Abstract P5-07-10: Quantification of circulating tumor cells using NanoFlares in breast cancer patients
Authors: Jain S , Angeloni NL , McMahon KM , Rademaker A , Cheng C , Thaxton CS 
Published: May 2016

Abstract
Background: NanoFlares enable the detection of intracellular mRNA targets in live-cells and can be used to fluorescently detect genetic markers of circulating tumor cells (CTCs) in whole blood. Unlike the current technology which tracks CTCs by protein markers located on the cell surface, NanoFlares allow for the detection of mRNA targets in live-cells at the single-cell level. This technique was recently validated in a murine model of metastatic breast cancer. In this study, we aimed to utilize the NanoFlares to quantify the number of CTCs in patients with breast cancer.
Methods: We enrolled a prospective cohort of patients with stage 1-4 breast cancer. After obtaining informed consent, donation of blood samples for CTC analysis was collected at study entry. Blood samples were treated with control scrambled NanoFlares or vimentin NanoFlares to a final concentration of 6nM. Samples were incubated for approximately seven hours at 37°C then processed to remove red blood cells and stained with a fluorescent anti-CD45 antibody. Data was acquired via flow cytometry on a LSRFortessa cell analyzer. CD45+ peripheral blood mononuclear cells were gated out, and vimentin-positive cells were enumerated by identifying populations in the vimentin Flare-treated samples which were above the baseline fluorescence found in the control Flare-treated samples. Vimentin is a reported marker of mesenchymal cells and aggressive breast cancer cells. Cells with a high vimentin NanoFlare fluorescent response were presumed to be CTCs for this study. Wilcoxon rank sum test was used to compare median CTCs between subgroups.
Results: Fourteen patients were included in this analysis. The median age was 52.6 years (range 30.1-81.5). Eight patients had early stage disease (stage 1: 2, stage 2: 3, stage 3: 3) and 6 patients had stage 4 breast cancer. Among the stage 4 patients, all had visceral metastases and 5 had progression of disease noted on imaging at blood draw. In the entire cohort, 12 (86%) patients had estrogen receptor (ER) and/or progesterone receptor (PR)-positive disease and 5 (26%) were HER2-positive. CTCs were observed in all patients on study. Patients with stage 4 breast cancer had higher median number of CTCs compared to stage 1-3 patients (139 vs 42, p=0.09). Patients with ER/PR negative disease had a higher median number of CTCs compared to ER and/or PR positive (191 vs 62, p=0.58). HER2 status did not appear to affect the median number of CTCs (67 in HER2-positive vs 56 in HER2-negative, p=0.84). Among stage 4 patients, there were no differences in CTCs in those who had progression of disease at the time of the blood sample versus those who had stable disease.
Conclusions: NanoFlares was successful in detecting CTCs in each patient. Though our data set is currently limited by the small number of patients, this analysis suggests that differences exist in the amount of CTCs by stage and/or receptor status with a trend for higher CTCs in stage 4 patients and those with ER/PR-negative disease. This technology is the first approach for detecting, isolating, and characterizing live cancer cells in patient blood based upon intracellular mRNA targets, and may have the ability to impact clinical decisions in patients.
Citation Format: Jain S, Angeloni NL, McMahon KM, Rademaker A, Cheng C, Thaxton CS. Quantification of circulating tumor cells using NanoFlares in breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-07-10.