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

D Hasegawa,F Locatelli,A Bertaina,D Longoni,C Bugarin,C Micalizzi,G Te Kronnie,G Gaipa,M Giordan,A Biondi,G Basso,U Ramenghi,S Bresolin

doi:10.1038/bcj.2013.56

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.

Highlights

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of childhood characterized by uncontrolled proliferation of monocytic and granulocytic cells.[1,2]Hematopoietic precursor cells of JMML often show in vitro hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF).[3]
We analyzed separately JMML fresh samples (n 1⁄4 13) and JMML thawed samples (n 1⁄4 9), and we did not observe any significant difference in p-STAT5 response: mean 29.30%
To assess the best threshold for distinguishing JMML from control vs 29.04%, respectively specimens based on GM-CSF-induced p-STAT5, we analyzed a (P 1⁄4 0.968)

Summary

Introduction

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of childhood characterized by uncontrolled proliferation of monocytic and granulocytic cells.[1,2]. Hematopoietic precursor cells of JMML often show in vitro hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF).[3] GM-CSF binds to the alpha and beta subunits of its cell surface receptor, triggering two distinct signaling pathways: the Ras-MAPK (mitogen-activated protein kinase) pathway and the JAK-STAT (Janus-activated kinase–signal transducer and activator of transcription factor) pathway. In patients with JMML, Ras-MAPK signaling is constitutively activated by a spectrum of usually mutually exclusive mutations affecting genes, such as NRAS, KRAS,[4,5] NF1,6 PTPN117 and CBL.[8,9]. GM-CSF hypersensitivity has been reported to be induced by human herpes virus-612 and cytomegalovirus (CMV)[13] infections

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Conclusion