Detection of Circulating Tumour Cells from Blood of Breast Cancer Patients via RT-qPCR

Abstract

Breast cancer is still the most frequent cause of cancer-related death in women worldwide. Often death is not caused only by the primary tumour itself, but also by metastatic lesions. Today it is largely accepted, that these remote metastases arise out of cells, which detach from the primary tumour, enter circulation, settle down at secondary sites in the body and are called Circulating Tumour Cells (CTCs). The occurrence of such minimal residual diseases in the blood of breast cancer patients is mostly linked to a worse prognosis for therapy outcome and overall survival. Due to their very low frequency, the detection of CTCs is, still a technical challenge. RT-qPCR as a highly sensitive method could be an approach for CTC-detection from peripheral blood of breast cancer patients. This assumption is based on the fact that CTCs are of epithelial origin and therefore express a different gene panel than surrounding blood cells. For the technical approach it is necessary to identify appropriate marker genes and to correlate their gene expression levels to the number of tumour cells within a sample in an in vitro approach. After that, samples from adjuvant and metastatic patients can be analysed. This approach may lead to new concepts in diagnosis and treatment.

Quantitative PCR marker genes for endometrial adenocarcinoma

“…Therefore, in order to successfully detect CTCs from blood samples of patients with endometrial adenocarcinoma, the primary challenge is to establish a suitable set of marker genes, which would enable the detection of CTCs with a high sensitivity. These results are in accordance with previous findings using breast cancer cell line cells and blood samples from patients with breast cancer for CTC detection (21)(22)(23).…”

“…The advantage of this method is that it is more sensitive, less expensive than other methods in the field and may be performed quickly in nearly every laboratory. However, it is challenging to find suitable marker genes for the detection of CTCs, which are able to distinguish between tumor and blood cells (21)(22)(23). Therefore, the expression of a set of genes was compared between cells isolated from healthy endometrium and endometrial adenocarcinoma cell lines.…”

Gene expression profiling and DNA methylation analyses of CTCs

“…Molecular assays for studying gene expression on CTCs take full advantage of the extreme sensitivity and specificity of RT‐qPCR. However it is important to note that gene expression analysis of CTCs require special precautions concerning the preanalytical steps; clinical samples should be treated in a way that intact and viable CTCs will isolated in a reasonable time, before proceeding to the isolation of total RNA so that false negative results, due to RNA loss during sample treatment will be avoided (Lianidou and Markou, 2011; Sieuwerts et al., 2011; Andergassen et al., 2013) (Figure 2).…”

“…Molecular assays can be quantitative, high throughput and usually require a very small volume for analysis. Measurements are objective, and quantifiable, and are not subjected to personal estimations as in the case of imaging approaches used for CTCs enumeration and molecular characterization (Andergassen et al., 2013; Ignatiadis et al., 2014). Molecular assays can be subjected to a quantifiable quality control system, and have the potential to be distributed in clinical labs in the form of IVD kits, in case that their clinical significance is proven.…”

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

“…Our research group has already worked on the topic of CTC detection from blood samples [21]. We were able to establish and validate this method in an in vitro model system [22,23].…”