Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, with a relapse rate of 15%. The presence of lymphoblasts in the central nervous system (CNS) is a negative prognostic indicator and a frequent site of disease relapse. CNS disease is defined as more than five white blood cells in the cerebrospinal fluid (CSF) with positive lymphoblast cytomorphology. Often leukemic blasts are detected by flow cytometry of the CSF prior to reaching the five white blood cell threshold, requiring repeat lumbar punctures at 2-4 week intervals until the arbitrarily defined clinical criteria for diagnosis are met. Additionally, more than 50% of autopsied brains from ALL patients show evidence of CNS disease, despite non-detectable CNS involvement via cytology of CSF. These data suggest that patients may be harboring CNS disease before it expands enough to be clinically diagnosed. We have developed a new assay that may allow for earlier detection of CNS disease and relapse by quantifying cell-free, circulating tumor DNA (ctDNA) in the CSF. ctDNA is a proven biomarker of relapse and metastasis in solid tumors in pre-clinical testing; however, its utility at predicting CNS disease in ALL has not been examined.
We examined two possible methods for using ctDNA as a biomarker: leukemia cell clonality and DNA methylation profiling. We collected bone marrow aspirate, blood, and CSF samples from 11 newly diagnosed patients prior to the start of chemotherapy treatment to use as a training data set. ctDNA was isolated from blood and CSF samples at diagnosis and throughout treatment. Genomic DNA was isolated from bone marrow and peripheral blood mononuclear cell (PBMC) samples at diagnosis. For leukemia cell clonality assays, 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 in the clones comprising each leukemia. Throughout the course of treatment, ctDNA samples were run on the MinION sequencer, examining the abundance of major clones present and ctDNA quantification was done on patient plasma and CSF samples over time.
While this assay relies on patient specific VDJ sequencing, we are also in the process of developing a more universal assay that utilizes recurrent methylation changes in T-ALL or B-ALL, compared to normal PBMCs. We performed methylation sequencing on 3 control PBMC samples and 7 ALL patient samples to identify differentially methylated regions (DMRs) present specifically in ALL samples. This sequencing identified 9,222 DMRs present in the ALL samples. These sites were then compared with publicly available datasets, yielding 55 overlapping regions and 19 specific overlapping methylation sites commonly present in ALL samples and not in PBMCs. We are now in the process of validating these differentially methylated sites by droplet digital polymerase chain reaction (ddPCR) to come up with a panel of biomarkers to track ALL disease over time. The end goal of our study is to develop an assay to rapidly detect relapse and CNS disease in ALL that is more sensitive and less invasive than the current clinical assays. Earlier detection of relapse and CNS disease will provide patients with more treatment options, which may ultimately improve patient outcome. Results will ultimately be correlated with patient response to therapy, the presence of CNS disease, and overall outcomes as determined by standard clinical diagnostic procedures.
Disclosures
No relevant conflicts of interest to declare.
Integrin-Mediated Adhesion and Chemoresistance of Acute Lymphoblastic Leukemia Cells Residing in the Bone Marrow or the Central Nervous System
