Minimal Residual Disease (MRD) Detection By Flow Cytometry Complements Molecular MRD Assessment in AML
Authors: Bücklein Veit , Köhnke Thomas , Schnorfeil Frauke , Metzeler Klaus H. , Konstandin Nikola P , Dufour Annika Maria , Sauerland Cristina , Hiddemann Wolfgang , Spiekermann Karsten , Subklewe Marion 
Published: October 2019

Abstract
The prognosis of patients with AML is determined by a multitude of recurrent genetic alterations, and treatment algorithms heavily rely on risk stratification by genetic characterization of the disease at the time of diagnosis. However, this a priori risk stratification does not integrate information on treatment susceptibility of the individual patient. Assessment of Minimal Residual Disease (MRD) aims to implement this information in the patient-specific treatment management. AML cells with aberrant phenotypes can be detected at sensitivities below 1:104 by flow cytometry in the majority of patients. Therefore, flow cytometry MRD assessment (flow MRD) enables determination of MRD status in patients without suitable molecular markers (e.g. NPM1, CBFß-MYH11, and RUNX1-RUNX1T1). Here, we validate the role of flow MRD in AML patients receiving intensive chemotherapy with and without available molecular markers.
Flow MRD was analyzed in patients with AML (excluding APL) diagnosed between 2012 and 2017 receiving intensive induction chemotherapy (sHAM or 7+3). Flow MRD analysis was performed during aplasia (on day 16 after treatment initiation) as well as post induction. Presence of ≥0.1% Leukemia-associated immunophenotype (LAIP)-positive cells was defined as flow MRD positivity. Molecular MRD was analyzed post induction for NPM1 and CBF, and post consolidation for RUNX1-RUNX1T1. Kaplan-Meier estimators and log-rank tests were used to analyze survival data. Cox's proportional hazards regression model was used to determine the influence of individual factors in multivariate analyses.
A total of 161 patients were included. In 5 cases (3.1% of all cases), no LAIP could be identified, and these patients were excluded from further analyses. Flow MRD assessment during aplasia was available in 145 cases. 122 patients had flow MRD assessments available post induction. 114 patients achieving CR or CRi after induction therapy had flow MRD assessments available at both time points. Flow MRD positivity during aplasia was associated with shorter event free survival (EFS, 6.1 months vs. 19.1 months, p<0.001). Similarly, flow MRD positivity post induction was associated with shorter EFS (11.9 months vs. median not reached, p=0.007). For both timepoints, flow MRD was an independent risk factor in multivariate analysis compared to known risk factors such as age, genetic/molecular risk profile as determined by the ELN2017 risk categories as well as early blast clearance by morphology. Persistent flow MRD positivity at both timepoints (combined flow MRD) identified patients with particularly short EFS (8.2 months), whereas patients with flow MRD negativity at both time points had the best outcome in our cohort (median not reached, p=0.002). Combined flow MRD status was an independent predictor of EFS and RFS (HR 1.9 and 1.8, p=0.001 and p=0.007, respectively), whereas blast clearance by morphology had no significant prognostic impact (p>0.05 for all endpoints).
64/161 patients (39%) had molecular MRD assessment available for analysis. In these patients, molecular MRD positivity predicted a significantly shorter EFS (9.3 months vs. median not reached, p=0.01). Indeed, molecular MRD positivity was an independent risk factor for adverse EFS and RFS (HR 1.7 and 1.6, p=0.008 and p=0.018, respectively). In this subgroup, flow MRD was not an independent prognostic factor. However, for patients without available molecular MRD marker (97/161 patients), flow MRD positivity at aplasia (p=0.004), post induction (p=0.015) or as combined status (p=0.004) was associated with a significantly shorter EFS and remained an independent risk factor in multivariate analysis (HR 2.5 and 2.6, p=0.016 and p=0.012 for EFS and RFS, respectively).
Taken together, we demonstrate that both flow MRD as well as molecular MRD strongly correlate with survival. While molecular MRD assessment was only available in 39% of patients, MRD assessment by flow cytometry was feasible in >95% of AML patients. Flow MRD positivity both during aplasia and post induction was an independent risk factor, confirming the superiority of flow MRD compared to early morphologic response assessment. In conclusion, molecular and flow cytometric MRD assessment are complementing methods for the estimation of treatment response, and will be integrated in clinical trials to validate their significance for patient-specific treatment management.

Disclosures
Metzeler: Celgene: Consultancy, Research Funding; Novartis: Consultancy. Hiddemann:F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Subklewe:Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Roche AG: Research Funding; AMGEN: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees.