Abstract 2693

Background:

Single Cell Network Profiling (SCNP) is used to measure simultaneously the effects of multiple modulators (including drugs) on signaling cascades at the single cell level. Using this technology, ECOG in collaboration with Nodality is developing several novel biomarker assays with the aim to find blast functional signaling profiles predictive of response to induction therapy and risk of relapse in AML patients. To date, such assays utilized patient bone marrow (BM) as the sample source of blasts. However, in about 65% of patients with AML, circulating peripheral blasts are detected and peripheral blood (PB) sampling is easier and less invasive for patients than BM sampling.

Objectives:

The objective of this study was to compare by SCNP the functional effects of a panel of compounds simultaneously on different signaling pathways (such as the phosphoinositide 3-kinase (P13K )and the Janus Kinases (Jak) signal transducers and activators of transcription (Stat) pathway) relevant both to the biology of the disease and the development of new therapeutics, in paired, diagnostic, cryopreserved PB mononuclear cells (PBMC) and BMMC samples from 44 AML patients. A paired sample was defined as a BMMC and PBMC specimen collected from the same patient on the same day.

Methods:

Modulated SCNP using a multiparametric flow cytometry platform was used to evaluate the activation state of intracellular signaling molecules in leukemic blasts under basal conditions and after treatment with specific modulators (Table 1). The SCNP phosphoflow assay was performed on 88 BMMC/PBMC pairs from ECOG trial, E3999. The relationship between readouts of modulated intracellular proteins (“nodes”) between BMMC and PBMC was assessed using linear regression, Bland-Altman method or Lin's concordance correlation coefficient.

Results:

Table 1 shows the goodness of fit (R2) values from the linear regression analysis for both the basal levels and the modulated levels of intracellular signaling proteins. Most of the signaling nodes show strong correlations (R2 >0.64) with several of the exceptions belonging to nodes with weak response to modulation (e.g. SCF -> p-Akt) or antibodies with dim fluorphores (i.e. Alexa 647). The lack of response is however, consistent between the tissue types for the weak response nodes. Using a rank based metric that is less sensitive to the absolute intensity levels seem to perform better for the antibodies with dim fluorophores. Results from other methods; Bland Altman and Lin's Concordance also show good concordance between the tissue types.

Conclusions:

The data presented here demonstrate: 1) Specimen source (BM or PB) does not affect proteomic signaling in patients with AML and circulating blasts. 2) PB myeloblasts can be used as a sample source for Nodality SCNP assays to identify functionally distinct leukemic blats cell populations with distinct sensitivities to therapy. 3) The ability to use PB as a sample source will greatly improve the utility of these assays. In particular, our results will facilitate the monitoring of cellular signaling effects following the administration of targeted therapies, e.g., kinase inhibitors, at time-points when BM aspirates are not clinically justifiable.

Table 1:

Goodness of fit (R2) values from regressing PB against BM. SCNP Nodes with R2 > 0.64 are highlighted Fold and Uu metrics measures increase (or decrease) in signaling protein levels due to modulation. Fold metrics measure the shift intensity, while Uu (rank based) metrics measure the proportion of cells that shift from baseline.

ModulatorAssay Read-outColorR2 for Metric
FoldUu
Ara-C + Daunorubicin cPARP FITC 0.72 0.68 
 p-Chk2 Alexa 647 0.73 0.81 
Etoposide cPARP FITC 0.48 0.66 
 p-Chk2 Alexa 647 0.76 0.74 
FLT-3 Ligand p-Akt Alexa 647 0.50 0.63 
 p-Erk 1/2 PE 0.67 0.64 
 p-S6 Alexa 488 0.73 0.73 
G-CSF p-Stat1 Alexa 488 0.64 0.57 
 p-Stat3 PE 0.62 0.76 
 p-Stat5 Alexa 647 0.63 0.78 
Hydrogen Peroxide p-Akt Alexa 488 0.83 0.72 
 p-PLCy2 PE 0.77 0.85 
 p-Slp76 Alexa 647 0.56 0.68 
IL-27 p-Stat1 Alexa 488 0.79 0.84 
 p-Stat3 PE 0.71 0.79 
 p-Stat5 Alexa 647 0.72 0.72 
PMA p-Creb PE 0.71 0.82 
 p-Erk 1/2 Alexa 647 0.51 0.74 
 p-S6 Alexa 488 0.74 0.77 
SCF p-Akt Alexa 647 0.48 0.77 
 p-Erk 1/2 PE 0.40 0.39 
 p-S6 Alexa 488 0.68 0.73 
ModulatorAssay Read-outColorR2 for Metric
FoldUu
Ara-C + Daunorubicin cPARP FITC 0.72 0.68 
 p-Chk2 Alexa 647 0.73 0.81 
Etoposide cPARP FITC 0.48 0.66 
 p-Chk2 Alexa 647 0.76 0.74 
FLT-3 Ligand p-Akt Alexa 647 0.50 0.63 
 p-Erk 1/2 PE 0.67 0.64 
 p-S6 Alexa 488 0.73 0.73 
G-CSF p-Stat1 Alexa 488 0.64 0.57 
 p-Stat3 PE 0.62 0.76 
 p-Stat5 Alexa 647 0.63 0.78 
Hydrogen Peroxide p-Akt Alexa 488 0.83 0.72 
 p-PLCy2 PE 0.77 0.85 
 p-Slp76 Alexa 647 0.56 0.68 
IL-27 p-Stat1 Alexa 488 0.79 0.84 
 p-Stat3 PE 0.71 0.79 
 p-Stat5 Alexa 647 0.72 0.72 
PMA p-Creb PE 0.71 0.82 
 p-Erk 1/2 Alexa 647 0.51 0.74 
 p-S6 Alexa 488 0.74 0.77 
SCF p-Akt Alexa 647 0.48 0.77 
 p-Erk 1/2 PE 0.40 0.39 
 p-S6 Alexa 488 0.68 0.73 
View Large
Disclosures:

Cesano:Nodality Inc.: Employment, Equity Ownership. Rosen:Nodality Inc.: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality Inc.: Employment, Equity Ownership.

Topics:
antibodies, biological markers, blast cells, cryopreservation, cyclic amp-responsive dna-binding protein, cytarabine, daunorubicin, etoposide, flow cytometry, flt3 ligand

Author notes

*

Asterisk with author names denotes non-ASH members.

© 2010 by The American Society of Hematology
2010
Sign in via your Institution