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Aberrant GM-CSF signal transduction pathway in juvenile myelomonocytic leukemia assayed by flow cytometric intracellular STAT5 phosphorylation measurement

Leukemia volume 23, pages 791–793 (2009)Cite this article

Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder of infancy and early childhood with an overproduction of myeloid cells that massively infiltrate hematopoietic and non-hematopoietic tissues.1, 2, 3 JMML is characterized by ‘spontaneous’ proliferation of marrow and peripheral blood mononuclear cells in vitro with exuberant growth of granulo-macrophage colonies in the absence of exogenous growth factors, because of a selective hypersensitivity of the hematopoietic precursor cells to granulocyte macrophage colony-stimulating factor (GM-CSF).4 Deregulation of GM-CSF signal transduction is driven by the RAS pathway because of oncogenic mutations in N-RAS, K-RAS, or PTPN11 genes or inactivation of the NF1 tumor suppressor gene.5, 6, 7 GM-CSF-RAS pathway in turn affects signaling cascades downstream of GM-CSF receptor.8 Multiparametric flow cytometric analysis has demonstrated new potentialities for assaying intracellular levels of phosphorylated proteins.9 Here, we report the flow cytometric analysis of intracellular phosphorylated signal transducer and activator of transcription (pSTAT5) and evaluate the possible impact of our findings in the diagnostic work-up of JMML.

The study was approved by the Institutional Review Board and written informed consent was obtained from the parents of the patient's. Bone marrow samples were taken from three consecutive patients with a new diagnosis of JMML (biological and clinical characteristics at presentation are reported in (Table 1) and from 11 control patients (healthy donors for hematopoietic stem cell transplantation (HSCT) or patients with non-hematological malignancies). Bone marrow mononucleated cells were stimulated with escalating doses (from 0.01 to 10 ng/ml) of GM-CSF (PeproTech Inc., Offenbach, Germany) for 15 min at 37 °C to allow signal transduction. Cells were then fixed with paraformaldehyde (1.5%) and permeabilized with ice cold 90% methanol. The cells were then incubated with anti-phospho-STAT5 Alexa 488 (BD Biosciences, San Jose, CA, USA), surface anti-CD33 PE and anti-CD34 APC (BD Biosciences) antibodies and analyzed on a FACSaria flow cytometer (BD Biosciences) equipped with 488, 633 and 405 nm lasers. The pSTAT5 response to stimulation at each GM-CSF dose was calculated as either proportion (%) of pSTAT5-expressing cells or as log2 of median fluorescence intensity stimulated/median fluorescence intensity unstimulated.9 The non-parametric Mann–Whitney U-test was used to determine the significance of differences among JMML and control patients. P-values less than 0.05 were considered statistically significant.

Table 1 Clinical and laboratory features of patients studied
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Acknowledgements

This study was initiated and promoted by the Fondazione Tettamanti and Associazione Italiana per la Ricerca sul Cancro, MIUR PRIN 2005.

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Authors and Affiliations

  1. Centro Ricerca M. Tettamanti, Clinica Pediatrica Università Milano-Bicocca, Ospedale San Gerardo, Monza (MI), Italy

    G Gaipa, C Bugarin, D Longoni, S Cesana, C Molteni & A Biondi

  2. Dipartimento di Medicina Clinica e Prevenzione, Facolta' di Medicina e Chirurgia, Università di Milano-Bicocca, Monza (MI), Italy

    A Faini

  3. Department of Pediatric Hematology-Oncology, University of Torino, Torino, Italy

    F Timeus

  4. Oncoematologia Pediatrica, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy

    M Zecca

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  1. G Gaipa
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Correspondence to G Gaipa.

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Gaipa, G., Bugarin, C., Longoni, D. et al. Aberrant GM-CSF signal transduction pathway in juvenile myelomonocytic leukemia assayed by flow cytometric intracellular STAT5 phosphorylation measurement. Leukemia 23, 791–793 (2009). https://doi.org/10.1038/leu.2008.265

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