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Letters to the Editor
AB
Figure 2. Outcomes according to NPM1-based minimal residual disease. (A) Disease-free survival and (B) overall survival in acute myeloid leukemia patients with rare NPM1 mutations according to post-induction log reduction in minimal residual disease in peripheral blood (<4-log reduction or >4-log reduction). Disease-free and overall survival were censored at allogeneic stem cell transplantation.
ability of droplet dPCR to quantify non-A/B/D NPM1 mutations and to retrospectively evaluate the prognostic impact of post-induction NPM1-based MRD in AML patients with a “rare” NPM1 mutation (i.e. non-A/B/D) enrolled in the ALFA-0702 trial.
From March 2009 to September 2013, 713 patients aged 18–59 years old with de novo AML were included in the phase II randomized multicenter ALFA-0702 trial (Eudra-CT, 2008-000668-18; ClinicalTrials.gov, NCT00932412).13 The study was approved in December 2008 by the Institutional Review Board of the French Regulatory Agency and the Ethics Committee Sud-Est IV, France. All patients gave informed consent for both treat- ment and genetic analyses, according to the Declaration of Helsinki. NPM1 mutations and FLT3-internal tandem duplications (ITD) were determined by fragment analysis and Sanger sequencing as part of the patients’ care.13 Overall, NPM1-mutated AML accounted for 36% of the cases of AML (234 patients), of which 13% (31 patients) had non-A/B/D mutations. Among them, 22 patients with available RNA-complementary DNA (cDNA) extracted from peripheral blood after induction therapy were selected for this study. Additionally, 28 AML patients with a NPM1-type A mutation were selected to compare the performance of droplet dPCR versus stan- dard RT-qPCR performed as previously described.6
Peripheral blood samples were collected at diagnosis and after induction (MRD1) for patients in complete remission. Droplet dPCR was performed on cDNA using the Bio-Rad QX200TM droplet dPCR system with FAM- and HEX-labeled probes (Online Supplementary Figure S1, Online Supplementary Table S1). NPM1 mutations and ABL1 transcripts were quantified in multiplex. Each sam- ple was partitioned into 20,000 uniform droplets allow- ing a random distribution of the target cDNA. End-point PCR amplification of the nucleic acid target was carried out within each droplet using the high-performance T100TM Thermal Cycler (Bio-Rad). PCR products were then subjected to the QX200 Droplet Reader (Bio-Rad), which measures the fluorescence of each droplet using a two-color detection system. Raw data were analyzed using QuantaSoftTM software (Bio-Rad). Data were shown as a one-dimension plot with each droplet from a sample plotted on the graph of fluorescence intensity ver- sus droplet number. The fraction of positive droplets was then estimated using a Poisson distribution model. Assays performed to optimize and validate the quantifi-
cation of NPM1-mutated transcript levels are described in the Online Supplementary Appendix (Online Supplementary Methods, Online Supplementary Figures S2-S7, Online Supplementary Tables S2-S4). For statistical analyses, over- all survival and disease-free survival were estimated by the Kaplan-Meier method and compared by cause-specif- ic hazard Cox models. Overall survival was measured from the date of diagnosis until death from any cause. Disease-free survival was measured from the date of complete remission until the date of relapse. Patients were censored at the time of allogeneic stem cell trans- plantation in first remission. A P-value <0.05 was consid- ered statistically significant.
NPM1-type A mutation transcript levels in 28 AML samples were quantified using both RT-qPCR and droplet dPCR and produced concordant results, showing that dPCR could be considered as an alternative for monitor- ing type A mutations3 (Figure 1). Subsequently, 22 AML patients enrolled in the ALFA-0702 trial who achieved complete remission and harbored 16 different rare NPM1 mutations were studied by droplet dPCR (Online Supplementary Table S5). Although the number of subjects was very small, AML patients with rare NPM1 mutations who did not achieve a 4-log reduction of NPM1-based MRD in peripheral blood had a significantly shorter dis- ease-free survival (3-year disease-free survival: 43.8% vs. 100%; P=0.004) (Figure 2A) as described for classical NPM1-type A, B and D mutations.6 The difference did not reach statistical significance for overall survival (3-year overall survival: 71.4% vs. 100%; P=0.065) (Figure 2B), perhaps due to low numbers. FLT3-ITD was found in one poor responder (ratio 0.95) and three good responders (ratios 0.4, 0.4 and 1.0). Interestingly, some studies have found that rare NPM1 mutations (i.e., non-A/B/D) have different clinical or biological behaviors compared to clas- sical NPM1 mutations (i.e., type A, B or D).10,14 This could result from different amino-acid substitutions or accom- panying alterations in commonly mutated genes such as FLT3, DNMT3A or IDH1/IDH2. In current practice, it can be assumed that the ability to monitor classical NPM1 mutations by RT-qPCR in most laboratories could lead to earlier detection of relapses, better selection of patients with an indication for allogeneic stem cell transplanta- tion, and easier administration of pre-emptive therapy. Although focused on a small subgroup of patients, our results extend those previously published by the ALFA group6 and suggest that a post-induction
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haematologica | 2021; 106(6)