STAT5 Phosphorylation Status by Flow Cytometry Is a Rapid and Reliable Tool for Diagnosis and Follow-up of Juvenile Myelomonocytic Leukemia
Authors: Gaipa Giuseppe , Bugarin Cristina , Giordan Marco , Bresolin Silvia , Hasegawa Daisuke , Zecca Marco , Longoni Daniela , Menna Simone , Basso Giuseppe , Kronnie Geertruy te , Biondi Andrea 
Published: October 2019

Abstract
Abstract 2751

Introduction.
Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder of infancy and early childhood characterized by overproduction of myeloid cells (Aricò M et al., Blood, 1997) and selective hypersensitivity of the hematopoietic precursor cells to GM-CSF (Emanuel PD et al., Blood, 1991). Current diagnostic criteria are based on matched clinical presenting features and laboratory findings according to established international criteria (reviewed in Emanuel PD, Leukemia, 2008). Sometimes though, in the absence of some of these specific conditions, arriving to a conclusive diagnosis may be challenging. When a novel rapid phospho-specific flow cytometric assay (phospho-flow) is used, we and others have reported in vitro specific phosphorylated STAT5 (p-STAT5) signaling signature in JMML. (Gaipa G et al., Leukemia, 2008; Kotecha N et al., Cancer Cell, 2008).

Aim and Methods.
Here, in order to validate the p-STAT5 phospho-flow assay as a new integrated tool in the diagnostic work-up of JMML we analyzed mononuclear cells from 14 JMML patients at diagnosis, 39 control subjects and 6 patients diagnosed with suspected JMML which were subsequently not confirmed. Samples were stimulated with GM-CSF at 0, 0.01, 0.1, 1.0, and 10 ng/mL. p-STAT5-responsive cells were identified within the CD34+/CD33+ subset, and quantified by scaling the maximum % of p-STAT5+ cells at 100 and the % of unstimulated p-STAT5+ cells to 0 (Kotecha N et al.). JMMLs and controls were compared at each dose using Wilcoxon's test in order to identify the best dose with lowest significative p-value after correction for multiplicity with a Bonferroni's method. Discriminating p-STAT5 % value was identified as the mean between the lowest of the JMML p-STAT5 values and the highest of the control subjects.

Results.
We found that a threshold of 18.9 % of p-STAT5+ cells, after stimulation with 0.1 ng/mL GM-CSF (p <0.01), was the best condition to discriminate JMMLs (n 8) from control subjects (n 27). This algorithm was then applied on an independent cohort of JMMLs (n 6), control subject (n 12) and patients with suspected diagnosis of JMML subsequently not confirmed (n 6) and reached concordant results with a sensitivity of 0.83 and a specificity of 1.0 was reached. Positive and negative predictive values were 1.0 and 0.94, respectively. We also applied p-STAT5 phospho-flow assay in bone marrow aspirates from 3 JMML patients during one-year post- transplantation follow-up. Two of these 3 patients showed p-STAT5 value below the diagnostic threshold at any of time point (mean 9.71% [range 4.3%-10.6%], and 4.26% [range 0.26%-13.6%]), chimerism analysis and morphology examinations confirmed the remission status. The third patients relapsed 3 months after transplantation with a p-STAT5 value of 21.5% which increased to 41.1% one month later together with clinical disease progression.

Conclusions.
Patients with JMML show p-STAT5 hyper-responsiveness, and this condition can be rapidly assayed by phospho-flow technology in routine diagnostic work-up with high sensitivity and specificity, under appropriated technical standardization. Although we tested a very limited series of patients, our results also show that p-STAT5 response may represent a surrogate marker of disease activity in post-transplantation follow-up of JMML patients with potential clinical impact.

Disclosures:
No relevant conflicts of interest to declare.