D'Angelo, J.

Introduction: NPM1 mutations, seen in up to 30% of adults with acute myeloid leukemia (AML), often co-occur with FLT3 internal tandem duplication (FLT3-ITD). Measurable residual disease (MRD) testing before allogeneic hematopoietic cell transplant (alloHCT) for persistence of mutated NPM1 and/or FLT3-ITD can identify patients with AML in complete remission (CR) at highest risk of relapse and death. Quantitative RT-PCR assays are currently recommended for NPM1 MRD testing, while FLT3-ITD MRD testing is DNA-based using next generation sequencing (NGS). DNA-based NGS assays for mutated NPM1 MRD testing may have several advantages but require validation. We performed NPM1 MRD detection in blood collected from patients with AML during first CR (CR1) prior to alloHCT on a subset of patients from the Pre-MEASURE study (PMID: 36881031) using a highly-sensitive, commercially available DNA-based NPM1 NGS MRD assay. Methods: Of the 531 patients aged 18 or older from the Pre-MEASURE study who received an alloHCT for NPM1mutated AML during CR1 at a CIBMTR reporting site in the USA between the years of 2013-2019, a subset of 190 were randomly selected to be included in this analysis. A commercially available research testing kit (IVS, Invivoscribe, San Diego, CA), which can detect DNA-based NPM1 MRD, was used to establish a workflow following clinical testing standards and validated to detect NPM1 insertion variants down to a variant allele fraction (VAF) of at least 0.005%. Results were compared to those previously reported using an anchored multiplex PCR-based (AMP) targeted NGS assay (detection limit 0.01%) and FLT3-ITD MRD results, where applicable. Overall survival (OS) and cumulative incidence of relapse were estimated with the day of transplant as time 0 using Kaplan-Meier estimation (log-rank tests), Fine and Gray model, and Cox proportional hazards models. Results: NPM1 NGS MRD testing was successful for 186 (98%) patients, of which 48 (26%) relapsed (median: 4.0 months; range: 0.7-52.5) and 64 (34%) died (median: 9.7 months; range: 0.4-58.9) post-alloHCT. The IVS assay detected NPM1 insertion variants in 71 patients with a median VAF of 0.0026% (range:0.0002-2.1%). Using a VAF threshold of ≥0.01% for MRD positivity, the IVS test showed that 24 patients tested positive for NPM1 MRD pre-alloHCT, which was associated with significantly increased rates of relapse (52% vs 20% at 3yrs; HR=4.3; P<0.001) and decreased OS (34% vs 71% at 3yrs; HR=3.6; P<0.001) compared to testing negative, in close alignment with those previously reported using the AMP assay. After removing the VAF threshold, an additional 47 patients were classified as NPM1 MRD positive, which remained associated with increased rates of relapse (40% vs 15% at 3yrs; HR=3.4; P<0.001) and decreased OS (50% vs 75% at 3yrs; HR=2.9; P<0.001). In multivariable analysis, residual NPM1 MRD burden prior to alloHCT was associated with risk of relapse and death in a dose-dependent manner. Patients were further subdivided based on their reported FLT3-ITD mutational status at baseline. Increased risk of relapse was seen for NPM1 MRD positive patients regardless of baseline FLT3-ITD. 123 (66%) patients were co-mutated for FLT3-ITD at baseline and previously tested for FLT3-ITD MRD using the IVS assay (PMID: 38696205). Defining MRD based on the presence of NPM1 vs NPM1 and/or FLT3-ITD revealed equivalent levels of relapse (45% vs 44%) and OS (45% vs 46%) at 3 years, with NPM1 MRD evaluation identifying 5 additional relapses. These results were confirmed in the full Pre-MEASURE cohort (n=317) using the AMP assay. NPM1 MRD positive patients receiving nonmyeloablative conditioning or reduced-intensity conditioning (RIC) without melphalan (mel) had increased risk of relapse or death compared to patients receiving myeloablative conditioning or RIC with mel, regardless of FLT3-ITD co-mutational status (3yrs: relapse 87% vs 55%, P=0.006; OS 15% vs 42%, P=0.013). Conclusions: In patients with NPM1 mutated AML from the Pre-MEASURE study, we show that detection of residual NPM1 variants in pre-transplant blood during CR1 using a highly sensitive DNA-based assay is associated in a dose-dependent manner with a significantly increased risk of relapse and death after allo-HCT, which can be mitigated in part by conditioning regimen. In patients co-mutated for both FLT3-ITD and NPM1 at diagnosis, NPM1 should be prioritized as a target for NGS-MRD if only one test is available.

ImportancePersistence of FLT3 internal tandem duplication (ITD) in adults with acute myeloid leukemia (AML) in first complete remission (CR) prior to allogeneic hematopoietic cell transplant (HCT) is associated with increased relapse and death after transplant, but the association between the level of measurable residual disease (MRD) detected and clinical outcome is unknown.ObjectiveTo examine the association between pre–allogeneic HCT MRD level with relapse and death posttransplant in adults with AML in first CR.Design, Setting, and ParticipantsIn this cohort study, DNA sequencing was performed on first CR blood from patients with FLT3-ITD AML transplanted from March 2013 to February 2019. Clinical follow-up was through May 2022. Data were analyzed from October 2022 to December 2023.ExposureCentralized DNA sequencing for FLT3-ITD in pre–allogeneic HCT first CR blood using a commercially available kit.Main Outcomes and MeasuresThe primary outcomes were overall survival and cumulative incidence of relapse, with non–relapse-associated mortality as a competing risk post–allogeneic HCT. Kaplan-Meier estimations (log-rank tests), Cox proportional hazards models, and Fine-Gray models were used to estimate the end points.ResultsOf 537 included patients with FLT3-ITD AML from the Pre-MEASURE study, 296 (55.1%) were female, and the median (IQR) age was 55.6 (42.9-64.1) years. Using the variant allele fraction (VAF) threshold of 0.01% or greater for MRD positivity, the results closely aligned with those previously reported. With no VAF threshold applied (VAF greater than 0%), 263 FLT3-ITD variants (median [range] VAF, 0.005% [0.0002%-44%]), and 177 patients (33.0%) with positive findings were identified. Multivariable analyses showed that residual FLT3-ITD was the variable most associated with relapse and overall survival, with a dose-dependent correlation. Patients receiving reduced-intensity conditioning without melphalan or nonmyeloablative conditioning had increased risk of relapse and death at any given level of MRD compared with those receiving reduced-intensity conditioning with melphalan or myeloablative conditioning.Conclusions and RelevanceThis study provides generalizable and clinically applicable evidence that the detection of residual FLT3-ITD in the blood of adults in first CR from AML prior to allogeneic HCT is associated with an increased risk of relapse and death, particularly for those with a VAF of 0.01% or greater. While transplant conditioning intensification, an intervention not available to all, may help mitigate some of this risk, alternative approaches will be necessary for this high-risk population of patients who are underserved by the current standard of care.