Single Cell Sequencing of Pediatric Acute Myeloid Leukemia Reveals Clonal Evolution to Relapse on Combination Chemotherapy with Sorafenib

Introduction: Relapse of pediatric acute myeloid leukemia (AML) remains a leading cause of childhood cancer mortality, and leukemias with activation of the Fms-like tyrosine kinase 3 (FLT3) are particularly susceptible to relapsed disease. Risk-directed therapy to prevent relapse is based both on genetic changes known to drive drug resistance, and measurable residual disease (MRD) at the end of induction therapy (EOI). In adult AML, resistance to type II FLT3-inhibitors, like sorafenib, is primarily driven by on-target FLT3 kinase domain (KD) mutations. However, the resistance mechanisms for pediatric leukemias, which are treated on combination therapies, have not been fully elucidated. MRD is considered the among the most predictive markers of future relapsed disease. It has been assumed that the major clone at the time of MRD assessment will predict the majority clone at relapse. However, this assumption has not been proven. The definition of the most specific genetic and MRD markers of relapse are essential to prognosticate and personalize therapy to prevent relapsed disease. Methods: We performed single cell sequencing (SCS) with a high-throughput DNA sequencing platform, Mission Bio Tapestri, on bone marrow or peripheral blood samples from 24 samples from 8 pediatric patients treated on COG AAML1031 with serial samples from diagnosis, EOI, and relapse. Results: We analyzed a total of 94,833 cells from 8 pediatric patients (median cells per patient 12,428) all treated on AAML1031. SCS revealed a sensitive and specific description of clonal evolution on the combination of sorafenib with cytotoxic chemotherapy. The FLT3 internal tandem duplication (ITD) was controlled by the therapy in only half of the patients. In five of the patients, the FLT3-ITD was present in multiple clones. The FLT3-ITD co-mutated with additional mutations (NRAS, SH2B3, WT1, TET2, or NPM1) in half of the patients. However, the presence of a co-mutation did not necessarily correlate with whether or not the ITD-containing clone persisted at the time of relapse. Of the leukemias whose relapse was not driven by FLT3, the most likely mutational driver of resistance was NRAS. Notably, however, despite the fact that FLT3 KD mutations make up the bulk of mutational resistance to type II FLT3i such as sorafenib in adult patients, there were no on-target FLT3 mutations found in any of these pediatric patients. Further, SCS allows for an unprecedented depth of analysis of the genetic complex