A Novel Assay for Juvenile Myelomonocytic Leukemia Based on Aberrant Signaling Networks Measured Via Phospho-Specific Flow Cytometry Reduces Diagnosis Time from Weeks to Days.

Juvenile myelomonocytic leukemia (JMML) is an aggressive clonal malignancy characterized by over-production of myeloid lineage cells that infiltrate hematopoietic and non-hematopoietic tissues. The diagnosis and treatment of JMML can present great challenges for clinicians. Infants or young children are suspected of having this disease when presenting with hepatosplenomegaly and a peripheral blood monocytosis - criteria that are often present in more prevalent but potentially less lethal conditions such as viral infections. The diagnostic workup includes several clinical tests as well as the confirmation of abnormal numbers of granulocyte-macrophage colony forming units (CFU-GM) grown in low concentrations of granulocyte-macrophage colony stimulating factor (GM-CSF). The colony assays are laborious and can take up to 4–6 weeks for results. Using phospho-specific flow cytometry, a technique that allows simultaneous single cell measurements of cell type and signal, we have developed an assay that shows a specific cell signature in JMML patients that can reduce diagnosis time from weeks to days. The assay combines traditional surface marker phenotyping with signaling responses in low doses of GM-CSF to identify a population of CD38 intermediate cells that hyper-phosphorylate Stat 5 (pStat5). We applied this assay to primary cells obtained from 10 JMML patients and demonstrate that the cell signature is specific to JMML with respect to a wide panel of other diagnoses. For comparison purposes, we developed a statistical measure to summarize the signature and monitor its expression across varying doses of GM-CSF. The measure also ensures consistency by eliminating the need for additional manual data processing steps (e.g. gating) prior to comparison—an important step towards developing a diagnostic tool. In addition, this characteristic phospho-specific flow signature disappeared in patient samples in remission and reappeared at relapse—indicating that the signature may indeed be representative of the malignant population in JMML. Further characterization of these cells is ongoing. We conclude that phospho-specific flow cytometry is a robust methodology that can be used in primary human cells as a method to rapidly diagnosis JMML as well as to follow patients on therapy. Importantly, it is a powerful technical tool that will allow improved understanding of the complex signaling pathways that are perturbed in cancer cells.