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Summary Progress in understanding the molecular pathogenesis of human myeloproliferative disorders (MPDs) has led to guidelines incorporating genetic assays with histopathology during diagnosis. Advances in flow cytometry have made it possible to simultaneously measure cell type and signaling abnormalities arising as a consequence of genetic pathologies. Using flow cytometry, we observed a specific evoked STAT5 signaling signature in a subset of samples from patients suspected of having juvenile myelomonocytic leukemia (JMML), an aggressive MPD with a challenging clinical presentation during active disease. This signature was a specific feature involving JAK-STAT signaling, suggesting a critical role of this pathway in the biological mechanism of this disorder and indicating potential targets for future therapies. Significance Recent advances have enabled simultaneous measurement of cell type and cell signals in primary populations using flow cytometry. This technique enables the question, "Can we track oncogenic cell populations from diagnosis through disease evolution via signaling?" Doing so in an era of using specific inhibitors against components of key signal transduction pathways will be necessary to assess treatment effects in human patients and adapt as cancer cells alter their signaling in response to these treatments. This work uses such an approach to follow patients over time and shows that disease status in juvenile myelomonocytic leukemia (JMML) -- at diagnosis, remission, relapse, and transformation -- is indicated by a subset of cells with an abnormal signaling profile.

Juvenile myelomonocytic leukemia is an aggressive and frequently lethal myeloproliferative disorder of childhood. Somatic mutations in NRAS, KRAS, or PTPN11 occur in 60% of cases. Monitoring disease status is difficult because of the lack of characteristic leukemic blasts at diagnosis. We designed a fluorescently based, allele-specific polymerase chain reaction assay called TaqMAMA to detect the most common RAS or PTPN11 mutations. We analyzed peripheral blood and/or bone marrow of 25 patients for levels of mutant alleles over time. Analysis of pre-hematopoietic stem-cell transplantation, samples revealed a broad distribution of the quantity of the mutant alleles. After hematopoietic stem-cell transplantation, the level of the mutant allele rose rapidly in patients who relapsed and correlated well with falling donor chimerism. Simultaneously ana- IntroductionJuvenile myelomonocytic leukemia (JMML) is an aggressive clonal malignancy of young children characterized by overproduction of myeloid lineage cells that infiltrate hematopoietic and nonhematopoietic tissues. 1,2 More than 70% of patients have a mutation in the NF1, RAS, or PTPN11 genes, which encode proteins involved in Ras signaling. 3 Animal models demonstrate that Nf1 Ϫ/Ϫ , Kras G12D , and Ptpn11 D61G mutant mice all develop fatal myeloproliferative disorders that model JMML in vivo and in vitro. [4][5][6][7][8] These observations strongly support the role of hyperactive Ras in initiating JMML.The diagnosis and treatment of patients present great challenges to physicians. Currently, the diagnosis is made by meeting a constellation of clinical and laboratory criteria, 1,9 including the demonstration of fewer than 20% blasts in the bone marrow (BM). Historically, patients with JMML treated with conventional chemotherapy have an event-free survival of less than 15%. 9,10 Allogeneic hematopoietic stem cell transplantation (HSCT) will cure up to 50% of patients. 11 Novel treatment approaches are urgently needed for this disease, given the dismal survival with traditional chemotherapy and the severity of late effects that HSCT confers on these young children.However, defining response to therapy is another major challenge for clinicians. 12 Current response criteria are broadly defined as a decreasing white blood cell count and lessening organomegaly. Detecting minimal residual disease (MRD) in JMML relies on tracking donor chimerism after HSCT. One quantitative donor chimerism tracking method used and reported in JMML involves polymerase chain reaction (PCR) amplification of short tandem repeats with comparison of informative loci. 13 Thus, delivering chemotherapy or novel agents before HSCT is severely limited by our inability to follow disease burden in these patients.Allele-specific oligonucleotide-PCR has been developed to detect single nucleotide substitution levels in various cancers 14 and involves designing primers that will preferentially amplify the mutant allele over the wild-type (WT) allele by exploiting differences in the amplification efficie...

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