Abstract P4-01-20: A novel microfluidic system for the detection, enumeration and molecular analysis of circulating tumor cells (CTCs) in metastatic breast cancer (MBC)
Authors: Paolillo Carmela , Mu Zhaomei , Toss Angela , Gogoi Priyadarshini , Sepehri Saedeh , Zhou Yi , Handique Kalyan , Zhong Ye , Yang Hushan , Capoluongo Ettore , Cristofanilli Massimo , Fortina Paolo 
Published: May 2015

Abstract
Background: In recent years blood testing for circulating tumor cells (CTC) has gained increasing interest in cancer research. CTC detection and enumeration can serve as a ‘liquid biopsy’ and an early marker of response to systemic therapy. Different analytical systems for CTC detection and isolation have been developed, but the CellSearch® is currently the only FDA-approved technology. We aimed to evaluate CTCs detection by a novel microfluidific technology, a size- and deformability-based capture system. This unique platform not only allows flex[not]ibility in the selection of antibody markers but also segregates the CTCs in their own chambers, thus, enabling morphological, immunological and genetic characterization of each CTC at the single cell level.
Methods: We performed a prospective study to compare the detection of CTCs using the CellSearch® (Janssen Diagnostics) vs. the new microfluidic platform. Enumeration by the CellSearch® was performed according to standard protocol. For the microfluidific-device capture (De Novo Sciences, MI) peripheral blood from MBC patients was diluted 1:1 with PFA 0.8% and PBS 1%. Prior to sample loading, the microfluidic device was coated with priming buffer. After cells were fixed using 4.0% PFA and were subsequently stained with pancytokeratin, Zym 5.2 and CD45. Nuclei were coun[not]terstained with Hoechst-33342. CTCs were identified as round and bright green cells not stained with red (CK+/CD45-/DAPI+). Data were analyzed using non-parametric methods: Cohen’s kappa, Chi2 test, Spearman rank correlations and Mann-Whitney test. Associations with CTCs were calculated in two ways: CTCs as a continuous variable normalized for ml of blood and CTCs categorized as &lt; 5 versus ≥ 5.
Results: The two methods was concordant in 88.2% of patients with a Cohen’s kappa of 0.743 when the detection of a single CTC is considered like positive. We also found a concordance of 85% (k=0.70) when we use the CTC cut-off level of ≥5 cells per 7.5 ml of blood to identify patient with higher risk for disease progression. Thirty-one patients with MBC were tested for CTCs using both methods. CTC detection by microfluidific platform was positively associated with Her2 positive patients if we consider as categorical variable; a weak association was seen also with the CellSearch®.
CTCs AssociationCTC/ml of blood ContinuousCTC categorized (≥5)DeNovomedian [min, max]p-valueNn (%)OR ( 95% CI)p-valueHer 20.5620.038No0.5 [0, 62]197 (36.8)refYes1.75 [0, 4]129 (75.0)5.14 (1.03, 25.6)CellSearch®median [min, max]p-valueNn (%)OR ( 95% CI)p-valueHer 20.7040.056No0.13 [0, 335.7]196 (41.2)refYes0.95 [0, 3.33]128 (34.7)4.33 (0.93, 20.2)
Conclusions: The enumeration of CTCs showed strong prognostic significance in MBC raising interest in a more accurate molecular characterization to achieve the possibility for a dynamic molecular monitoring. Introducing novel methodologies should demonstrate comparable detection rate for epithelial cells. This new microfluidic system showed accuracy and flexibility to move to the next phase of molecular testing with the intent of assessing the value as liquid biopsy.
Citation Format: Carmela Paolillo, Zhaomei Mu, Angela Toss, Priyadarshini Gogoi, Saedeh Sepehri, Yi Zhou, Kalyan Handique, Ye Zhong, Hushan Yang, Ettore Capoluongo, Massimo Cristofanilli, Paolo Fortina. A novel microfluidic system for the detection, enumeration and molecular analysis of circulating tumor cells (CTCs) in metastatic breast cancer (MBC) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-20.