Ultrasensitive Detection of Genomic Minimal Residual Disease before or after Allogeneic Hematopoietic Cell Transplantation for Adult AML is Associated with Inferior Survival (2018)

Background: Genomic minimal residual disease (MRD) detection of AML in the setting of allogeneic transplantation (HSCT) has been limited by inadequate sensitivity for most genes. Recently, the feasibility of high sensitivity mutation detection of most recurrently mutated genes in AML using droplet digital PCR (ddPCR) has been demonstrated. Here we seek to apply ddPCR for AML MRD detection prior to and after HSCT. Methods: This study included 48 AML patients from 2005-12 who underwent HSCT in 1st or 2nd morphological complete remission and for whom both diagnostic bone marrow or peripheral blood mononuclear cells (PBMC) as well as PBMC specimens prior to (“Pre-HSCT”, N = 40) or day +30 after HSCT (“D30”, N = 44) were available. DNA from diagnostic samples underwent Sanger sequencing of 13 recurrently mutated genes in AML, or in cases with <2 mutations, targeted next gen sequencing of 36 more genes to identify somatic mutations. ddPCR was used to measure the variant allele frequency of each mutation to as low as .005% in 500 ng of genomic DNA from PBMCs sampled Pre-HSCT and at D30. Results: A median of 2 mutations was measured with ddPCR per case (range 1-4). Pre-HSCT, at least 1 mutation per case was detected in 85% (34 of 40) of cases, and all mutations measured per case were detected in 68% (27 of 40) of cases. ASXL1 (5 of 5), RUNX1 (3 of 3), TP53 (3 of 3), DNMT3A (7 of 8), and IDH2 (5 of 6) mutations were frequently detected Pre-HSCT, while NPM1 (7 of 13), FLT3 (1 of 3), and WT1 (1 of 3) were less likely to be detected. At D30, at least 1 mutation per case or all mutations measured per case were still detected in 34% (15 of 44) and 16% (7 of 44) of cases, respectively. ASXL1 (4 of 5), RUNX1 (2 of 3) and TP53 (2 of 3) mutations frequently persisted at D30, but ≤25% of cases with mutant DNMT3A (2 of 8), TET2 (2 of 8), IDH1 (1 of 5) or NPM1 (2 of 12) and no cases with mutant FLT3, IDH2 or WT1 had detectable mutations. Detection of all measured mutations in Pre-HSCT PBMC associated with inferior leukemia free survival (LFS) (HR 3.0 [95% CI 1.3-7.4], P = .03) and overall survival (OS) (HR 2.67 [1.1-6.6], P = .04) with a relapse rate (RR) of 66% at 1 yr. At D30, detection of any measured mutation associated with substantially worse LFS (HR 5.3 [1.9-14.7], P < .001) and OS (HR 6.5 [2.2-18.1], P < .001) and RR of 71% at 1 yr. D100 specimens (N = 21) were available for 8 cases that relapsed post-HSCT and 13 who did not relapse. ≥1 mutations were detected in 5 of 8 relapsed cases compared with 1 of 13 non-relapse cases (63% vs 9%, respectively; P < .01). Of the 5 relapsed cases with detectable mutations, the time from sampling to relapse was 46-92 d versus 148-264 d for the 3 relapsed cases without detectable D100 mutations. Conclusion: In this cohort, ASXL1, RUNX1 and TP53 mutations frequently persisted 30 days after HSCT, and detection of gene mutations in PBMC of AML patients measured either just prior to conditioning or 30 days after HSCT was associated with inferior LFS and OS.