The denominator variable in the quantitation of <scp>MRD</scp> in acute myeloid leukemia
Authors: Neil Came, David Westerman, Surender Juneja
Journal: American Journal of Hematology (2015)

To the Editor: The recent paper published in this journal by Ouyang et al. adds to a growing number of high quality clinical studies that discuss the impact of minimal residual disease (MRD) assessment of acute myeloid leukemia (AML) by multiparameter flow cytometry (MPFC) on clinical outcome 1. The authors should be complimented for providing methodological details of MPFC analysis that have not always been as clear in similar publications. An underplayed but important determinant for the calculation of the “percent MRD” (the only information that the clinician really wants) is the cell population/s used to calculate the denominator. While the number of denominator events determines the assay's sensitivity, knowledge of the cell type/s enumerated to arrive at this number is crucial when comparing assessment of response and prognostication across studies, but this is not uniform across clinical studies of AML that measure MRD by MPFC. The potential effect on final MRD percentage using different denominator bone marrow cell populations becomes particularly relevant when the level of MRD is close to meaningful thresholds such as 0.01% and 0.1% 2 because important therapeutic decisions are being made based on these levels. Other variations in pre-analytical and analytical steps aside 3, the denominator population is one of the few pieces of information available to the astute reader that enables critical evaluation of the clinical study for application to one's own patients or comparison with other clinical studies that report MRD values using MPFC. However, there is no global harmonized assay for the monitoring of MRD in AML by flow cytometry. Regulatory bodies such as the United States Food and Drug Administration (FDA) are trying to address this in the context of “a series of FDA workshops to establish processes and procedures necessary to qualify a prognostic biomarker, MRD, as a possible response or efficacy biomarker in a group of hematological malignancies” 4. The fruition of a similar workshop and cooperation between transatlantic flow cytometry societies resulted in consensus for assessment of MRD in plasma cell myeloma 5, and there is no reason why this cannot be achieved with AML 3. The potential impact of using different denominators to calculate the percent MRD is illustrated in the accompanying figure which shows the change in proportion of a CD34+ precursor cell population (for example) relative to different denominators, and explains the common terminology used in clinical studies of AML MRD in bone marrow aspirate samples analyzed by MPFC (Fig. 1). Referring to the figure, each box represents a bivariate dot plot of light scatter data derived from the same bone marrow aspirate sample analyzed on a flow cytometer that has counted a total of 1,773,161 putative cells (“events”) as they pass in single file through a laser beam. “Singlets” (top left-hand box) refers to the population of cells that show a proportionate relationship between the area (A) versus height (H) of the signal produced by forward scatter of light (FSC), thus enabling the “gating out” of “doublets” which show a disproportionate FSC-A vs. FSC-H. “Viable Cells” (top row of boxes) are those singlets that also show signal characteristics plausible for “real” or “live cells,” thus alternatively referred to in clinical studies as either: “Nucleated cells” (either because this is implied by the light scatter characteristics, or because they have been more accurately demonstrated using an intracellular DNA binding dye); “total events”; or simply “events” (the latter two referring to the number of cells left over after excluding doublets and non-viable cellular debris from the initial number of events counted by the analyzer). White blood cells (WBC's) also referred to in studies as leukocytes, or “non-erythroid cells”) are counted as all viable events that also express CD45. “Mononuclear cells” (top right-hand box) are counted by excluding the cluster of CD45+ cells that exhibit increased side-scatter (SSC) characteristic of the maturing granulocytic compartment in the bone marrow. This may also be refined with the aid of a DNA binding dye to better differentiate true nucleated cells from other extraneous events such as non-lyzed erythrocytes, platelets, and cellular debris in the CD45-negative (non WBC) region; or alternatively, partitioned during the pre-analytical phase by ficoll density gradient centrifugation. At each stage that a cell population is selected (“gated”), the percentage of the numerator population changes. The bottom row of boxes illustrate how the proportion of a small population of cells in the bone marrow (CD34+ precursor cells in this example) changes depending on which cellular compartment is used to calculate the denominator. Notably, the proportion of CD34+ cells varies in this sample by approximately 10% depending on whether viable cells or WBC's are used. Excluding granulocytes and using mononuclear cells as the denominator raises the proportion of CD34+ cells by more than double—a factor that can vary greatly depending on the degree of myeloid regeneration at the time point of MRD assessment. On closer inspection of the eight clinical studies each involving 50 or more patients that were included in a recent review of the prognostic and therapeutic implications of MRD detection in AML by MPFC, there were differences in how each study defined and named the cell population/s used to calculate the denominator as follows: events, total events, nucleated cells, leukocytes, mononuclear cells, and total events within a live gate of cells in the lymphomonocytic/granuloblastic region, excluding debris and platelet aggregates 2. Consensus with all aspects of AML MRD testing by MPFC, including the denominator, will result in better reproducibility between studies and facilitate drug-discovery and regulatory approval. While each method of denominator enumeration has pros and cons, “total nucleated cells” has been proffered as the likely best parameter around which to standardize in the future 3. A global effort is urgently needed to standardize the definition of denominator. Until such time, it is recommended that laboratories report explicitly which method is used for calculating the denominator 6, enabling the clinician-reader to extrapolate therapeutically meaningful information. Variation in the percentage of a CD34+ progenitor sub-population, for example, is demonstrated using different denominator cell populations in a bone marrow sample by MPFC. Neil Came1,2,3*, David Westerman1,3, Surender Juneja2,3 1Pathology Department, Peter MacCallum Cancer Centre, Melbourne, Australia; 2Diagnostic Haematology, the Royal Melbourne Hospital, Parkville, Australia; 3Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Australia