A refined approach to detect and measure minimal residual disease in childhood acute myeloid leukemia by flow cytometry
Authors: Federica Capolunghi, Claudia Capponi, Barbara De Stefani, Matteo Luciani, Franco Locatelli, Maurizio Muraca, Rita Carsetti
Journal: American Journal of Hematology (2013)

Event-free survival for children with acute myeloid leukemia (AML) ranges between 40% and 60% 1, 2. Risk stratification of patients with AML mainly relies on cytogenetic/genetic classification and morphological response to induction therapy. However, assessment of response to chemotherapy by conventional morphology is subjective and lacks sensitivity. Thus, high-resolution methods such as real-time quantitative polymerase chain reaction (RQ-PCR) and flow cytometry (FCM) have been recently proposed to detect minimal residual disease (MRD) 3. Notwithstanding its high sensitivity, RQ-PCR can be used only in a fraction of patients and cannot precisely calculate MRD because the amount of PCR product does not accurately reflect cell number 3. MRD quantification by FCM has a significant prognostic value in predicting both risk of relapse and survival 4. Recently, FCM has been successfully used for the final risk assignment determining the timing and the intensity of therapy in childhood AML 2. The main limitation of MRD monitoring by FCM is the requirement of highly specialized skills and experience to distinguish MRD among the different populations arising in the bone marrow in the reconstitution phase following chemotherapy or transplantation 3, 5, 6. Limited information is available on FCM characterization of myeloid developmental stages during normal hematopoiesis 5, 6. This knowledge is indispensable to discriminate MRD by FCM in AML 3, 6. Here, we describe a simple and most effective approach to detect MRD in AML, based on the characterization and distinction of normal precursors from leukemia cells. Figure 1 illustrates a prototypical characterization of the phenotype of myeloid precursors using a 6-color staining with antibodies anti-CD33, CD34, CD117, CD71, CD45, and HLA-DR. In Fig. 1a, we show a healthy child bone marrow (upper panels), a steroid-treated child with rheumatic disease (middle panels) and a 1 month-old infant with anemia associated to absence of bone marrow erythroid progenitors (lower panels). Monocyte-myeloid precursors reside in the CD34posCD117pos population (blue cells, gate A) whereas committed precursors of the granulocytes lineage and erythroid lineage are identified as CD34dullCD117pos cells (light blue cells, gate B). The CD34posCD117neg population (asterisk) corresponds to CD19pos pro-B cells (data not shown). Using our combination of markers, cells included in gate B can be dissected into three CD33pos myeloid precursor stages, based on the progressively down-regulation of the HLA-DR component (dark blue, green and pink cells). The location of these three stages within the CD45-side scatter plot (right columns, dark blue, green, and pink areas), showing the increasing intracellular complexity, confirms their maturation toward granulocytes. The pro-erythroblast (pro-e) are CD33negHLA-DRneg and can be easily identified by their high levels of CD71 (gate B, orange cells). Figure 1b shows an example of childhood AML followed from onset until relapse, first detected 7 months after bone marrow transplantation (MRD 0.3% of live cells gate) and confirmed 2 months later by relapse (10%). The immunophenotype of blast cells (red cells) at onset is showed in the upper panels. Leukemia cells closely resembled normal precursors based on the expression of CD34 and CD117 (first plot in the left column) but lacked CD33, HLA-DR, and CD71 (upper plots panel). Cells with this phenotype do not exist during normal development (compare with examples shown in Fig. 1a). In the middle panels, blast cells (red cells) appear among the normal precursors in the reconstituting bone marrow 7 months after transplantation (MRD 0.3%). Leukemic blasts increased to 10% in bone marrow taken two months later, when normal precursors had become undetectable. Thus, our approach allows identification and precise quantification of rare leukemia cells based on their distinction from normal precursors. This method also allows to detect and track leukemia cells when information on leukemia-associated phenotype (LAP) at onset is unavailable and when leukemia cells at relapse are not identical to the previously defined LAP. Further studies are necessary to evaluate whether our method to detecting and measuring MRD in AML can be used to improve patient stratification and assign them to personalized treatment protocols. Federica Capolunghi1* Claudia Capponi1 Barbara De Stefani2 Matteo Luciani3 Franco Locatelli,3 Maurizio Muraca1 Rita Carsetti1,2 1Department of Laboratories, Bambino Gesù Children's Hospital (IRCCS), Rome, Italy 2B-cell Development Laboratory, Research Center Bambino Gesù Children's Hospital (IRCCS), Rome, Italy 3Department of Pediatric Oncology and Hematology, Bambino Gesù Children's Hospital (IRCCS), Rome, Italy