Abstract:
Acute myeloid leukaemia (AML) is an aggressive blood cancer associated with impaired differentiation and abnormal expansion of blood-producing cells in the bone marrow. Despite the modern medical advances, this disease remains highly lethal, particularly for the elderly patients.
To offer the appropriate therapeutic strategy, haematologists need to be able to track changes in leukaemia cell numbers occurring throughout treatment. Measurable residual disease (MRD) is the number of cancer cells that survive induction chemotherapy. It is the strongest independent prognostic marker of relapse and is therefore in the spotlight for physicians offering care to AML patients. Numerous methods have been trialled to detect MRD with high sensitivity. Of those, the next generation sequencing (NGS)-based assays have recently gained attention due to their flexibility and applicability.
We proposed a novel type of NGS-based MRD assay, MRD-Seq. It is a PCR-based test which can be applied to any somatic point mutation, provided there are appropriate gene-specific primers and PCR conditions. We used the MRD-Seq method to establish assays targeting Type A mutations in the NPM1 gene, internal tandem duplications in the FLT3 gene (FLT3-ITDs), as well as point mutations in the IDH1 and IDH2 genes. For the IDH1/2, we aimed to greatly improve the sensitivity by using unique molecular identifiers (UMIs) to barcode individual DNA molecules during PCR to reduce amplification-borne errors. To detect the point mutations in the NPM1 gene, a working MRD-Seq assay has previously been successfully established in our laboratory from gDNA. We hypothesized that MRD-Seq starting from cDNA will have greater sensitivity as the number of mRNA molecules is often much bigger than the number of DNA molecules, allowing more targets to be investigated.
Interestingly, our results suggest that cDNA-based assays have lower sensitivity than gDNA-based assays. This is dependent on the gene being investigated and the bioinformatic pipeline used for NGS data analysis. Thus, more genes recurrently mutated in AML should be investigated for NGS-based MRD monitoring. Given the great flexibility and wide applicability of the method, it will undoubtedly prove useful by individualising AML treatment based on a unique mutational profile of every patient.