A multiplexed marker-based algorithm for diagnosis of carcinoma of unknown primary using circulating tumor cells

// Elizabeth M. Matthew 1,2,* , Lanlan Zhou 1,2,* , Zhaohai Yang 3 , David T. Dicker 1,2 , Sheldon L. Holder 4 , Bora Lim 1,4,6 , Ramdane Harouaka 5 , Si-Yang Zheng 5 , Joseph J. Drabick 4 , Nicholas E. Lamparella 4 , Cristina I. Truica 4 and Wafik S. El-Deiry 1,2 1 Laboratory of Translational Oncology and Experimental Cancer Therapeutics, Division of Hematology-Oncology, Penn State Hershey Cancer Institute, Hershey, PA, USA 2 Laboratory of Translational Oncology and Experimental Cancer Therapeutics, Department of Medical Oncology and Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA, USA 3 Department of Pathology, Penn State Milton S. Hershey Medical Center, Hershey, PA, USA 4 Division of Hematology-Oncology, Penn State Milton S. Hershey Medical Center and Penn State Hershey Cancer Institute, Hershey, PA, USA 5 Department of Biomedical Engineering, Penn State University, University Park, PA, USA 6 The University of Texas MD Anderson Cancer Center, Houston, TX, USA * These authors have contributed equally to this work Correspondence to: Wafik S. El-Deiry , email: // Keywords : circulating tumor cells (CTCs), carcinoma of unknown primary (CUP), immunofluorescence, diagnosis Received : November 14, 2015 Accepted : November 23, 2015 Published : December 18, 2015 Abstract Real-time, single-cell multiplex immunophenotyping of circulating tumor cells (CTCs) is hypothesized to inform diagnosis of tissue of origin in patients with carcinoma of unknown primary (CUP). In 20 to 50% of CUP patients, the primary site remains unidentified, presenting a challenge for clinicians in diagnosis and treatment. We developed a post-CellSearch CTC assay using multiplexed Q-dot or DyLight conjugated antibodies with the goal of detecting multiple markers in single cells within a CTC population. We adapted our approach to size-based CTC enrichment protocols for capturing CTCs and subsequent immunofluorescence (IF) using a minimal set of markers to predict the primary sites for common metastatic tumors. The carcinomas are characterized with cytokeratin 7 (CK7), cytokeratin 20 (CK20), thyroid transcription factor 1 (TTF-1), estrogen receptor (ER) or prostate-specific antigen (PSA. IF has been optimized in cultured tumor cells with individual antibodies, then with conjugated antibodies to form a multiplex antibody set. With IF, we evaluated antibodies specific to these 5 markers in lung, breast, colorectal, and prostate cancer cell lines and blood from metastatic prostate and breast cancer patients. This advanced technology provides a noninvasive, diagnostic blood test as an adjunct to routine tissue biopsy. Its further implementation requires prospective clinical testing.