Abstract
KMT2A-rearranged (KMT2A-r) leukemia represents a high-risk subtype marked by aggressive biology and high early relapse risk. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is commonly used for remission consolidation. Our prior work showed that detectable KMT2A transcripts by real-time quantitative PCR (RT-qPCR) before HSCT was associated with inferior 3-year outcomes: OS (51% vs. 82%, p < .001), LFS (42% vs. 81%, p < .001), and CIR (33% vs. 12%, p < .001) (Cancer, 2025). Post-HSCT, KMT2A transcript–positive patients experienced poor outcomes: relapse rate 93.5%, 3-year OS 12.5%, and LFS 0% (Br J Haematol, 2025; BBMT, 2014). Notably, 77.8% of transcript reappearance occurred within 3–4 months post-HSCT, with a median interval of 109 days (range, 44–305) from re-positivity to relapse. Given this high relapse risk, whether droplet digital PCR (ddPCR)—with greater sensitivity and absolute quantification—can improve MRD monitoring and relapse prediction remains unknown.
In this prospective trial (NCT06211166), we enrolled patients with KMT2A-r acute leukemia undergoing allo-HSCT. Serial MRD monitoring was performed post-HSCT using both RT-qPCR and ddPCR targeting patient-specific KMT2A fusion transcripts. Patients were categorized into three cohorts based on longitudinal MRD results: (1) Double-negative (DN): persistently negative by both assays; (2) Discordant (Dis): persistently RT-PCR–negative but with ≥1 ddPCR-positive result; (3) Double-positive (DP): positive by both assays before discordant conversion. The primary endpoint was 2-year cumulative incidence of relapse (CIR); secondary endpoints included 2-year MFC-detected MRD recurrence, LFS, and OS.
From June 12, 2023, to August 1, 2025, 158 patients underwent 872 paired RT-PCR and ddPCR MRD assessments (median 7 per patient, range 1–16). Median age was 34 years (range, 1–68); 46.8% were male. AML accounted for 92.4% of diagnoses, and 91.8% received HSCT in CR1. Pre-HSCT, 61.4% (97/158) had undetectable KMT2A transcripts; for the remainder, the median transcript level was 0.11% (range, 0.0012%–25%). After a median follow-up of 18.4 months, 110 patients (69.6%) were classified as Dis, 35 (22.2%) as DN, and 13 (8.2%) as DP (excluded from primary comparisons). Compared to the DN group, Dis patients showed worse outcomes: 2-year CIR (7.69% vs. 0%, p = 0.0759), MFC recurrence (13.2% vs. 0%, p = 0.0232), and LFS (86.3% vs. 100%, p = 0.027); OS was comparable (95.6% vs. 100%, p = 0.20). Among ddPCR-positive patients (n=110), 14 (12.7%) later converted to RT-PCR positivity; ddPCR predicted conversion a median of 73.5 days earlier (range, 9–392). Among Dis patients, 14 developed MFC-confirmed recurrence, with a median of 89.5 days (range, 16–394) from ddPCR positivity to MFC conversion. Eight patients developed hematologic or CNS relapse, with a median lead time of 163.5 days (range, 16–392) from ddPCR positivity to clinical relapse. In both univariate and multivariate analyses, pre-HSCT KMT2A MRD positivity was independently associated with relapse (HR = 46.5, 95% CI: 7.81–277.0, p < 0.001), MFC recurrence (HR = 6.45, 95% CI: 2.46–16.9, p < 0.001), and inferior LFS (HR = 18.3, 95% CI: 5.02–67.1, p < 0.001). The excluded DP group had poor outcomes (2-year CIR 35.4%, MFC recurrence 64.5%, LFS 55.4%).
ddPCR enables earlier and more sensitive detection of residual disease than conventional RT-PCR and MFC, and identifies high-risk patients misclassified as MRD-negative by standard assays. These findings support ddPCR-based MRD surveillance to optimize post-transplant risk stratification and guide preemptive interventions in KMT2A-rearranged leukemia.
