Development of leukemia-specific RT-qPCR assays to track treatment response is dependent upon molecular characterization of diagnostic material to determine the most appropriate assay, with MRD monitoring strategies informed by maximal achievable sensitivity, optimal sample type, and typical kinetics of disease relapse. (A) Analysis of diagnostic material is critical to determine the appropriate assay primer and probe set to detect MRD in any given patient because of heterogeneity in chromosomal breakpoints (eg, PML-RARA, CBFB-MYH11, MLL fusions) or mutation type (NPM1). For example, in ∼5% of acute promyelocytic leukemia cases, the standard EAC assays are not suitable because of occurrence of rarer breakpoints within the PML locus requiring design of patient-specific forward primers to be used in conjunction with the standard EAC probe and reverse primer (both located in RARA).⁴⁴  Figure panel adapted from Grimwade et al⁴⁶  with permission. (B) A key determinant of the sensitivity for MRD detection is the relative level of expression of the leukemia-specific transcript (ie, fusion gene, NPM1 mutant) as indicated by comparison with that of an endogenous control gene (eg, ABL). This can be measured as the difference in the number of PCR cycles (ΔCt) to detect fluorescence above background from amplification of the leukemic transcript and the control gene at the threshold (set at 0.05 according to EAC criteria³⁹ ); see left panel. The detection limit of PCR is taken as 40 cycles (equivalent to ∼1 copy), with 1-log being equivalent to 3.45 cycles, as determined from the slope of the plasmid standard curve. Assuming ABL amplification at cycle threshold (Ct) value of 24, the observed Ct value for amplification of the leukemic target in blasts at diagnosis indicates the maximal theoretical sensitivity for detection of MRD in that particular patient. The Ct value of the MRD target equating with a given level of sensitivity (10⁻¹ to 10⁻⁵) is marked based on an ABL Ct value of 24. For example, MRD can be detected at a sensitivity of at least 1 in 10⁴, where ΔCtTarget-ABL is ≤2.2. Detection of MRD at a sensitivity of 1 in 10⁵ is possible when the MRD target is more highly expressed than ABL, with a ΔCt of −1.2. ΔRn, normalized reporter signal (change in fluorescence intensity). Figure panel adapted from Freeman et al²⁶  with permission. Examination of diagnostic BM samples from primary leukemia samples using standardized assays developed within the EAC program demonstrates marked variation in the level of leukemic transcripts both between and within different molecular subsets, which impacts on the sensitivity to detect MRD in any given patient (right panel). Figure adapted from Gabert et al³⁹  with permission. (C) Apart from maximal assay sensitivity, a further parameter to take into account in determining MRD sampling schedules is the kinetic of disease relapse. For example, in APL, the median increment in PML-RARA fusion transcripts is ∼1-log/month. Reproduced from Grimwade et al⁴⁴  with permission. (D) Parallel tracking of MRD status by RT-qPCR in PB and BM in a patient with NPM1 mutant AML, with filled and unfilled data points indicating that disease transcripts were detectable or undetectable, respectively. For PCR-negative samples, data points are plotted according to the maximal sensitivity afforded by the follow-up sample based on the respective level of ABL control gene expression and taking into account the difference in expression between the NPM1 mutant allele and ABL in leukemic cells at diagnosis (ΔCtNPM1mut-ABL), as described in panel B. In this patient, rapid PCR negativity was achieved in the PB. However, serial BM samples afforded greater sensitivity, revealing that the patient failed to achieve molecular remission after frontline therapy, with relapse preceded by a rapid rise in NPM1 mutant transcripts. The PB MRD assay only converted to PCR positivity at the time of diagnosis of clinical relapse.