for monitoring CEBPA mutations in normal karyotype acute myeloid leukaemia

The persistence of minimal residual disease (MRD) posttreatment may provide an early indicator of impending relapse in acute myeloid leukaemia (AML) (Raanani & Ben-Bassat, 2004). The absence of an appropriate molecular signature in 40–60% of AML patients who have normal cytogenetics associated with intermediate risk has limited the analysis in this group. Through advances in mutation analysis a list of recurring gene mutations as potential MRD targets are now being compiled. Many AML cells have more than one recurring mutation, indicating that there is a multistep pathogenesis of disease. Mutation events in AML have been detected by analysis of the blast cells; therefore, chronology of mutational events within the stem cell and/or early progenitor populations has not been established. Many of these mutations result in the insertion or deletion of a small number of additional nucleotides that should allow for the design of a specific primer against the unique sequence generated by the mutation (Pabst et al, 2001). Found in approximately 20% of normal karyotype AML, the presence of CEBPA mutation segregates with favourable risk among this otherwise intermediate risk group. The CEBPA gene encodes the granulocytic differentiation factor CCAAT Enhancer Binding Protein-alpha and plays a crucial role in granulocytic differentiation of haematopoietic stem cells and has antiproliferative activity in various cell types. Mutation in CEBPA was investigated as a potential MRD marker because of its prevalence in normal karyotype AML, the frequency of insertion and deletion mutations enabling a straightforward design of mutation-specific primers and concordance between presentation and relapse (Tiesmeier et al, 2003). Four patients with normal karyotype AML FAB type M1 were selected for study based on the availability of diagnostic (patients 1, 3 and 4) or relapse (patient 2) bone marrow (BM) and on the presence of CEBPA mutations identified during previous screening (Snaddon et al, 2003). Samples were collected and analysed in accordance with the requirements of the local ethics committee. Five real-time quantitative polymerase chain reaction (RQ-PCR) assays were developed that selectively amplified mutated CEBPA DNA from four patients. Figure 1A and B demonstrates the RQ-PCR strategy used in this study. Three patients (1, 2 and 3) had an insertion mutation in the region of the gene that codes for the C-terminal DNA-binding/basic leucine zipper domain of the protein, the fourth contained mutations corresponding to both the Nand C-terminal regions of the C/EBP-a protein (4a and 4b respectively). Mutations in 1, 2, 4a, and 4b were all tandem duplications ranging from 3 to 57 base pairs (bp) in length while patient 3 had a 3-bp insertion. Mutation-specific forward primers were designed by making use of the unique sequences generated by the insertion mutations along with the introduction of single nucleotide changes in the primers to prevent annealing to the wild-type (WT) sequence. Patient-specific primer sequences are given in Fig 1C. Real-time PCR analysis was performed on 25 ll reaction mixture in duplicate using Taqman chemistry on the ABI PRISM 7700 Sequence Detector (Applied Biosystems, Warrington, UK) with default settings. b-2-Microglobulin was used as the endogenous reference gene to assess total cell number per well. The specificity of each RQ-PCR assay was tested by amplifying 750 ng (equivalent to 125 000 cells) of each patient’s diagnostic BM DNA, wt DNA, patient diagnostic BM DNA serially diluted in wt DNA and no template controls, in duplicate. An assay was deemed to be specific if no amplification was observed with wt DNA. To test the sensitivity of each assay a standard curve was generated by five serial dilutions of diagnostic DNA in water (ranging from 125 000 to 4 cells per well). In all amplification plots the same threshold was maintained to allow comparison between experiments. The correlation coefficient of the standard curves was at least 0Æ95, indicating that the assays had a high probability of precise quantification. The mean Ct for DNA equivalent to 125 000 cells was 24Æ6 (range 24–27). The mean slope of the dilution curves was )3Æ2 (range )3Æ5 to )2Æ8), indicating good amplification efficiencies (the perfect theoretical slope is )3Æ3). As shown in Fig 1C, the maximum reproducible sensitivity (defined as the dilution for which duplicates were both positive) for all the patient mutation assays was 10 (8Æ0 · 10; 10 in 125 000) and therefore were of the required order of sensitivity, >10, for an MRD monitoring assay. Insufficient material was available for retrospective MRD monitoring in these patients. Instead we have taken advantage of the assay’s ability to quantify mutated cells for the determination of presence or absence of mutation within the leukaemic stem cell (LSC) population. The LSC generally accounts for approximately 1% of leukaemic cells (Hope et al, 2004). As with the normal haematopoietic stem cell (HSC) compartment, LSCs are not functionally homogeneous and correspondence