Abstract 3625: A multiplexed marker-based algorithm for diagnosis of carcinoma of unknown primary (CUP) using circulating tumor cells
Authors: Matthew Elizabeth M. , Zhou Lanlan , Yang Zhaohai , Lamparella Nicholas , Liu Yingqiu Y. , Dicker David T. , El-Deiry Wafik S. 
Published: August 2012

Abstract
Metastasis is responsible for the majority of cancer-related deaths. A limiting step in metastasis is access to circulation, thus circulating tumor cells (CTCs) should be present in all patients with metastatic tumors, which may have both diagnostic and prognostic value. Accounting for only 2-3%, carcinoma of unknown primary (CUP) presents a challenge for clinicians in diagnosis and therapy. Tissue biopsy is the standard for diagnosis but there is need for improved methodology including a non-invasive blood test that may rely on CTC analysis. We have been developing a post-Veridex CTC analysis method using multiplexed Q-dot conjugated antibodies with the goal of detection of multiple markers in each single cell within a CTC population to aid in CUP diagnosis including tissue of origin. We have developed an immunofluorescence (IF) protocol with a minimal set of markers to predict the primary sites and provide prognostic information for common metastatic tumors: melanoma, lung, colorectal, upper gastrointestinal organs, and breast or prostate cancer. Two kits are available to isolate CTCs through Veridex CellSearch: melanoma kit (using melanoma antigen), and epithelial or carcinoma kit (using EpCam). A patient's blood is initially tested with both kits, and positive CTCs from the melanoma kit supports a diagnosis of melanoma, whereas positive CTCs from the epithelial kit supports carcinoma. For carcinomas, we use cytokeratin 7 (CK7), cytokeratin 20 (CK20), thyroid transcription factor 1 (TTF-1), estrogen receptor (ER) or prostate-specific antigen (PSA) as the minimal set of markers. IF has been optimized in cultured tumor cells, first with individual antibodies, then quantum dots (Q-dot) conjugated multiplex quadra-antibody set. We evaluated antibodies by IF specific to these 5 markers in lung, breast, colorectal, and prostate cancer cell lines. The CK7 and ER antibodies conjugated to Qdot 605 and 705 respectively demonstrated expression in the appropriate cell lines. CK7-Qdot 605 and ER-Qdot 705 expression in cells that were recovered after being spiked into normal blood samples and isolated by the Veridex CellSearch system was similar to that observed with unconjugated antibodies. Our data show that it is feasible to detect expression using Qdot-labeled multiplexed antibodies on tumor cells isolated from blood by Veridex CellSearch system. Successful application of this protocol to CTCs in patient samples is expected to provide a noninvasive, relatively quick and inexpensive blood test that can be both diagnostic and prognostic, which may be a valuable adjunct to routine biopsy.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3625. doi:1538-7445.AM2012-3625