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[Figure][1] A 73-year-old man without prior personal or family history of anemia developed a chronic hemolytic anemia characterized by an absolute reticulocyte count of 347 000/mm3 (13%) and spherocytic red-cell morphology (see panel A). Antiglobulin testing was negative but osmotic

Background FLT3 is expressed in most human acute leukemias. When activated by FL, wild type (wt) FLT3 dimerizes and initiates downstream signals that result in proliferation and inhibition of apoptosis and differentiation. Activating FLT3 mutations (internal tandem duplications (ITDs) or point mutations) are common in AML and rare in ALL. ITD mutations confer a poor outcome in AML. In vitro, mutant FLT3 signaling can be further enhanced by binding of FL. Peripheral blood (PB) plasma FL levels rise in adults with AML, peaking about two weeks after initiation of chemotherapy. We sought to determine plasma levels of FL in pediatric patients after chemotherapy, and the functional effect of various levels of FL on both wt and mutant FLT3 leukemia cells. Methods FL levels were measured using FL ELISA on plasma samples (n=352) isolated from PB of children (n=75) enrolled on 4 multi-center acute leukemia clinical trials. Functional studies were performed on AML and ALL cell lines with wt FLT3 (HL60, RS4;11, SEMK2, and KOPN-8) and mutant FLT3 (MOLM14, MV4-11, and HB-1119). 72 hr etoposide IC50 was determined by WST-1 for each line. Cells were plated (250,000 cell/mL) for 72hr at etoposide IC50 in RPMI 1640 along with increasing concentrations of recombinant human FL (62.5 to 4,000 pg/ml). Cell cycle and apoptosis were analyzed using propidium iodide staining and annexin V/7-AAD binding, respectively. To explore the mechanism of FL effects, Ba/F3-ITD cells were incubated for 72hr in serum-free conditions with either 4,000 pg/mL (“high”), 62.5 pg/mL (“low”), or no FL. After washing, total and phosphorylated FLT3 protein levels were determined by Western blot. Results Pediatric patients receiving chemotherapy for the treatment of acute leukemia demonstrate a pattern of plasma FL rise with low levels at baseline (mean 41 pg/ml) and peak levels at day 11-14 following initiation of therapy (mean: 1,190 pg/mL; max: 5,783 pg/mL)(Fig 1A). Cell lines with FLT3 activating mutations selectively demonstrate resistance to etoposide-induced apoptosis (Fig 1B) and G2/M cell cycle arrest (Fig 1C) at low concentrations of FL (62.5 pg/mL). Dose-dependent reduction of etoposide resistance is seen with increasing concentrations of FL up to 4,000 pg/mL, suggesting that optimal etoposide-induced killing of FLT3-mutant leukemias may occur when FL plasma levels are at their peak. Ba/F3-ITD cells pre-incubated with peak concentrations of FL showed diminished baseline FLT3 phosphorylation, suggesting that the interaction of FL and FLT3/ITD exhibits substrate inhibition kinetics and results in a loss of FLT3/ITD-induced activation with high level FL exposure, thus providing a mechanistic basis for the observed loss of etoposide resistance. Conclusions Plasma FL rises to peak levels 11-14 days after initiation of chemotherapy. Through substrate inhibition of mutant FLT3 enzymatic activity, peak FL levels may reduce the etoposide resistance that characterizes FLT3-mutant leukemia cells exposed to pre-chemotherapy levels of FL. Thus, introduction of etoposide in a “time sequential” manner during periods of peak plasma FL levels may enhance killing of residual chemoresistant FLT3-mutant leukemia cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 568 Background: Infant acute lymphoblastic leukemia (ALL) is clinically and biologically distinct from ALL in older children. About 80% of infant ALL cases harbor MLL rearrangements (MLLr). MLLr infant ALL is an aggressive disease with poor prognosis, particularly in cases diagnosed at Methods: We prospectively characterized 54 consecutive diagnostic leukemia specimens from infants with ALL enrolled on AALL0631 with respect to: 1) Quantitative surface FLT3 protein expression (s-FLT3) using FACS CD135 mean fluorescence index (MFI); 2) FLT3 inhibitor (lestaurtinib: 0 to 100 nM) in vitro sensitivity using 48 hour WST-1 cytotoxicity assays; samples defined as “sensitive” if 100 nM produced ≥40% inhibition of cell viability compared to vehicle controls; 3) Identification and quantification of putative leukemia stem cell (LSC) subpopulation by flow cytometric immunophenotyping (% CD34+, CD38− viable cells). Studies were performed blinded to clinical information and MLL status; data were later correlated with age at diagnosis ( Results: Of 54 cases, 42 (78%) are MLLr and 12 (22%) are MLLwt. For MLLr cases, fusion partners are AF4 (n=13), ENL (n=17), AF9 (n=3), AF1p (n=2), and other (n=7). Of MLLr cases, 10 (24%) were 90days (mean MFI 62.6 vs. 22.0, p We analyzed FLT3 inhibitor sensitivity according to MLL genotype and s-FLT3. All 42 of the MLLr samples were evaluable, with 35 (83%) sensitive and 7 (17%) resistant; 11 of 12 MLLwt samples were evaluable (1 had excessively poor viability), with 6 (55%) sensitive and 5 (45%) resistant. We thus found a significant association between MLLr and FLT3 inhibitor sensitivity (Chi square 4.125, p=0.042). In the s-FLT3 analysis, the sensitive samples trended towards higher s-FLT3 than the resistant samples (mean MFI 31.3 vs. 16.7, p=0.17). Finally, we compared s-FLT3 in the putative LSC-like subpopulation fraction (if present) with the “bulk” leukemia population. Of the 54 samples analyzed, 35 (65%) had a clearly identifiable LSC-like population (i.e., comprising >0.5% of the total viable leukemia population). Within these 35 samples, s-FLT3 was significantly higher in the LSC-like fraction than in the bulk leukemia population (mean MFI 40.4 vs. 31.2, p=0.05). Conclusions: We have for the first time prospectively compared FLT3 expression and FLT3 inhibitor sensitivity in unselected representative cohorts of MLLr and MLLwt infants, confirming that MLLr cases express significantly higher levels of surface FLT3 protein and are more sensitive to the cytotoxic effects of FLT3 inhibition. Novel findings include higher surface FLT3 protein expression in MLL-AF9 cases relative to MLL-AF4 cases, and the strikingly higher surface FLT3 protein expression in MLLr cases in the youngest infants. An intriguing finding is the presence of phenotypically-defined subpopulations of “LSC-like” cells in the majority of cases, and that this LSC-like population expresses significantly higher levels of FLT3 protein than the “bulk” population. These findings provide further evidence that FLT3 overexpression plays a role in MLLr leukemogenesis. If these findings correlate with clinical responses in COG AALL0631, they may prove to be useful predictive biomarkers in selecting patients for whom FLT3-targeted therapy is most appropriate. Disclosures: No relevant conflicts of interest to declare.