Abstract Acute leukemia is the most common childhood cancer, and remains one of the leading causes of death in children under 15 years of age. Prevention, therefore, remains the ultimate goal. An essential part of future preventive efforts will involve identifying children harboring preleukemic clones at birth. Several leukemia-initiating genetic lesions have been shown to arise prenatally; however, many other leukemia-initiating lesions have not been examined at birth. Several critical questions remain to be answered: 1) What subtypes and what proportion of childhood leukemia develops in utero? 2) What is the specific cell(s) of origin in which preleukemic clones arise? and 3) Are some newborns more prone to developing leukemia-initiating lesions than others? In this project, we will answer these questions, with a focus on the ~70% of patients with known translocation or point mutation driver events. This project has three main aims. Aim 1: To identify the presence and clonal frequency of prenatal leukemia-initiating lesions at birth. We will obtain matched cord blood (CB) and diagnostic leukemia samples from ~250 childhood leukemia patients in the Children’s Oncology Group Project:Every Child study. Backtracking will focus on ~182 patients with translocation/mutation-driven subtypes. We derive patient-specific somatic mutations from tumor profiling, then use droplet digital PCR (ddPCR) in flow-sorted CB cells to confirm the presence and frequency of leukemia-initiating lesions at birth, and at different hematopoietic stages. We will also use ddPCR to backtrack lesions in available newborn dried bloodspots (N ~45). Aim 2: To determine the cell of origin of leukemia- initiating lesions, the transcriptomic changes from preleukemia to overt leukemia, and whether secondary mutations arise prenatally, across childhood leukemia subtypes. In 50 childhood leukemia patients where CB ddPCR is positive we will conduct single-cell TARGET-seq on flow-sorted cell populations to simultaneously detect the presence of each patient-specific leukemia-initiating events, secondary mutations, and gene expression in genotypically- and immunophenotypically-defined populations at a single cell level. Aim 3: To determine whether the presence and frequency of preleukemic clones correlate with known risk factors for leukemia. Demographic, perinatal, and genetic risk factors for childhood leukemia will be obtained through parental survey, birth records, and sequencing data. For overall ALL and AML, and for common subtypes, we will test for association between risk factors and the presence and clonal frequency of preleukemic clones at birth, as measured in Aim 1. Identifying a specific cell of origin of preleukemic genomic alterations, and their frequency in neonatal blood, will shed light on childhood leukemia etiology and have important implications for precision prevention efforts. This study will identify key steps required for leukemogenesis by directly comparing the ge...