Nanopore sequencing methods detect cell-free DNA associated with minimal residual disease and central nervous system infiltration in pediatric Acute Lymphoblastic Leukemia

Acute Lymphoblastic Leukemia (ALL) patients that have minimal residual disease (MRD) after therapy or infiltration of ALL into the central nervous system (CNS) are considered high risk. These patients are often given intensified and/or additional rounds of chemotherapy in the hopes of eliminating their disease. Current methods to diagnose MRD and CNS infiltration rely on detecting ALL cells in patient samples using pathology, flow cytometry, or isolation of ALL genomic DNA for next-generation sequencing. However, blasts may be present in the patient but not detectable in a bone marrow biopsy or cerebrospinal (CSF) fluid sample, leading to incorrect or delayed patient diagnosis. We have developed a nanopore sequencing assay to detect B-ALL-associated cell-free DNA in patient blood and CSF samples. Quantitation of B-cell specific VDJ recombination events in cell-free DNA samples defined B-ALL clonal heterogeneity. Monitoring cfDNA in blood and CSF samples allowed us to track the response of individual B-ALL clones throughout the course of treatment for each patient. Detection of cell-free DNA predicted the clinical diagnosis of MRD and CNS disease. We also identified patients diagnosed as CNS negative who had low levels of cell-free DNA in their CSF sample. These data suggest that cell-free DNA assays may be useful in detecting the presence of ALL in the patient even when blasts are not in the biofluid sample. In total, nanopore analysis of cell-free DNA is a simple, rapid, and inexpensive assay that can serve as a useful complement to traditional clinical diagnostic approaches in the treatment of ALL.