Euroflow-Based Immunophenotypic Characterization of CD34+ Cell Compartment in Juvenile Myelomonocytic Leukemia (JMML): A New Tool for Differential Diagnosis
Authors: Gaipa Giuseppe , Bugarin Cristina , Matarraz Sergio , Buracchi Chiara , Sedek Lukasz , van der Velden Vincent H.J. , Szczepanski Tomasz , van der Sluijs Alita , Sobral da Costa Elaine , Novakova Michaela , Mejstrikova Ester , Aanei Carmen , van Dongen Jacques JM , Biondi Andrea , Orfao Alberto 
Published: October 2019

Abstract
Background
Juvenile myelomonocytic leukemia (JMML) is a lethal myeloproliferative disease (MPD) of young childhood characterized by an overproduction of myelomonocytic cells and an increased in vitro sensitivity of hematopoietic progenitors to granulocyte-macrophage colony-stimulating factor [GM-CSF] (Emanuel PD et al, Blood 1991). Diagnostic criteria for JMML are currently well-established and based on clinical features and laboratory findings. However, in some patients diagnosis of JMML vs other overlapping disease entities, still remains a challenge, immunophenotyping being not part of the diagnostic work-up of JMML. Here, we aimed at detailed characterization of the CD34+ cell compartment in JMML bone marrow (BM) using the standardized EuroFlow myeloid panel in combination with innovative EuroFlow software maturation tools. Our major goal was to determine the potential utility of immunophenotyping of CD34 cells in the diagnostic work-up of JMML.
Methods
Overall, we analyzed BM cells from 10 JMML patients at diagnosis (age range: 0-7 years), 17 control subjects (age range: 0-15 years) and 5 patients (age range: 0-5 years) with a suspected diagnosis of JMML that was subsequently not confirmed following standardized EuroFlow antibody combinations:
1) cyCD3/ CD45/ cyMPO/ cyCD79a/ CD34/ CD19 / CD7/smCD3 (for early lineage assignement);
2) HLADR/CD45/CD16/CD13/CD34/CD117/CD11b/CD10 (neutrophilic maturation);
3) HLADR/CD45/CD35/CD64/CD34/CD117/CD300e (IREM2)/CD14 (monocytic maturation);
4) HLADR/CD45/CD36/CD105/CD34/CD117/CD33/CD71 (erythroid vs plasmacytoid dendritic cell maturation).
Samples were processed and analyzed according to the Euroflow standard operating protocols (van Dongen JJM et al, Leukemia 2012, Kalina T et al, Leukemia 2012). Data analysis was specifically focused on the immunophenotypic profile of CD34+ gated cells.
Results
Within the CD34+ BM cell compartment the proportion (mean % ± 1SD) of granulocytic and monocytic precursors were not significantly different in JMML as compared to controls: 33% ± 15% vs 25% ± 12% (p = 0.16) and 14% ± 6.3% vs 12% ± 7.1% (p = 0.68) respectively. Otherwise we observed a slightly decreased in erythroid CD34+ progenitors in JMML vs controls (1.0% ± 1.2% vs 2.8% ± 1.7%, p<0.05). Moreover, a significantly different distribution of lymphoid precursors was observed: B-cell precursors were strongly reduced in JMML vs controls (3.0% ± 3.5% vs 53% ± 16%, p<0.0001), while CD7+ lymphoid precursors resulted significantly enhanced (28% ± 18% vs 2.3% ± 1.2%, p<0.0001). We then investigated the presence of unusual immunophenotypes in JMML CD34+ BM cells, including CD7+/MPO+, CD79a+/CD7+, and CD79a+/MPO+ cells. Interestingly, we consistently found CD7+/MPO+ and/or CD79a+/CD7+ cells in 7/7 JMML patients analyzed (mean 7.9% ± 6.4%), while in control subjects these cells were virtually absent (0.02% ± 0.00%, p<0.0001). In contrast, no CD79a+/MPO+ cells were detected among CD34+ precursors. Those 5 patients suspected of having JMML showed a CD34+ BM cell immunophenotypic profile that was not significantly different from that of normal subjects. These patients were finally diagnosed as not having JMML (two had CMV infection, one a Leukocyte Adhesion Deficiency II, one a Noonan Syndrome, the final diagnosis is the other patient being still pending). Of note, JMML peripheral blood (PB) CD34+ cells from 6 JMML patients (3 paired BM-PB samples and 3 additional PB samples) fully confirmed the aberrant immunophenotypic signature seen in BM-derived samples.
Conclusions
CD34+ precursor cells from JMML patients display a unique immunophenotypic profile characterized by an inverted ratio of CD19+ B/CD7+ lymphoid precursors, associated with unusual marker coexpressions, which might contribute to fast and more precise diagnostic work-up of JMML. Further studies in larger patient series are required to confirm our observations.

Disclosures
Biondi: Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; BMS: Membership on an entity's Board of Directors or advisory committees; Cellgene: Other: Advisory Board.