Abstract P3-01-16: Micro-cavity array system for size-based enrichment of circulating tumor cells and circulating cancer associated fibroblasts from blood of patients with breast cancer
Authors: Parajuli R , Ly R , Ziogas A , Eapen A , Lane K , Chen J , Lin E , Mehta R , Tsai A 
Published: May 2019

Background:Circulating Tumor Cells(CTCs) have prognostic implications in patients with metastatic breast cancer(MBC).During the Epithelial Mesenchymal Transition(EMT), CTCs acquire a more mesenchymal phenotype. Hence, methodologies such as the Cell search that rely on the expression of an epithelial marker EpCAM in CTCs fail to capture a subset of CTCs undergoing the process of EMT and therefore do not adequately represent the true circulatory metastatic load. Hitachi chemicals has invented a size based micro cavity array (MCA) system that allows for the isolation of tumor cells based on the differences in size and deformability between tumor and blood cells. Photolithography and the metal plating can precisely control the filter pore size of our system. Our platform is more sensitive than the Cell Search method in detecting CTCs in Lung Cancer. Cancer Associated Fibroblasts (CAFs) are a major component of the breast tumor microenvironment. Using a micro filter capture technique, our co-authors have demonstrated that CAFs can be enumerated as circulating CAFs (cCAFs). Hitachi's Micro cavity Array System has not been evaluated in the detection of CTCs and cCAFs in patients with Breast Cancer. The purpose of this study is to demonstrate that CTCs and cCAFs can be enumerated using our platform and the cCAFs can serve as biomarkers of metastasis simultaneously with CTCs.

Method:We undertook a Pilot study of 20 patients each with breast cancer across Stage I, Stage II, Stage III and Stage IV. A total of 10ml of peripheral blood was obtained from each patient. Enumeration of CTCs and cCAFs was carried out by the size based mircocavity array system invented by Hitachi Chemicals. Identification of these cells was done by a triple Immunofluorescence staining for pan-CK (cytokeratin), FAP (Fibroblast Activated Protein) and CD45. CTCs were identified as CK+, CD45-, FAP- cells and cCAFs were identified as FAP+, CK- and CD 45 negative cells.

Result:Our method had a high cell recovery rate (90%or higher) and efficient white blood cells depletion rate (99.99%). We present the data from a total of 13 patients in this abstract, (two with stage III and eleven with stage IV breast cancer) . Data from rest of the subjects will be presented at the actual meeting. We detected the presence of CTCs in 11/11(100%) in patients with stage IV(mean of 44) and in 2 out of 2 (100%) patients with Stage III Breast Cancer. We detected the presence of cCAFs in 1 out of 2 patients( 50%) with stage III and in 8 of 11(81.8%) (mean of 9)patients with stage IV breast cancer( Fisher's exact test p-value= 0.42). The number of CTCs and cCAFs was significantly elevated in patients with MBC and the number was clinically associated with a high metastatic burden.

Conclusions:CTCs and cCAFs can be enumerated using a size based size based micro cavity array invented by Hitachi Chemicals that does not rely on the expression of epithelial markers in CTCs. CTCs and cCAFs can be detected in patients with stage III and stage IV breast cancer. CTCs and cCAFs were associated with high metastatic burden and their numbers were significantly elevated in patients with MBC. cCAFs could serve as biomarkers alongside of CTCs in MBC.

Citation Format: Parajuli R, Ly R, Ziogas A, Eapen A, Lane K, Chen J, Lin E, Mehta R, Tsai A. Micro-cavity array system for size-based enrichment of circulating tumor cells and circulating cancer associated fibroblasts from blood of patients with breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-16.