A Single Mouse Trial Platform for Evaluation of Novel Agents in Acute Lymphoblastic Leukemia By the Pediatric Preclinical Testing Consortium

Introduction: While children diagnosed with the most common pediatric malignancy, acute lymphoblastic leukemia (ALL), now experience close to a 90% likelihood of cure, the outcome for several high-risk subtypes remains poor. Furthermore, since standard-of-care drugs are extremely effective in this disease, and there are relatively few patients eligible for early phase clinical trials, only the most promising new agents are advanced for clinical evaluation following rigorous preclinical testing. However, conventional preclinical testing of novel agents is not sufficiently resourced to be able to encompass the vast heterogeneity of pediatric ALL, and new approaches to preclinical testing are required in this disease. The purpose of this study was to evaluate the utility of a single mouse trial (SMT) platform for preclinical assessment of novel agents on an almost clinical trial scale, to encompass the broad heterogeneity of pediatric ALL in a single experiment, and to identify molecular biomarkers associated with in vivo drug responses when carried out using molecularly-annotated patient-derived xenografts (PDXs). Methods: Eighty pediatric ALL PDXs broadly representative of all pediatric ALL subtypes were characterized in terms of engraftment kinetics in immune-deficient NSG mice, and molecularly annotated by RNA-seq, exome-seq and DNA copy number analysis. Between 2-5 million cells from each PDX were inoculated via the tail vein into 2 NSG mice/PDX. Starting at 2 weeks post inoculation engraftment was monitored by flow cytometric enumeration of the proportion of human CD45+cells in the murine peripheral blood (%huCD45+). When the %huCD45+ for each PDX reached >1% one mouse/PDX was treated with the established drug and topoisomerase I inhibitor topotecan (Tpt, 0.6 mg/kg IP daily x 5 x 2 weeks, repeated at 21 days) and the other mouse was treated with the experimental drug and second mitochondria-derived activator of caspases (SMAC)-mimetic birinapant (Bpt, 15 mg/kg IP every 3 days x 5). Treatment response was monitored using stringent objective response criteria modeled after the clinical setting, by mouse event-free survival (EFS where an event was defined as 25% huCD45+), and by waterfall plots comparing the maximum decrease in %huCD45+at any point post treatment initiation. The authenticity of each PDX was verified using a 60-allele SNP array both at the time of inoculation and at relapse post drug treatment for all mice. Results: Retrospective analysis o