Measurable Residual Disease (MRD) in Elderly Acute Myeloid Leukemia (AML): Results from the Pethema-Flugaza Phase III Clinical Trial
Authors: Simoes Catia Patricia , Paiva Bruno , Martínez-Cuadron David , Bergua Juan-Miguel , Vives Susana , Algarra Jesús Lorenzo , Tormo Mar , Martinez Pilar , Serrano Josefina , Herrera Pilar , Ramos Fernando , Salamero Olga , Lavilla Esperanza , Gil Cristina , Lopez Lorenzo Jose Luiz , Vidriales Maria-Belen , Labrador Jorge , Falantes José F. , Sayas Maria Jose , Ayala Rosa , Martinez-Lopez Joaquin , Villar Sara , Calasanz María José , Prosper Felipe , San-Miguel Jesús F. , Sanz Miguel A. , Montesinos Pau 
Published: November 2020

Background: AML is the most common acute leukemia in adults and its prevalence significantly increases in the elderly. The 5-year survival rate for adults younger than 60 is around 40% and decreases to 10% in patients above this age. Even those patients who tolerate intensive induction chemotherapy and achieve complete remission (CR) have poor outcome. Detection of MRD in younger AML refines outcome prediction of patients in CR after intensive chemotherapy. However the value of MRD in elderly patients is inconsistent between those treated with intensive vs hypomethylating drugs, and unknown after semi-intensive therapy.
Aim: Define the role of MRD assessment by multidimensional flow cytometry (MFC) for therapeutic decision making in elderly AML patients treated with semi-intensive chemotherapy vs hypomethylating agents (HMA).
Methods: Two-hundred eighty-three elderly AML patients were included in the PETHEMA phase III FLUGAZA clinical trial and randomized to receive three induction cycles with fludarabine and cytarabine (FLUGA) followed by six consolidation cycles with reduced intensity FLUGA, or three induction cycles with 5-azacitidine (AZA) followed by 6 consolidation cycles with AZA. After consolidation, patients continued with the same treatment if MRD ≥0.01% or stopped if MRD &lt;0.01%. MRD was prospectively assessed using MFC after induction and consolidation. Patient-specific aberrant phenotypes were used for highly-purified FACSorting of leukemic cells at diagnosis and after treatment in MRD positive patients, or CD34 progenitors in MRD negative cases.
Results: Patients in CR/CRi with undetectable MRD (N = 13/72) had significantly improved 1-year cumulative incidence of relapse (CIR, 38% vs 81%; HR: 0.43, P =.030) and relapse free survival (RFS, median of 13 vs 5 months; HR: 0.40, P = .012) as compared to cases with persistent MRD (N = 59/72), though not overall survival (median of 19 vs 11 months; HR: 0.56, P =.097). Achieving undetectable MRD significantly improved RFS of patients with adverse genetics (HR: 0.32, P =.013). On multivariate analysis including genetic risk and treatment arm, MRD status in patients achieving CR was the only independent prognostic factor for CIR (HR: 2.95; P =.002) and RFS (HR: 3.45; P =.002). Eleven of the 13 patients with detectable MRD did relapse or died after the last update of the follow-up, notwithstanding these patients showed a trend for longer overall survival.
To better understand the limitations of MFC-based MRD assessment in identifying patients with long-term survival and driven by the paucity of data about immunophenotyping in elderly AML, we investigated the level of phenotypic divergence/overlap between leukemic cells vs their normal maturation-stage counterpart in healthy adults (N=10). Using an unbiased scoring approach based on principal component analysis of merged data, we found that only 7/265 (3%) patients harbored leukemic cells showing phenotypic profiles fully overlapping with their normal maturation-stage counterpart. We thus hypothesized that in patients with undetectable MRD, phenotypically normal CD34 progenitors would contain cells with leukemic-initiating-potential that could be identified on genetic grounds. Using whole exome sequencing, we systematically identified mutations and copy-number abnormalities (CNA) consistent with genetic MRD (gMRD). These findings were similar to that observed when the genomic landscape of leukemic cells at diagnosis were compared to persistent MRD cells, underpinning the extent of gMRD in patients achieving CRMRD by MFC. Interestingly, there was a significant increase in the number of genetic alterations from diagnosis to MRD stages in patients treated with FLUGA vs AZA (1.9-fold vs 1.1-fold, P =.002), which could be related to the mutagenic potential of antimetabolites.
Conclusions: Our results show that older AML patients achieving undetectable MRD after semi-intensive therapy or HMA have lower risk of relapse and a trend for longer survival. However, this study unveiled that phenotypically normal CD34 progenitors possess substantial genetic abnormalities in cases with negative MRD. Thus, improved sensitivity of MRD testing by MFC and possibly the combined use of this method with NGS, might be warranted to identify elderly AML patients in CRMRD that may experience long-term survival and could benefit from treatment individualization based on MRD status.

Disclosures
Paiva: SkylineDx: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Roche: Research Funding; Adaptive: Honoraria; Amgen: Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Kite: Consultancy; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding. Tormo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; MSD: Honoraria; Daiichi Sankyo: Honoraria; Servier: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Ramos:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: travel and research grants; Novartis: Consultancy, Other: travel grant; Amgen: Consultancy, Other: travel grant; Abbvie: Consultancy, Other: travel grant; Jannsen: Other: travel grant; Roche: Other: travel grant; Rovi: Other: travel grant; Merck-Sahrp & Dohme: Other: travel grant; Daiichi-Sankyo: Other: travel grant; Takeda: Consultancy, Other: travel grant . Vidriales:Janssen, BMS, Novartis, Roche, Astellas Pharma, and Jazz Pharmaceuticals.: Honoraria, Other: Advisory boards. Martinez-Lopez:Novartis: Consultancy; Janssen-cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; BMS: Consultancy, Research Funding; Incyte: Consultancy, Research Funding. San-Miguel:Bristol-Myers Squibb, Celgene, Novartis, Takeda, Amgen, MSD, Janssen, and Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche, AbbVie, GlaxoSmithKline, and Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sanz:Teva, Daiichi-Sankyo, Orsenix, AbbVie, Novartis, and Pfizer: Other: Consulting or Advisory Role.