Abstract 3523: Quantitative measurements of EGFR pathway signaling and modulation in pleural effusion samples from non-small cell lung cancer (NSCLC) patients using single cell network profiling (SCNP).
Authors: Westfall Matt , Hawtin Rachael , Covey Todd , Gulrajani Michael , Atallah Michelle , Cholankeril Michelle , Vora Reena , Cesano Alessandra , Fields Scott Z. , Raftopoulos Haralambos 
Published: August 2014

Abstract
Background: SCNP is a multiparametric flow cytometry-based assay that quantitatively and simultaneously measures, at the single cell level, both extracellular surface markers and activation levels of intracellular signaling proteins in response to modulation. SCNP has been successfully applied to hematologic malignancies in both prognostic and predictive analyses. Previous work using bladder washes from bladder cancer patients (pts) established the feasibility of applying SCNP to the functional characterization of epithelial cell signaling and response to modulation and targeted kinase inhibition in non-conventional samples.
Objectives: Assess the feasibility of applying SCNP to detect and functionally characterize epithelial cells, including sensitivity to targeted kinase inhibition, in pleural effusion (PE) samples from NSCLC pts.
Methods: PE from confirmed NSCLC pts (n=4) were collected at the NSUH and shipped overnight to Nodality for processing on day of receipt. Antibodies to CD45, CytoKeratin (CK), EpCAM, and cPARP were used to differentiate non-apoptotic epithelial cells from leukocytes. DNA aneuploidy was evaluated using the lymphoid population in the patient samples as a diploid G0/G1 reference. Signaling in PI3K and MAPK pathways was quantified through measurement of p-AKT and p-ERK levels at baseline and after in vitro exposure to EGF +/- PI3K inhibitor GDC-0941. The epithelial bladder carcinoma cell line HT-1376 was used as a positive control for epithelial phenotypic staining and EGF-induced signaling.
Results: In 3 of 4 PE samples, epithelial cells (DAPI+, CD45-, CK+, and EpCAM+) were identified (range 0.35% to 15.87% of all nucleated cells). Epithelial cells had a DNA index &gt;1.3, indicating aneuploidy and therefore tumor origin. The majority of epithelial cells were cPARP negative (&gt;80%) and suitable for SCNP analysis. In all 3 samples with epithelial cells: 1) EGF modulation induced increased p-AKT and p-ERK; 2) Pre-treatment with GDC-0941 inhibited EGF induced p-AKT but not p-ERK; 3) Constitutive activation of the PI3K pathway in the tumor epithelial cells was suggested by reduction in p-AKT levels compared to basal (no EGF exposure) following GDC-0941 treatment.
Conclusion: This study demonstrates the feasibility of applying SCNP to the functional characterization of PE samples from NSCLC pts. Ongoing analyses will expand upon these data, including both additional NSCLC PE samples and the evaluation of compounds targeting alternative signaling pathways relevant to NSCLC (eg: MAPK, EGFR). The feasibility of extending such SCNP analyses to other solid tumor indications, using alternative tissue sources and circulating tumor cells, is under further investigation for both prognostic and response predictive utility.
[S.Z. Fields and H. Raftopoulos are senior co-authors.]
Citation Format: Matt Westfall, Rachael Hawtin, Todd Covey, Michael Gulrajani, Michelle Atallah, Michelle Cholankeril, Reena Vora, Alessandra Cesano, Scott Z. Fields, Haralambos Raftopoulos. Quantitative measurements of EGFR pathway signaling and modulation in pleural effusion samples from non-small cell lung cancer (NSCLC) patients using single cell network profiling (SCNP). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3523. doi:10.1158/1538-7445.AM2013-3523