Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

Abstract

To diagnose juvenile myelomonocytic leukemia (JMML) is sometimes challenging, because around 10% of patients lack molecular abnormalities affecting Ras-MAPK (mitogen-activated protein kinase) pathway and other diseases such as cytomegalovirus infection can mimic clinical signs of JMML. In order to validate a phospho-specific flow cytometry assay assessing phospho-signal transducer and activator of transcription factor 5 (p-STAT5) as a new diagnostic tool for JMML, we examined 22 samples from children with JMML and 47 controls. CD33+/CD34+ cells from 22 patients with JMML showed hyperphosphorylation of STAT5 induced by sub-saturating doses of granulocyte-macrophage colony-stimulating factor (GM-CSF). Using a training set of samples (11 JMML and 23 controls), we identified a threshold for p-STAT5-positive after stimulation with 0.1 ng/ml GM-CSF (17.17%) that discriminates JMML from controls. This threshold was validated in an independent series (11 JMML, 24 controls and 7 cases with diseases other than JMML) where we demonstrated that patients with JMML could be distinguished from other subjects with a sensitivity of 91% (confidence interval (CI) 59–100%) and a specificity of 87% (CI 70–96%). Positive and negative predictive values were 71% (CI 42–92%) and 96% (CI 82–100%), respectively. In conclusion, flow cytometric p-STAT5 profiling is a reliable diagnostic tool for identifying patients with JMML and can contribute to consistency of current diagnostic criteria.

How I treat juvenile myelomonocytic leukemia

“…1 CD38 low cells has been found in a proportion of patients with JMML, 22,23 and thus could also be helpful in supporting the diagnosis of children without any detectable molecular lesion ( Table 1).…”

RAS diseases in children

“…However, for a subpopulation of CD33+CD14+CD38low JMML cells, a distinct pattern of STATA5 hyperphosphorylation in response to GM-CSF could be validated. 47,48 Most recently, induced pluripotent stem cells (iPSCs) from malignant cells of 2 JMML patients with somatic heterozygous p.E76K missense mutations in PTPN11 have been generated. 49 In vitro differentiation of iPSCs produced myeloid cells with increased proliferative capacity, constitutive activation of GM-CSF, and enhanced STAT5/ERK phosphorylation, similar to what has been seen in primary JMML cells.…”

Bedside to bench in juvenile myelomonocytic leukemia: insights into leukemogenesis from a rare pediatric leukemia

“…This phospho-STAT5 signature was recently validated as a diagnostic tool in 83 specimens (22 JMML, 47 controls, 7 cases with diseases other than JMML) and demonstrated a sensitivity of 91% (95% confidence interval [CI], 59%-100%) and a specificity of 87% (95% CI, 70%-96%), with positive and negative predictive values of 71% (95% CI, 42%-92%) and 96% (95% CI, 82%-100%), respectively. 98 …”

“…113 We and others described phosphorylation of STAT5 to low-dose GM-CSF in subsets of JMML cells. 90,98,114 Whole-exome sequencing has also identified JAK3 mutations in patients with JMML.…”