Optimizing Acute Myeloid Leukemia Monitoring: A Novel Multi-Target NGS Application for Measurable Residual Disease Assessment
Authors: Condorelli Annalisa , Spinelli Orietta , Stavola Roberta , Kubik Slawomir , Liu Yuanlong , Bieler Jonathan , Frigeni Marco , Lussana Federico , Rambaldi Alessandro , Salmoiraghi Silvia 
Published: December 2024

Background: Measurable residual disease (MRD) is becoming a crucial tool used in clinical practice for prognostic stratification and treatment planning in Acute Myeloid Leukemia (AML) patients. Different approaches for MRD detection are available including multiparametric flow cytometry (MFC) and molecular evaluation by quantitative PCR (qPCR). Recently, the updated ELN guidelines added Next Generation Sequencing for MRD quantification (NGS-MRD) even though the optimal targets and threshold (provisionally defined as ≥0.1% VAF) have not been established yet. A major advantage of NGS-MRD is the possibility to detect, simultaneously by a single test, several genetic variants, at diagnosis or emerging during the disease evolution. This characteristic makes NGS-MRD applicable to the large majority of AML patients, including those without a targetable marker by PCR or MFC. However, NGS-MRD remains more subject to a high error rate (false positive) and less sensitive than other available approaches. A tailored NGS-MRD technique overcoming these limitations and leading to better standardization in the interpretation and reporting of NGS-MRD results is required to strengthen its applicability in AML management.
Methods: We performed a single-center, NGS-MRD-based analysis on a cohort of 74 AML patients treated with allogeneic hematopoietic stem cell transplantation (alloHSCT) in morphological and immunophenotypic remission between 2010 and 2024. We applied a novel error suppression Unique Molecular Identifiers (UMI)-based NGS-MRD method designed by Sophia Genetics using a panel including 23 commonly mutated genes in AML (CALR, CEPBA, DDX41, ETV6, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PTPN11, RAD21, RUNX1, SF3B1, SRSF2, STAG2, TP53, U2AF1, WT1). For the analysis, 800 ng of DNA should be available before starting the conditioning regimen to warrant a high level of sensitivity (10-4). A total of 62 genetic variants (1-6 per patient) were detected at diagnosis by a conventional NGS approach. We obtained a high number of good-quality sequences allowing us to reach a uniform coverage greater than 500000 X, a key premise for a high sensitivity level. We evaluated a positive control obtained by diluting the Horizon Myeloid Mix (HD-829) in GIAB sample NA24385, used in turn as a negative control, to have the majority of variants at VAF of ~0.02%. All the mutations in the positive control were detected by this NGS-MRD assay. In the negative control, a very low VAF level could be occasionally detected due to a high noise signal related to the deamination phenomenon. A bioinformatic tool to reduce these errors was applied as well as UMI CUMIN adapters to minimize PCR error propagation.
Results: Among the first 23 patients, tested at conditioning, 16 (70%) proved positive by MRD-NGS, defined as the presence of at least a known variant with a VAF ≥ 0.01%, with a median VAF of 0.09%. Clonal hemopoiesis mutations (DTA) as well as germinal genetic markers were excluded as not considered for MRD monitoring. In this cohort, ten patients experienced relapse: eight of them resulted NGS-MRD positive before transplant.
To validate this approach, the results obtained were compared with the current gold standard RT-PCR in NPM1 mutated patients (9/23) and with digital droplet PCR (ddPCR) (12/23) for patients harboring other point mutations detectable by this technique. In 83% of the samples, the qualitative results were concordant. Three samples proved positive (VAF=0.01%) by MRD-NGS and negative by ddPCR. This discrepancy can be explained by the different detection limits of each method (0.01% vs. ̴ 0.03%, depending on the mutation analyzed). Similarly, one patient was found NPM1 negative by NGS-MRD and weakly positive by qPCR. A comparison of VAF values obtained from NGS with those obtained by ddPCR in 12 patients provided a Pearson correlation index of 0.999. The comparison of quantification was not possible between the results from NGS-MRD, expressed as variant allele fraction (VAF), and qPCR data, expressed as cDNA copies or as logarithmic reduction of leukemic burden at diagnosis.
Conclusion: This novel UMI-based NGS-MRD method is widely applicable to AML patients and allows the detection of multiple variants at very low frequencies. An extended analysis of a larger cohort of AML patients receiving an alloHSCT is ongoing.