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

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.

Evaluating prognosis by CK7 differentiating renal cell carcinomas from oncocytomas can be used as a promising tool for optimizing diagnosis strategies

“…We summarized CK7 staining in the majority of RCC diffusely expressing membranous and Oncocytoma being typically negative or, at most, focally positive in scattered cells[12,14,18]. Matthewreported that CK7 is helper for diagnosis in circulating tumor cells (CTCs) of tissue of origin in breast cancer, prostate cancer and more expression in gastrointestinal, respiratory and gynecological malignancies[38,39,40]. Kinney proved that CK7 differentiate from Metanephric adenoma and papillary renal cell carcinoma[41].…”

Evidence on the cost of breast cancer drugs is required for rational decision making

“…Biomarkers enable the identification of patients who are highly likely or unlikely to benefit from a particular treatment or can be used as response markers to establish treatment effectiveness very rapidly after treatment initiation. Several studies have been focusing on the use of liquid biopsies, which are tests for biomarkers traceable in body fluids such as blood and urine, to be able to further target and personalise treatment [ 11 ]. One of the advantages of using these liquid biopsies is that tests can be cheaper, less invasive, and might even be more sensitive in targeting the treatment population than expensive imaging diagnostics [ 12 – 15 ].…”

Circulating tumor cells: silent predictors of metastasis

“…For example, multiplexed immunofluorescence staining of isolated CTCs with CK7, CK8, CK20, thyroid transcription factor 1 (TTF-1), estrogen receptor (ER), or prostate-specific antigen (PSA) could reveal the tissue of origin as the lung, differentiating it from breast, colorectal, and prostate cancer. CTCs provide a non-invasive diagnostic blood test as an adjunct to a tissue biopsy 35 . Individual CTCs, CTC clusters, EMT-CTCs, and cancer-associated macrophage-like cells (CAMLs) all give support for metastatic disease.…”