Abstract 565: Circulating tumor cell (CTC) detection and 5T4 characterization in breast, ovarian, and lung cancer patients on active therapy
Authors: Pirie-Shepherd Steven R. , Jilani Iman , Tucker Eric , Valenta David , Graf Ryon , Anderson Amanda , Marrinucci Dena , Sapra Puja , Powell Eric L. 
Published: August 2015

Abstract
Introduction:
Monitoring CTCs from a blood draw could enable a minimally invasive, longitudinal observation of an evolving disease. Antibody drug conjugates (ADC) are a promising approach to deliver highly potent chemotherapeutics to cells expressing target proteins. An ADC that targets 5T4, an oncofetal antigen expressed on tumor initiating cells, is currently in clinical development. Patients with tumors or CTCs expressing 5T4 may benefit from this therapeutic. We sought to develop a 5T4 CTC test and to evaluate the incidence of 5T4 expressing CTCs in breast, ovarian, and lung cancer patients.
Experimental Procedures:
Blood samples from breast (n = 10), ovarian (n = 10) and NSCLC (n = 25) patients on active therapy were collected and shipped to Epic Sciences. Upon receipt, nucleated blood cells were plated onto glass microscope slides and stored at -80C. Two slides per patient were thawed and stained for cytokeratins (CK), CD45, 5T4, and DAPI. CTCs were detected using a combination of CD45 exclusion, CK and 5T4 expression, and morphology parameters. CTC enumeration and 5T4 expression were analyzed alongside pathology, staging and treatment information.
Results:
Assay Development: A novel CTC assay utilizing an antibody against 5T4 was developed. Dilutions of high, medium and low/negative 5T4 expressing cell lines were spiked into normal whole blood to assess the linearity (R2 = 0.99) of CTC detection. These cell lines as well as 5T4 high and 5T4 low patient samples were used to assess intra- and inter-assay reproducibility of% 5T4(+) CTCs. All patient and calibration control samples met acceptance criteria of ≤ 30% CV and ≤ 20% CV, respectively.
Breast Cancer: 7 of 10 patients had traditional CTCs (median: 6/mL range: 0-23/mL). 9 of 10 patients had additional subpopulations of CK(-) CTCs, apoptotic CTCs, and CTC clusters. Among non-apoptotic CTCs, 5T4 positivity was detected in 6 of 10 patient samples, with the highest% positivity seen in patients with corresponding ER(+) primary tumors.
Ovarian Cancer: 3 of 10 patients had traditional CTCs (median: 0/mL, range: 0-41/mL). Additional subpopulations were identified in the same 3 of 10 patients.
NSCLC: 14 of 25 patients had traditional CTCs (median: 1/mL, range: 0-45/mL). 20 of 25 patients had additional subpopulations.
Conclusions:
We have developed a robust, sensitive and specific assay to quantify 5T4 expression on CTCs and tested this assay on small cohorts of breast, ovarian and lung cancer patients on active therapy. CTCs expressing 5T4 were detected in a subset of patients from each indication.
Citation Format: Steven R. Pirie-Shepherd, Iman Jilani, Eric Tucker, David Valenta, Ryon Graf, Amanda Anderson, Dena Marrinucci, Puja Sapra, Eric L. Powell. Circulating tumor cell (CTC) detection and 5T4 characterization in breast, ovarian, and lung cancer patients on active therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 565. doi:10.1158/1538-7445.AM2015-565