Abstract P2-02-14: Detection and characterization of CTCs isolated by ScreenCell®-Filtration in metastatic breast cancer
Authors: Mu Z , Benali-Furet N , Uzan G , Ye Z , Austin L , Wang C , Nguyen1 T , Avery T , Jaslow R , Yang H , Cristofanilli M 
Published: May 2016

Background: Circulating Tumor cells (CTCs) detection has prognostic and predictive implications in patients with metastatic breast cancer (MBC). Genomic and phenotypic analysis of CTCs hold enormous promise as blood-based molecular characterization and monitoring disease progression and treatment benefit with a strong potential to be translated into more individualized targeted treatments. FDA-approved CellSearch™ detection allows only enumeration of CTCs expressing EpCAM without molecular characterization. CTCs represent very heterogeneous populations of tumorigenic cancer cells and some subpopulations have undergone epithelial-Mesenchymal transition (EMT), which is associated metastasis process and an unfavourable outcome. EpCAM-based enrichment technique has failed to detect EMT subpopulations due to the decreased expression or loss of epithelial markers. Non-EpCAM-based approaches are needed for identifying EMT CTCs. The ScreenCell® devices are single-use and low-cost innovative devices that use a filter for enrichment-free isolation of CTCs by a two-steps combining size-based separation and staining using different markers. The DEPArray™ system is the ideal downstream isolation system to collect single or pooled CTCs for molecular and genetic analysis. In this study, we evaluated the feasibility of achieving CTCs detection/enumeration using ScreenCell® filtration followed by single cell isolation with the DEPArray™ in MBC patients.

Methods: The first part of the study consisted in evaluating CTCs detection/enumeration in 30 patients with stage III and stage IV breast cancer. 3 mL of whole blood in an EDTA or Transfix tubes was collected and processed on the ScreenCell® Cyto device following the instructions of the supplier. CTCs were stained with cytokeratin (CK-8, 18, and 19), leukocyte antigen (CD45), and a nuclear dye (DAPI) and counted under fluorescence microscope. CTCs were identified as positive staining for CK and DAPI and negative staining for CD45 (CK+/DAPI+CD45-). In the second part, After enrichment, CTCs were stained with CK, CD45, and DAPI and sorted with DEPArray™ Platform (Silicon Biosystems, Inc). Single CTCs were collected and the DNA of each single CTCs was amplified with Ampli1™ WGA kit, and the genome integrity index (GII) was assessed by Ampli1™ QC kit (Silicon Biosystems, Inc). Library was constructed and whole exome sequencing (WES) of DNA mutations was conducted.

Results: Twenty patient samples had CTCs detected (66.7%), the number of CTCs was 1 to 347 per 3.0 ml of whole blood. CTC-clusters were detected in 7 patient samples (23.3%). Single CTCs were collected on DEPArray™ platform after enrichment with ScreenCell filtration. GII was confirmed with the presence of short, medium, and long DNA fragments (3 to 4 PCR bands) in the WGA library by PCR-based assay. All collected CTCs showed high GII as measured by Ampli1™ QC kit (GII ≥ 3) for WES of DNA mutations. The data analysis of WES results is under processing.

Conclusions: ScreenCell® filtration is simple and effective devices to isolate CTCs and identify CTC-clusters. Isolation of single cells for molecular analysis using the combination of ScreenCell® filtration and DEPArray™ Platform is feasible for genetic characterization of CTCs.

Citation Format: Mu Z, Benali-Furet N, Uzan G, Ye Z, Austin L, Wang C, Nguyen1 T, Avery T, Jaslow R, Yang H, Cristofanilli M. Detection and characterization of CTCs isolated by ScreenCell®-Filtration in metastatic breast cancer. [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 P2-02-14.