Abstract 5385: Standardization and automation of volume reduction procedure for circulating tumor cells and rare cell workflows with VRNxT technology
Authors: Mariano Di Trapani, Alex Calanca, Fabrizio Alberti, Gianpiero Parisi, Valeria Stivani, Chiara Maranta, Francesca Dattolo, Francesco Grasso, Michele Ricci, Alex Marchi, Gianni Medoro
Journal: Cancer Research (2020)

Abstract Tumor heterogeneity is a hallmark of cancer and a major driving force for tumor progression, and emergence of resistance to cancer therapy. Single-cell studies have greatly contributed to unravelling tumour heterogeneity but wider adoption has been prevented by technical challenges. Isolation and analysis of single circulating tumor cells (CTCs) offer a unique minimally invasive approach to characterize and monitor dynamic changes in tumor heterogeneity at the genomic level. Standardization and reproducibility of the workflows are mandatory for translating CTC-based assays to the clinic and meet the regulatory requirements to enter routine practice. Recently, we and other groups have demonstrated the applicability of the CellSearch®/DEPArray™/ Ampli1™ workflow for the enumeration, isolation and molecular characterization of single CTCs. This workflow combines the high sensitivity and specificity of the CellSearch platform for CTC detection and enrichment with the DEPArray technology, which ensures a precise and accurate image-based isolation of single CTCs, 100% pure. Here, we validate the VRNxT™, an innovative technology that further harmonizes the CellSearch/DEPArray workflow by automating the volume reduction steps. The working principle of VRNxT instrument uses centrifugal forces to reduce the sample volume, eliminating the use of pipettes in washing steps and consequently, the risk of cell loss. Precision and accuracy of VRNxT were tested by comparing automatic and manual volume reduction (VR) procedures in a typical workflow. Briefly, after single cell isolation with the DEPArray, standard sample preparation for whole genome amplification analysis were carried out by four different skilled operators and compared to automatic VRNxT procedure. Each operator sorted 90 single CTCs using DEPArray and then performed manual VR in order to obtain a final volume of about 2 µl, compatible with the whole genome amplification procedure. Significant differences were observed in the volume reduction procedure performed by operators (2.65±0.79; 1.16±0.50; 1.26±0.96; 1.58±0.67, respectively). Conversely, VRNxT exhibited a higher reproducibility of data (1.82 µl±0.23 SD), by eliminating the user-dependent variability. Moreover, automatic VR exponentially reduced the time required to VR (about 30 minutes compared to 218 minutes of manual VR, for 96 single cell recoveries), and minimized single cell loss with a success rate never achieved so far (100% respect to 92.8% manual VR). VRNxT is thus an easy-to-use technology, that minimizes the variability and error rate in rare cell workflow and consequently reduces user-dependent variability and improve standardization. VRNxT technology coupled to CellSearch, DEPArray and Ampli1, offers an effective solution for a streamlined liquid biopsy workflow to characterize and monitor tumor heterogeneity. Citation Format: Mariano Di Trapani, Alex Calanca, Fabrizio Alberti, Gianpiero Parisi, Valeria Stivani, Chiara Maranta, Francesca Dattolo, Francesco Grasso, Michele Ricci, Alex Marchi, Gianni Medoro. Standardization and automation of volume reduction procedure for circulating tumor cells and rare cell workflows with VRNxT technology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5385.