P432: validation of limit of quantification approach based flow cytometry for measurable residual disease assessment in acute myeloid leukemia in the hovon-sakk-132 trial

Background: Measurable residual disease (MRD) detected after intensive induction chemotherapy by multiparameter flow cytometry (MFC) in patients with acute myeloid leukemia (AML) in complete remission (CR), is associated with a worse event-free survival (EFS) and overall survival (OS). However, around 30% of MRD-negative patients relapse and these false negative results may indicate that MRD levels below the clinically validated 0.1% threshold could have prognostic value when correctly defined. A recent post-hoc analysis by the GIMEMA group in the AML-1310 trial showed that this lower level of MRD can be assessed using the limit of detection and limit of quantification (LOQ). Aims: In this study, we aim to validate the clinical relevance of low level MRD assessment using the objective LOQ approach in AML patients by determining the association with outcome parameters. Methods: For 482 de novo AML patients aged 18-65 years from the HOVON-SAKK 132 multicenter prospective phase III trial, MRD was assessed using MFC after two cycles of standard induction chemotherapy with or without the addition of Lenalidomide. Using the rules in rare event acquisition, we considered 50 relevant events enough to classify a rare population as ‘quantifiable’. We subsequently calculated the LOQ as “(50/ total number of cells analysed) × 100%”. Patients with a MRD value above the LOQ and below the 0.1% MRD cut-off, were considered LOQ-pos/MRD-neg. Since the calculation depends on the total number of cells acquired and the European Leukemia-Net (ELN) advises to collect at least 500,000 CD45-expressing events, we only included samples that fulfilled this requirement in the analysis. The primary endpoints of the study were EFS and OS and differences between MRD groups were assessed using Log-Rank test with Bonferroni adjustment in pairwise comparisons and visualized via Kaplan-Meier curves. Results: A total of 438 (91%) samples met the inclusion criteria, of which 63/438 (14%) were MRD-positive based on 0.1% MFC-MRD cut-off. These patients had a significantly worse EFS (3-year EFS 39% vs 59%; p=0.004) and OS (5-year OS 44% vs 63%; p<0.001) compared to MRD-negative patients, respectively. The 375 MRD-negative patients were reclassified based on the LOQ-status, with 146/375 (39%) LOQ-neg patients (33% of total cohort) and 229/375 (61%) LOQ-pos/MRD-neg (52% of total cohort). The 3-year EFS (LOQ-neg 66%; LOQ-pos/MRD-neg 55%; MRD-pos 39%, p=0.001; Figure 1A) and 5-year OS (LOQ-neg 74%; LOQ-pos/MRD-neg 59%; MRD-pos 44%, p<0.001; Figure 1B) was significantly different for the three groups. Also in pairwise comparison, a statistical significant difference in 3-year EFS and 5-year OS was found. Summary/Conclusion: By separating the MRD-negative patients based on the LOQ-status, we were able to identify a low level MRD group (LOQ-pos/MRD-neg), which had a statistical significantly worse EFS and OS compared to LOQ-neg patients. The clinical relevance remains to be elucidated, but implementation of this objective cut-off may increase sensitivity and aid clinical decision making.Keywords: Minimal residual disease (MRD), Flow cytometry, Acute myeloid leukemia