Increased Sensitivity of Minimal Residual Disease Monitoring using Peripheral Blood in Pediatric Patients with Acute Lymphoblastic Leukemia

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Molecular & Cellular Analysis Technologies
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AbstractThe primary cause of death in patients with acute lymphoblastic leukemia (ALL) is sudden disease relapse (80%of patients with minimum residual disease, MRD, will relapse). For better outcome, onset of relapse must bedetected early. Therefore, frequent monitoring of MRD is needed. In pediatric ALL, monitoring MRD utilizes multi-parameter flow cytometry (MFC) or qPCR from bone marrow aspirates (BMA), which is painful and an invasiveprocedure, hence not performed frequently. MFC/BMA detects MRD in ~40% of patients; most patients areclassified as MRD negative in spite of residual cancer cells present in their blood. qPCR detection in B-ALL islabor intensive/expensive as it requires patient-specific target identification via sequencing. In some B-ALLpatients, specific DNA markers cannot be identified. Although MRD status has proven to be an effectiveprognostic indicator allowing for risk assessment and improving survival with treatment intensification, MFC andqPCR require a BMA due to limited ability to detect rare leukemia cells in peripheral blood. Bone marrow is notthe only niche where leukemia cells are found. Circulating leukemia cells (CLCs) are present in patient?speripheral blood. Therefore, if sensitive methods for MRD detection from blood could be used, frequent MRDmonitoring could improve patient outcome. Built from a successful R21 IMAT project for MRD monitoring in adultacute myeloid leukemia (AML) patients, an innovative and highly sensitive MRD platform will be delivered herein,which will consist of a fluidic cartridge that isolates and analyzes enriched CLCs from peripheral blood in anautomated fashion. The cartridge is comprised of two modules, one for the affinity selection of B- or T-type CLCsfrom blood and the other module used to ?trap? enriched cells to allow for immunophenotyping and/or FISHanalyses (FISH performed on high-risk patients). In this R33 application, the utility of this microfluidic assay willbe demonstrated in pediatric B-ALL and T-ALL patients. Appropriate antibodies (anti-CD19 and anti-CD7monoclonal antibodies for B-ALL and T-ALL, respectively) will be immobilized to a surface of the cell selectionmodule with the aid of a photocleavable linker to affinity-select cells expressing CD19 or CD7 surface antigens.Cells will be photolytically released from the capture surface and CLCs identified by the expression of aberrantmarkers, such as Terminal deoxynucleotidyl Transferase (TdT), in a unique microtrap (µTRAP) module. Thismodule allows for automated cell staining and phenotyping. The CLC microfluidic cartridge will be clinicallyvalidated by monitoring MRD status in B-ALL and T-ALL pediatric patients and CLC burden will be tracked. Thecartridge will perform CLC FISH analysis for high-risk patients to gain information on chromosomal aberrationsthat provide information to allow precision treatment. Given the strong data generated to date in AML (100% testpositivity) and the urgent diagnostic need for an improved easy-to-implement MRD assay that permits frequentsampling using peripheral blood, broad patient coverage, and early detection of relapse, the proposed cartridgewill fill an unmet clinical need in pediatric oncology as well as other leukemic diseases.