Novel methods to assess cell-free circulating tumor DNA in acute lymphoblastic leukemia.

Abstract

10034 Background: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, with a relatively high relapse rate, which is associated with poor prognosis. Currently, minimal residual disease (MRD) at the end of induction and consolidation therapy is the best predictor of patient relapse, however obtaining bone marrow aspirate is invasive and not always accurate. Another major concern in ALL is the presence of central nervous system (CNS) disease, which is often present long before clinical diagnosis can be made by flow cytometry. To circumvent these clinical challenges, we developed a new assay quantifying cell-free, circulating tumor (cfDNA) as a biomarker of disease progression, which can be correlated with MRD status as a predictor of relapse. cfDNA is frequently used to monitor progression of solid tumors, but pediatric leukemias lack common mutations that can be used to distinguish leukemic cfDNA from normal cfDNA. Methods: We examined two possible methods for using ctDNA as a biomarker: leukemia cell clonality and DNA methylation profiling. We developed a novel workflow for identifying VDJ rearrangements in leukemia cells and tracking their presence in cfDNA. We collected bone marrow, blood, and CSF samples from newly diagnosed patients, and cfDNA was isolated from blood and CSF samples throughout treatment. Invivoscribe Lymphotrack PCR assays combined with MinION (Oxford Nanopore Technologies) sequencing were used to identify the VDJ sequence of the immunoglobulin (B-ALL) or T-cell receptor (T-ALL) rearrangements of leukemic clones in genomic DNA. The MinION assay relies on patient-specific sequencing. We are also in the process of developing a universal assay that utilizes recurrent methylation changes in ALL to identify leukemic cfDNA in patient samples. Results: The MinION workflow was used to follow leukemic cfDNA throughout the course of treatment, and accurately identified MRD and CNS disease in patients. This workflow performed equivalent or better at detecting leukemic clones compared to MiSeq and droplet digital polymerase chain reaction (ddPCR), and is faster and less expensive than traditional Illumina sequencing. Methylation analysis of 865 ALL and 79 healthy samples yielded 55 regions and 19 specific methylation sites that were uniquely present in ALL samples. We are validating these sites by ddPCR to establish a panel of biomarkers to track ALL over time via cfDNA. Conclusions: The end goal of our study is provide a more sensitive and less invasive method for tracking MRD and CNS disease than current approaches. Results will ultimately be correlated with patient response to therapy, the presence of relapse or CNS disease, and overall outcomes determined by standard clinical diagnostic procedures.