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.
Highlights
Metastasis is responsible for the majority of cancer-related deaths [1]
We have developed an immunofluorescence (IF) protocol with a minimal set of markers to predict the primary sites for common metastatic tumors from lung, colon, breast or prostate cancer (Figure 1), and this algorithm may be expanded to include additional cancer types (Supplemental Figure S1)
When the chosen primary antibody coupled with a fluorescence-labeled secondary antibody was successful as defined by appropriate positive and negative staining in respective plated cancer cell lines, the primary antibody was subsequently conjugated to a Q-Dot or DyLight fluorophore
Summary
A limiting step in metastasis is access to circulation, theoretically circulating tumor cells (CTCs) should be present in all patients with metastatic tumors, which may have both diagnostic and prognostic values. Administration-cleared Janssen (Veridex) CellSearch® system, circulating tumor cells are an independent prognostic factor of progression-free survival (PFS) and overall survival in metastatic breast [2,3,4], colon [5] and prostate cancers [6]. Carcinoma of unknown primary (CUP) accounts for only 3-5% of metastatic tumors and presents a challenge for clinicians in diagnosis and therapy [9]. CUP patients can have early and unusually aggressive metastatic dissemination without a readily identifiable primary tumor site [9, 10]. A non-invasive blood test that takes advantage of CTC analysis may be a valuable adjunct to routine biopsy
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