Phosphoflow-Based Evaluation of Mek Inhibitors as Small-Molecule Therapeutics for B-Cell Precursor Acute Lymphoblastic Leukemia.

Upstream mutations that lead to constitutive activation of Erk in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are relatively common. In the era of personalized medicine, flow cytometry could be used as a rapid method for selection of optimal therapies, which may include drugs that target the Erk pathway. Here, we evaluated the utility of phospho-flow, compared to Western blotting, to monitor Erk pathway activation and its inhibition by targeted Mek kinase inhibitors in human BCP ALL. Because the Erk pathway is not only activated endogenously, by mutations, but also by normal extracellular stimulation through stromal contact and serum growth factors, we compared Erk activation ex vivo in ALL cells in the presence and absence of stroma and serum. Phospho-flow was able to readily detect changes in the pool of pErk1/2 that had been generated by normal microenvironmental stimuli in patient-derived BCP-ALL cells passaged in NSG mice, in viably frozen primary patient samples, and in fresh patient samples. Treatment with the Mek1/2 inhibitor selumetinib resulted in a rapid, complete and persistent reduction of microenvironment-generated pErk1/2. Imaging flow cytometry confirmed reduction of nuclear pErk1/2 upon selumetinib treatment. An ALL relapsing with an activating KRasG12V mutation contained higher endogenous as well as serum/stromal-stimulated levels of pErk1/2 than the matched diagnosis sample which lacked the mutation, but selumetinib treatment reduced pErk1/2 to the same level in both samples. Selumetinib and trametinib as Mek inhibitors were mainly cytostatic, but combined treatment with the PI3K∂ inhibitor CAL101 increased cytotoxicity. Thus phospho-flow cytometry could be used as a platform for rapid, individualized in vitro drug sensitivity assessment for leukemia patients at the time of diagnosis.

Circulating hematopoietic and endothelial progenitor cells in newborn infants: effects of gestational age, postnatal age and clinical stress in the first 3 weeks of life.

INTRODUCTION: Circulating endothelial progenitor cells (EPC) are bone marrow derived progenitors that can be mobilized by erythropoietin or in response to tissue injury, and participate in vascular repair. EPC are understudied in human neonates. Whether EPC frequency in newborn infants may be influenced by gestational age or postnatal stress is unknown. METHODS: Blood samples were collected on day 1 of life and weekly for 3 weeks from hospitalized neonates for plasma erythropoietin and flow cytometry analysis for CD34+, CD34+CD45-, CD34+VEGFR2+ and CD34+CD45-VEGFR2+ cells (EPC). Associations between CD34+ cell subsets and clinical parameters were studied. RESULTS: Forty five patients were enrolled. An inverse correlation with gestational age was observed for CD34+ and CD34+ VEGFR2+ cell frequencies in whole blood (WB) on day 1 (p<0.05). In preterm infants, CD34+ cell frequency decreased with increased postnatal age (p=0.0001) and CD34+VEGFR2+ cell frequency was higher at week 3 than on day 1 in WB (p=0.0002). On day one, CD34+ and CD34+CD45- cell frequencies in the mononuclear cell fraction (MNC) were higher in preterm than term infants (p=0.035 and p=0.049, respectively) but CD34+CD45-VEGFR2+ cell frequency (median 2.2/million MNC versus 3.8/million MNC) and erythropoietin levels were not significantly different. Transient increases in EPC were observed in five infants with infection. Four preterm infants who developed bronchopulmonary dysplasia had undetectable or low EPC through the first 3 weeks of life. CONCLUSIONS: Gestational age and postnatal age influenced circulating CD34+ and CD34+VEGFR2+ but not CD34+CD45-VEGFR2+ (EPC) cell frequencies. Circulating EPC in neonates may be influenced by clinical stress.

Human intrathymic lineage commitment is marked by differential CD7 expression: identification of CD7- lympho-myeloid thymic progenitors.

The identity and lineage potential of the cells that initiate thymopoiesis remain controversial. The goal of these studies was to determine, at a clonal level, the immunophenotype and differentiation pathways of the earliest progenitors in human thymus. Although the majority of human CD34(+)lin(-) thymocytes express high levels of CD7, closer analysis reveals that a continuum of CD7 expression exists, and 1% to 2% of progenitors are CD7(-). CD34(+)lin(-) thymocytes were fractionated by CD7 expression and tested for lineage potential in B-lymphoid, T-lymphoid, and myeloid-erythroid conditions. Progressive restriction in lineage potential correlated with CD7 expression, that is, the CD7(hi) fraction produced T and NK cells but lacked B and myelo-erythroid potential, the CD7(int) (CD10(+)) fraction produced B, T, and NK cells, but lacked myelo-erythroid potential. The CD7(-) fraction produced all lymphoid and myelo-erythroid lineages and expressed HSC-associated genes. However, CD34(+)lin(-)CD7(-) thymocytes also expressed early T lymphoid genes Tdt, pTalpha, and IL-7Ralpha and lacked engraftment capacity, suggesting the signals that direct lymphoid commitment and corresponding loss of HSC function are rapidly initiated on arrival of HSC in the human thymus. Thus, differential levels of CD7 identify the progressive stages of lineage commitment in human thymus, initiated from a primitive CD7(-) lympho-myeloid thymic progenitor.