Abstract P4-03-05: Characterization of circulating tumor cells identified by high definition assay in patients with benign and malignant breast disease
Authors: Lorizio W , Luttgen M , Marks J , Wilderman E , Bethel K , Kuhn P , Hwang S 
Published: August 2014

Abstract
Background: Identification of circulating tumor cells (CTC) has the potential for minimally invasive diagnosis and assessment of therapy response. Current CTC assay techniques rely on immunomagnetic bead isolation for CTC enrichment. However, these methods result in low cell yield, limiting the potential use in early stage where CTCs are thought to be less abundant. The high definition (HD)-CTC assay is a novel non-enrichment assay providing unique opportunities to identify single circulating epithelial cells (CepiCs) in patients before or at diagnosis. This study's goal is to determine feasibility and performance of the HD-CTC method for isolating CepiCs in women presenting for diagnostic breast biopsy. Methods: Women undergoing image-guided biopsy for BIRADS 4 and 5 mammographic abnormalities were selected from April 2012 onward at the Duke Breast Clinic. After obtaining informed consent, sample collection and shipping procedures were performed following specific standard operating procedures. Diagnosis was kept blinded during assay analysis. Quantitative measurements were used at the single-cell level to identify and enumerate CepiCs. We morphologically characterized specific high content screening variables including cytokeratin (CK) intensity and epithelial cell adhesion molecule (EpCAM) staining. Measurements included cellular size, shape, and staining pattern. For each draw, 4 monochrome images per frame from 4 stained slides were used for CK/EpCAM evaluation. Results: To date 34 women have been enrolled. Fourteen samples (41%) were drawn before biopsy and 20 (59%) after. Out of the 20, 2 (10%) were non-evaluable and a total of 18 (90%) have HD-CTC assay derived data available. The average diagnosis age was 54; 70% were Caucasian. Six (33%) out of 18 had benign disease (fibrocystic, fibroadenoma and apocrine), 10 (55%) had invasive (ductal or lobular), and 2 (12%) had atypical ductal hyperplasia and ductal carcinoma in situ. Five (83%) benign cases and 4 (40%) invasive had detectable CepiCs (Fisher exact test = 0.43). We found a borderline significant trend between the number of CepiCs and the nodes involved (P for trend = 0.06). No association found with ER, PR or ERBB2 status. The mean number of CepiCs/mL in invasive cases was 45.25 +/-96.65 (range 0 to 270.6) compared to 17.47 CepiCs/mL +/-27.8 (range 0 to 72.3) in benign. Additionally, all the CK(+) morphologically distinct cells were EpCAM(-). There is no significant difference between benign and malignant cases when analyzing draws after biopsy. Conclusion: HD-CTC technology is feasible for detection of CepiCs in early stage breast cancer, and in some cases, results in retrieval of a large number of them. Additional technology refinements including determination of appropriate thresholds are needed to improve discrimination of cancer patients from non-cancer, and follow up will help determine whether HD-CTC assay derived enumeration and characterization of CepiCs are associated with prognosis. These early results are promising and ongoing studies at Duke with TSRI will further evaluate the role of CepiCs in early stage breast cancer along with the isolation and analysis of DNA from single-CepiCs. New data will be available by time of presentation.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-03-05.