Real-time quantitative RT-PCR detection of circulating tumor cells from breast cancer patients

Abstract

Circulating tumor cells (CTCs) were recognized as novel tumor biomarker for prognostic and predictive purposes in various cancers. Various detection technologies and devices have been developed to enumerate and characterize CTCs. Most of those approaches are based on the positive enrichment strategy and immunocytological techniques. However, the sensitivity of these approaches proved to be limited in metastatic tumors and the detection of early tumor cell dissemination was problematic. In the present study, we developed a novel CTC detection method by real-time RT-PCR technique in combination of negative enrichment strategy. The developed enrichment approach could recover more than 75% of spiked breast cancer cells from peripheral blood. The detection limit of duplex real-time RT-PCR assay using KRT19 and ERBB2 as targeted genes was consistently one breast tumor cell. Moreover, CTC detection by duplex real-time RT-PCR assay had higher detection sensitivity than that by immunostaining, especially in early breast cancer. In summary, the results of the present study indicated the potential clinical utilities of CTCs identification on breast cancer by duplex real-time RT-PCR in combination with negative enrichment.

Metastasis of circulating tumor cells: Favorable soil or suitable biomechanics, or both?

“…[30][31][32] RT-PCR analysis of CTCs signature, enabling highly sensitive detection and is being widely used in clinical settings for many different types of cancer, given the ease of use. 31,[33][34][35][36][37] However, CTCs may have different progenitors within tumors, which are heterogeneous in nature, and experimental pooling could mask cell-to-cell variations in expression that are biologically interesting and important. Recently, several reports charting the molecular heterogeneity of the CTCs population, [38][39][40] at single cell level, in breast, prostate and pancreatic cancer patients have been published.…”

Real-Time qPCR-Based Detection of Circulating Tumor Cells from Blood Samples of Adjuvant Breast Cancer Patients: A Preliminary Study

“…Another important and interesting point would be to decide by the RT-qPCR results whether a certain blood sample came from a cancer patient or was withdrawn from a healthy donor. But for this purpose, a lot of work still needs to be done, especially in defining a set of marker genes on which this decision could be made with high reliability [34,35,36]. The analysis of more marker genes like Her-2 [37], MMP13 [38], UBE2Q2 [39], or Nectin-4 [40] for their use in CTC detection could help in this point, allowing a more sensitive detection with simultaneous characterization of the tumor cells as well.…”

Real-time RT-PCR systems for CTC detection from blood samples of breast cancer and gynaecological tumour patients (Review)

“…both studies found, that cytokeratins are suitable PCR marker genes for CTC quantification from blood samples, promising new possibilities in CTC detection, treatment assessment and follow-up (152). The sensitivity of the CTC quantification could even be increased by a duplex PCR-assay, carried out subsequently after a negative enrichment (153).…”