A Standard, 3-Tube, 10-Color Flow Cytometry Panel for Acute Myeloid Leukemia Diagnosis Can be Used for MRD Detection at Day 30 after Induction, and Is Predictive of Early Relapse
Authors: Alam Naheed , Tierens Anne , Yee Karen , Schimmer Aaron D. , Gupta Vikas , Minden Mark D. , Seftel Matthew , Porwit Anna , Schuh Andre C 
Published: October 2019

Abstract
Introduction:
As acute myeloid leukemia (AML) is a highly heterogeneous disease, strategies are needed to follow patients after induction and to predict relapse, so that second line treatments can be initiated early. Since assessment of remission status by morphology has known limitations, minimal residual disease (MRD) detection by flow cytometry (FC) has been used to evaluate remission status. In most publications, individually-tailored, patient-specific panels were applied at follow up. We evaluated MRD using a standard, 3-tube, 10-color antibody panel, which was applied at diagnosis and at day 30 post induction as the routine practice at Princess Margaret Cancer Center (PMCC).
Methods:
Patients who received front line induction chemotherapy for AML at PMCC from November 2012 to June 2013 and who were alive at Day 30 and able to undergo bone marrow aspiration were evaluated. All patients have had at least one year of follow-up.
The leukemia associated immunophenotype was determined at diagnosis by a standard panel [AML1: CD65 FITC, CD13 PE, CD14 ECD, CD33 PC5.5, CD34 PC7, CD117 APC, CD7 A700, CD11b A750, CD16 PB, AML2: CD36 FITC, CD64 PE, CD56 ECD, CD33 PC5.5, CD34 PC7, CD123 APC, CD19 A700, CD38 A750, HLA-DR PB, CD45 KO, AML3: CD71 FITC, CD11c PE, CD4 ECD, CD33 PC5.5, CD34 PC7, CD2 APC, CD10 A700, CD235a A750, CD15 PB, CD45 KO with NaviosTM flow cytometer and KaluzaTM analysis software (Beckman Coulter)]. The analysis protocol was created individually for each patient to follow-up aberrant phenotypes detected at diagnosis. At follow up, 250x103 events were acquired, allowing a sensitivity level of 0.05%. A level of <0.1% cells with the leukemia-associated phenotype was used as the cutoff for MRD positivity, following previously published data.
Results:
The characteristics of 51 patients who were followed are summarized in Table 1. The median age of patients was 52 (range 18-81) years. Overall survival of all patients at one year was 70%. The cumulative incidence of relapse at one year was 22% (95% CI 8.0-34.1).
Overall, 30 (59%) patients were positive for MRD by FC at day 30 after induction chemotherapy. Of these, 14 (47%) relapsed and 1 (3%) died in remission. Of 21 (41%) patients who were negative for MRD at day 30, only 4 (19%) relapsed, giving an odds ratio 3.72 [(95%CI 1.009-13.702), p-value 0.04].
Fifteen patients positive for MRD at day 30 remained in remission after 1 year. Of these, five (33%) were good risk patients with either inv(16) or t(8;21) abnormalities. Seven patients (47%) underwent allogeneic hematopoietic cell transplantation and remain in remission. The remaining three patients (20%) remain in remission without further intervention.
Conclusion:
MRD monitoring using standard, 3-tube, 10-color FC at day 30 after induction chemotherapy in AML is predictive of early relapse in standard and poor cytogenetic risk groups. In good risk cytogenetics patients, however, MRD positivity does not appear to be predictive.
Table 1: Patient Characteristics n=51 Age at diagnosis, median (range), years 52 (18-81) AML classification (WHO 2008) Acute myeloid leukemia with inv(16)(p13.1q22) or t(16;16)(p13.1;q22), CBFB/MYH11 4 (8%) Acute myeloid leukemia with t(9;11)(p22;q23);MLLT3-MLL 1 (2%) Acute myeloid leukemia, t(8;21)(q22;q22) RUNX1-RUNX1T1 5 (10%) Acute myeloid leukemia with myelodysplasia-related changes 8 (15%) Acute myeloid leukemia not otherwise specified 12 (23%) Acute myeloid leukemia, NPM1 mutated, FLT3 ITD 9 (18%) Acute myeloid leukemia, NPM1 mutated, FLT3 TKD 2 (4%) Acute myeloid leukemia, NPM1 mutated, FLT3 negative 10 (20%) Cytogenetic Risk Good 5 (10%) Standard 39 (76%) Poor 7 (14%) WBC count at diagnosis, median (range) 16.7 (0.4-237) Hemoglobin at diagnosis, median (range) 88 (50-131) Platelet count at diagnosis, median (range) 36 (5-340) Neutrophil count at diagnosis, median (range) 1.4 (0-34.9) Peripheral blasts at diagnosis, median (range) 4.72 (0-197) Bone marrow blasts at diagnosis, median (range) 51.5 (20-95) Performance status 0-1 51 (100%) MRD + ve 30 (59%) MRD - ve 21(41%) Allogeneic HCT 16 (31%)

Disclosures
Yee: Roche: Research Funding. Gupta:Incyte Corporation: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Minden:Celgene: Honoraria. Porwit:Beckman-Coulter: Speakers Bureau.