Abstract CN02-02: CTCs as pharmacodynamic and /or predictive biomarkers
Authors: Dive Caroline , Hou Jian Mei , Krebs Matthew , Ranson Malcolm , Hughes Andrew , Ward Tim , Blackhall Fiona 
Published: May 2019

Abstract
CTC analysis has potential utility in the development of novel therapeutics and particularly so for the management of those patients with cancer types for which obtaining tumor biopsies poses significant challenge, especially pre and serially post therapy. We have enumerated and begun to characterize CTCs from patients with lung cancer (both small cell lung cancer (SCLC) and non small cell lung cancer (NSCLC)) using two techniques; a) the Veridex CellSearch Platform wherein CTCs are identified based on EpCam and cytokeratin expression together with the absence of the white blood cell surface marker CD45, and b) the Metagenex ISET filter-based system that isolates CTCs on the basis of their size. SCLC accounts for ∼15% of all lung cancers, is highly aggressive with a rapid doubling time, high growth fraction and propensity to metastasise early. Primary SCLC biopsies are often of small volume, highly necrotic and thus present difficulties for biomarker analysis. As SCLC has neuro-endocrine characteristics, we examined initially, the ability of the CellSearch approach (with the epithelial cell marker EpCam as its primary discriminator) to detect SCLC CTCs. In chemo-naive SCLC, CTCs were detected in 86% patients (median 28, range 0–44896, n=50). Tumor stage correlated with CTC number (#) and was highest in patients with liver metastases. The median survival for patients with baseline CTC# &gt;300/7.5ml blood was 134 days compared to 443 days when CTC# &lt;2. We next asked whether the CTCs detected expressed the neuro-endocrine surface marker CD56 using the 4th immunofluorescence channel on the CellSearch platform. Expression of CD56 was heterogeneous but consistent with expression in primary tumors. CTC# was pharmacodynamic, decreasing after one cycle of etoposide/platinum chemotherapy mirroring radiological response. CTC# and % apoptotic CTCs (analyzed by nuclear morphology) are now being applied as pharmacodynamic biomarkers in early clinical trials of novel agents and in particular of pro-apoptotic agents. To test the hypothesis that CTCs could provide added value to novel drug development, we also assessed the feasibility of drug-target characterization in SCLC CTCs. Bcl-2, an antiapoptotic drug target, is amplified in ∼40% SCLC patients and was detectable in SCLC CTCs using fluorescence in situ-hybridization; potentially, Bcl-2 amplification will predict sensitivity to Bcl-2 targeted drugs. In a cohort of 93 chemo-naive stage III/IV NSCLC patients, 2 or more CTCs/7.5ml blood were detected using CellSearch in 6% patients (2/36) with stage IIIA/IIIB disease and 32% (18/57) patients with stage IV disease. A CTC# of &gt;5 at baseline predicted for a worse progression free and overall survival compared to patients with &lt;5 CTCs (p = &lt; 0.0001). For the 20 patients with 2 of more CTCs at baseline, 55 % exhibited reduction in CTC# following a single cycle of standard platinum based chemotherapy, suggesting that CTC# may be useful in early prediction of response to novel therapeutics. Matched blood samples were taken for ISET analysis for 30 of this cohort of NSCLC patients. CTCs were detected in patients in whom no CTCs were identified using CellSearch. This may be due to loss of, or reduction in EpCam expression perhaps during eptithelial to mesenchymal transition and this now requires further investigation. After optimization of immunohistochemical assays using ISET filters, a panel of NSCLC markers including Thyroid Transcription Factor 1 (TTF1, a routinely used diagnostic marker for NSCLC) and EGFR are being used to confirm the identity of NSCLC CTCs distinct from peripheral blood mononuclear cells. This approach is being applied for detection of protein targets of novel therapies. Overall, our data suggest that CTC analysis is a powerful addition to the biomarker portfolio for drug development in lung cancer.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):CN02-02.