Secondary transplantation of risk-stratified AML cells in NSGS mice. (A) Experimental design. 1ry, primary; 2ry, secondary. (B) Percentage of BM engraftment in secondary (2ry) recipients (n = 24) at euthanization (18 weeks or sacrifice point) for each patient sample grouped by risk (left panel). Only the highly engrafted primary samples/mice were selected for serial transplantation (1-2 mice). The dotted line indicates the cutoff for engraftment levels > 0.1%. Each circle represents a single mouse. Gray circles depict mice with <0.01% engraftment. Percentage of engrafted 2ry recipients (leukemic cells > 0.1%) transplanted with BM cells from primary recipients of FR, IR and HR samples (right panel). (C) Representative FACS analysis of CD45, CD33, CD19, and CD34 expression in AML patient samples and PDXs. CD34 expression within the leukemic blasts is heterogeneous. (D) Proportion of CD34⁺ AML blasts before and after xenotransplantation in NSGS mice for all risk-stratified AML primary samples (n = 28). For primary AML samples, each point represents a single patient. (E) Schematic diagram of the LTC-IC assays. Magnification 40×. Red arrowheads depict the colonies. (F) Number of wells plated with 250 cells showing CAs after 5 weeks of blast:MS5 LTC. (G) Estimated LICs in FR, IR, and HR AML primary samples for cell doses of 250 to 2000 AML blasts. For PDX samples, each dot represents the mean ± SEM from all mice transplanted with that sample (n = 112 mice). Data are mean ± SEM. **P < .01, ****P < .0001, unpaired Student t test. D, day; IBMT, intra-BM transplantation; ND, not detected; NK, normal karyotype; ns, not significant; W, week.