A pilot study to standardize MRD estimation in AML by flowcytometry at a cancer centre in India.
46 patients of AML in CR post-induction were enrolled during the period December 1, 2012 to May 13, 2014 (median follow-up 13.2 months,1.7 -19 months). For flow cytometric analysis of BM aspirate, at least 20,000 events at diagnosis and 200,000 at CR were acquired in all cases from each tube and data were stored. An extensive panel of fluorochrome-conjugated antibodies, selected based on published literature, was applied to bone marrow samples, at two time points (baseline and post-induction) to identify leukemia associated abberant immunophenotypes at baseline and quantify these post-induction. We used seven combinations, each containing five antibodies, with CD34 and CD45 as backbone (CD65FITC/CD2PE/CD34ECD/CD13PE-Cy5/CD45PE-Cy7, CD9FITC/HLA-DRPE/CD34ECD/CD33PE-Cy5/CD45PE-Cy7, CD11b/CD15PE/CD34ECD/CD117PE-Cy5/CD45PE-Cy7, CD34FITC/CD56PE/CD19ECD/CD33PE-Cy5/CD45PE-Cy7, CD15FITC/CD7PE/CD34ECD/CD33PE-Cy5/CD45PE-Cy7, CD38/CD117PE/CD34ECD/CD4PE-Cy5/CD45PE-Cy7 and CD36FITC/HLA-DRPE/CD14ECD/CD64PE-Cy5/CD45PE-Cy7).Serial dilution experiments with normal bone marrow samples were conducted at 10%, 1%, 0.1%, 0.01% and 0.001%. Ten BM aspirates were used as controls.
42/46 samples (91%), showed LAIPs (median= 4, range = 1-8). Cross lineage infidelity was the commonest LAIP (78%), followed by underexpression(71%), overexpression(69%) and asynchronous expression (43%).The individual most common aberrancies were underexpression of CD45 ( 52%), followed by cross-lineage expression of CD56 ( 43%) and CD 4(36%), asynchronous expression of CD65(40%), and underexpression of CD33(36%) and CD38 (36%). Median MRD value was 0.055% (range =0.001-4.1). 31/42 (74%) patients had detectable MRD post-induction.Five patients relapsed, of which four had a positive MRD value post-induction (0.04, 0.3, 0.01 and 0.2%). OS and RFS of the cohort was 87% and 72% respectively. The relapse-free survival was 90.9% in patients with no detectable MRD post-induction and 65.9% in patients with detectable MRD (p = 0.6, HR = 1.7).
MRD estimation by flowcytometry is feasible and applicable in our patients of AML.
Clinical trial identification
All authors have declared no conflicts of interest.