Abstract
BackgroundDetection of circulating tumor cells (CTC) in the blood of cancer patients may have prognostic and predictive significance. However, background expression of 'tumor specific markers' in peripheral blood mononuclear cells (PBMC) may confound these studies. The goal of this study was to identify the origin of Cytokeratin 19 (CK19) and HER-2 signal in PBMC and suggest an approach to enhance techniques involved in detection of CTC in breast cancer patients.MethodsPBMC from healthy donors were isolated and fractionated into monocytes, lymphocytes, natural killer cells/granulocytes and epithelial populations using immunomagnetic selection and fluorescent cell-sorting for each cell type. RNA isolated from each fraction was analyzed for CK19, HER2 and Beta 2 microglobulin (B2M) using real-time qRT-PCR. Positive selection for epithelial cells and negative selection for NK/granulocytes were used in an attempt to reduce background expression of CK19 and HER2 markers.ResultsIn normal PBMC, CK19 was expressed in the lymphocyte population while HER-2 expression was highest in the NK/granulocyte population. Immunomagnetic selection for epithelial cells reduced background CK19 signal to a frequency of <5% in normal donors. Using negative selection, the majority (74–98%) of HER2 signal could be removed from PBMC. Positive selection methods are variably effective at reducing these background signals.ConclusionWe present a novel method to improve the specificity of the traditional method of detecting CTC by identifying the source of the background signals and reducing them by negative immunoselection. Further studies are warranted to improve sensitivity and specificity of methods of detecting CTC will prove to be useful tools for clinicians in determining prognosis and monitoring treatment responses of breast cancer patients.
Highlights
Detection of circulating tumor cells (CTC) in the blood of cancer patients may have prognostic and predictive significance
The goal of this study is to identify the source of background signals for Cytokeratin 19 (CK19) and HER-2 in peripheral blood mononuclear cells (PBMC) and propose an approach to reduce the cells contributing to the background to improve the specificity of a currently available and sensitive method of detecting CTC
Sample 10 demonstrates the utility of the Beta 2 microglobulin (B2M) assay to assess RNA integrity as low B2M signal indicates that the RNA is not adequate and the CK19 result cannot be interpreted
Summary
Detection of circulating tumor cells (CTC) in the blood of cancer patients may have prognostic and predictive significance. Background expression of 'tumor specific markers' in peripheral blood mononuclear cells (PBMC) may confound these studies. In an effort to improve sensitivity, analysis of gene expression using reverse transcription polymerase chain reaction (RT-PCR) has been employed for detection of micrometastases. While these methods have increased sensitivity, and allow the detection of as few as one epithelial cell in 107 mononuclear blood cells, specificity remains an important problem [5]. One of the factors that compromises the specificity of RT-PCR methods in detecting micrometastases is the background expression of 'tumor markers' in normal peripheral blood. Understanding the origin of background and developing methods to selectively eliminate it is a critical step to improving the specificity of the RT-PCR method
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