Abstract Acute lymphoblastic leukemia (ALL) is the most common malignant disease in childhood and accounts for approximately 30% of all cancers diagnosed before the age of 18 years (1). The primary cause of death for ALL patients is disease relapse. Therefore, monitoring for minimal residual disease (MRD) is considered the most powerful predictor of outcome in acute leukemias, including B-type acute lymphoblastic leukemia (B-ALL). If clinicians could identify a patient’s MRD before the tumor cells rapidly expand to florid relapse, preemptive therapies could be undertaken with better patient outcome. For pediatric B-ALL, there are existing tests for monitoring relapse from MRD including PCR or multi-parameter flow cytometry, but require a bone marrow aspirate, which can be painful and limits the frequency of testing (2,3). If MRD could be detected in B-ALL patients from peripheral blood and not bone marrow, the corresponding assay could assist in guiding therapy to enable precision medicine resulting in better patient outcome. In this application, an innovative test that consists of a microfluidic assay and the associated hardware will be developed. The test can provide high clinical sensitivity for MRD testing and permits frequent minimally invasive sampling using peripheral blood (1 mL) as opposed to an invasive, especially for pediatric patients, bone marrow biopsy. The assay uses a microfluidic device to analyze peripheral blood and search for circulating leukemic cells (CLCs). Using this microfluidic assay in a longitudinal study of acute myeloid leukemia (AML) patients following stem cell transplantation, MRD via monitoring of CLCs was detected ~2 months earlier compared to both multi-parameter flow cytometry (MFC) and PCR, which used bone marrow aspirates; the microfluidic assay was 2-orders of magnitude more sensitive than PCR and MFC. Owing to the ability of the microfluidic assay to detect CLCs in blood, more frequent testing of a patients’ disease status was possible when compared to bone marrow biopsy testing. For B-ALL, anti-CD19 antibodies immobilized within a microfluidic device can affinity- select cells expressing CD19 surface antigen commonly expressed by B-ALL lymphoblasts (i.e., CLC) and normal B-cells. CLCs are identified by expression of aberrant markers, such as Terminal deoxynucleotidyl Transferase (TdT) and the number of CLCs tracked to determine the onset of relapse or the risk of relapse. In this SBIR Phase I/II fast track proposal, the CLC microfluidic test will be expanded and developed for commercialization to monitor MRD and potential relapse in B-ALL pediatric patients to provide coverage of 100%. Given the strong data generated to-date and the urgent diagnostic need for an improved easy-to-implement MRD assay for frequent monitoring, the proposed test fills an unmet clinical need in the area of pediatric oncology. As a note, the test can be reprogrammed to search for other pediatric oncological diseases such as T-cell ALL (requires only a change in the cell selection antibody).

Narrative Acute lymphoblastic leukemia (ALL) is the most common malignant disease in childhood and accounts for ~30% of all cancers diagnosed before the age of 18 years. The primary cause of death for ALL occurs due to disease relapse. Monitoring of minimal residual disease (MRD) is considered the most powerful predictor of outcome in acute leukemias, including B type acute lymphoblastic leukemia (B-ALL). If clinicians could pinpoint when a patient’s minimum residual disease (MRD) begins rapid expansion to relapse, preemptive therapies can be taken to dramatically improve patient outcome. In this Phase I/II application, a fully automated instrument that can process peripheral blood directly, search for circulating leukemic cells (CLCs) in pediatric ALL patients, and detect relapse earlier than current techniques will be developed. This application represents a significant improvement to standard-of-care that requires painful bone marrow aspirates in children to monitor MRD.