Cell signaling-based classifier predicts response to induction therapy in elderly patients with acute myeloid leukemia.

Single-cell network profiling (SCNP) data generated from multi-parametric flow cytometry analysis of bone marrow (BM) and peripheral blood (PB) samples collected from patients >55 years old with non-M3 AML were used to train and validate a diagnostic classifier (DXSCNP) for predicting response to standard induction chemotherapy (complete response [CR] or CR with incomplete hematologic recovery [CRi] versus resistant disease [RD]). SCNP-evaluable patients from four SWOG AML trials were randomized between Training (N = 74 patients with CR, CRi or RD; BM set = 43; PB set = 57) and Validation Analysis Sets (N = 71; BM set = 42, PB set = 53). Cell survival, differentiation, and apoptosis pathway signaling were used as potential inputs for DXSCNP. Five DXSCNP classifiers were developed on the SWOG Training set and tested for prediction accuracy in an independent BM verification sample set (N = 24) from ECOG AML trials to select the final classifier, which was a significant predictor of CR/CRi (area under the receiver operating characteristic curve AUROC = 0.76, p = 0.01). The selected classifier was then validated in the SWOG BM Validation Set (AUROC = 0.72, p = 0.02). Importantly, a classifier developed using only clinical and molecular inputs from the same sample set (DXCLINICAL2) lacked prediction accuracy: AUROC = 0.61 (p = 0.18) in the BM Verification Set and 0.53 (p = 0.38) in the BM Validation Set. Notably, the DXSCNP classifier was still significant in predicting response in the BM Validation Analysis Set after controlling for DXCLINICAL2 (p = 0.03), showing that DXSCNP provides information that is independent from that provided by currently used prognostic markers. Taken together, these data show that the proteomic classifier may provide prognostic information relevant to treatment planning beyond genetic mutations and traditional prognostic factors in elderly AML.

Functional pathway analysis in acute myeloid leukemia using single cell network profiling assay: effect of specimen source (bone marrow or peripheral blood) on assay readouts.

BACKGROUND: Single cell network profiling (SCNP) is used to simultaneously measure the effects of modulators on signaling networks at the single cell level. SCNP-based biomarker assays predictive of response to induction therapy and relapse risk in acute myeloid leukemia (AML) patients are being developed. Such assays have typically used bone marrow (BM) as the sample source of blasts. Because circulating peripheral blasts are detectable in ∼65% of AML patients and peripheral blood (PB) sampling is less invasive than BM sampling, this study was performed to assess the effect of sample source on AML blasts signaling as measured in SCNP assay. METHODS: SCNP using multiparametric flow cytometry was used to evaluate the activation state of intracellular signaling molecules in leukemic blasts under basal conditions and after treatment with modulators in 46 pairs of BM mononuclear cells/PB mononuclear cells. The relationship between readouts of modulated intracellular proteins ("nodes") was measured using linear regression, Bland-Altman method, and Lin's concordance correlation coefficient. RESULTS: The majority (156/161) of signaling nodes show strong correlations between paired PB and BM samples independently from the statistical method used. Notable exceptions were two PB samples with almost undetectable levels of circulating blasts compared with paired BM samples. CONCLUSIONS: Our results demonstrate that specimen source (BM or PB) does not significantly affect proteomic signaling in patients with AML and circulating blasts. The ability to use PB as a sample source will facilitate the monitoring of cellular signaling effects following administration of targeted therapies and at time points when BM aspirates are not clinically justifiable.

Dynamic single-cell network profiles in acute myelogenous leukemia are associated with patient response to standard induction therapy.

PURPOSE: Complete response to induction chemotherapy is observed in approximately 60% of patients with newly diagnosed non-M3 acute myelogenous leukemia (AML). However, no methods exist to predict with high accuracy at the individual patient level the response to standard AML induction therapy. EXPERIMENTAL DESIGN: We applied single-cell network profiling (SCNP) using flow cytometry, a tool that allows a comprehensive functional assessment of intracellular signaling pathways in heterogeneous tissues, to two training cohorts of AML samples (n = 34 and 88) to predict the likelihood of response to induction chemotherapy. RESULTS: In the first study, univariate analysis identified multiple signaling "nodes" (readouts of modulated intracellular signaling proteins) that correlated with response (i.e., AUC(ROC) > or = 0.66; P < or = 0.05) at a level greater than age. After accounting for age, similar findings were observed in the second study. For patients <60 years old, complete response was associated with the presence of intact apoptotic pathways. In patients > or =60 years old, nonresponse was associated with FLT3 ligand-mediated increase in phosphorylated Akt and phosphorylated extracellular signal-regulated kinase. Results were independent of cytogenetics, FLT3 mutational status, and diagnosis of secondary AML. CONCLUSIONS: These data emphasize the value of performing quantitative SCNP under modulated conditions as a basis for the development of tests highly predictive for response to induction chemotherapy. SCNP provides information distinct from other known prognostic factors such as age, secondary AML, cytogenetics, and molecular alterations and is potentially combinable with the latter to improve clinical decision making. Independent validation studies are warranted.

Substituent Effects on Rates and Stereoselectivities of Conrotatory Electrocyclic Reactions of Cyclobutenes. A Theoretical Study.

Substituent effects on the geometries and conrotatory electrocyclic ring openings of cyclobutenes were studied. This work extends the original investigations to many more substituents and provides a comprehensive theory of substituent effects on geometries and reaction rates. The effects of substitution at the 1 position are minimal; donor substituents raise the activation energy slightly, and powerful acceptor substituents slightly lower the activation energy. Substituents on C(3) cause small distortions of the cyclobutene geometry, in the same direction as the favored stereochemistry of reaction. Donors prefer outward rotation, while strong acceptors prefer inward rotation. The activation energy changes and cyclobutene geometrical perturbations were found to correlate with Taft sigma(R)(0) parameters. Amino, hydroxy, fluoro, chloro, methyl, cyano, formyl, and vinyl substituents were studied in the 1 position. Boryl, dimethylboryl, nitroso, formyl, nitro, carboxyl (neutral, protonated, and deprotonated), cyano, trifluoromethyl, sulfoxyl, sulfonyl, sulfinic acid, imino, N-protonated imino, ammonio, ethynyl, methyl, mercapto, chloro, fluoro, hydroxyl, amino, lithium oxy, vinyl, and acetyl were calculated as substituents in the 3 position. Comparisons with experimental results are given when available, and predictions are made in other cases.