Gene expression profile of circulating tumor cells in breast cancer by RT-qPCR

Abstract

BackgroundCirculating tumor cells (CTCs) have been associated with prognosis especially in breast cancer and have been proposed as a liquid biopsy for repeated follow up examinations. Molecular characterization of CTCs is difficult to address since they are very rare and the amount of available sample is very limited.MethodsWe quantified by RT-qPCR CK-19, MAGE-A3, HER-2, TWIST1, hTERT α+β+, and mammaglobin gene transcripts in immunomagnetically positively selected CTCs from 92 breast cancer patients, and 28 healthy individuals. We also compared our results with the CellSearch system in 33 of these patients with early breast cancer.ResultsRT-qPCR is highly sensitive and specific and can detect the expression of each individual gene at the one cell level. None of the genes tested was detected in the group of healthy donors. In 66 operable breast cancer patients, CK-19 was detected in 42.4%, HER-2 in 13.6%, MAGE-A3 in 21.2%, hMAM in 13.6%, TWIST-1 in 42.4%, and hTERT α+β+ in 10.2%. In 26 patients with verified metastasis, CK-19 was detected in 53.8%, HER-2 in 19.2%, MAGE-A3 in 15.4%, hMAM in 30.8%, TWIST-1 in 38.5% and hTERT α+β+in 19.2%. Our preliminary data on the comparison between RT-qPCR and CellSearch in 33 early breast cancer patients showed that RT-qPCR gives more positive results in respect to CellSearch.ConclusionsMolecular characterization of CTCs has revealed a remarkable heterogeneity of gene expression between breast cancer patients. In a small percentage of patients, CTCs were positive for all six genes tested, while in some patients only one of these genes was expressed. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known.

A versatile method for enumeration and characterization of circulating tumour cells from patients with breast cancer

“…Figure 6 shows representative images of two CTCs characterized as EpCAM and CK positive, with a well-defined nucleus and negative for the Determination by quantitative RT-PCR for CK 19, EpCAM, and MUC-1 in the enriched fraction was also a specific approach which was applied in validating CTCs numbers obtained by flow cytometry. Further, to overcome representation of results as expression in relative fold and to show absolute quantitation, the Strati et al [23] and Aerts et al [22] methods were adapted. These methods can overcome differences in expression of the selected genes across samples, estimate the quantities as copies/mL in blood samples, and enable direct comparison of samples across time points of collection.…”

“…Based on the Strati et al [23] method, we developed a CTC gene expression qRT-PCR assay by using quantification calibrators for four gene transcripts (CK-19, EpCAM, MUC1, Her2) containing a known number of copies, prepared as described in Methods. These were synthesized as described and aliquoted to eliminate experimental variation.…”

“…We also prepared qRT-PCR calibrators by adapting the Strati et al [23] method as a qRT-PCR requires analysis of samples across time frames. Individual PCR amplicons corresponding to gene-targets CK-19, EpCAM, MUC-1, and Her2Neu that could serve as quantification calibrators were generated.…”

“…Similar spiking experiments were done for QRT-PCR and expression of CK-19, MUC-1, EpCAM, and GAPDH. The methods described by others [22,23] have been adapted for quantitation of CK 19, EpCAM, MUC-1, and Her2neu. Data pertaining to these methods have been generated and any one of these methods can be used.…”

Analysing the Mutational Status of PIK3CA in Circulating Tumor Cells from Metastatic Breast Cancer Patients

“…Breast cancer cells in primary tumors, CTCs as well as disseminated tumor cells (DTCs) vary phenotypically and functionally in established prognostic factors such as HER2, ER, PR and in gene expression profiles (Bozionellou et al, 2004;Meng et al, 2004;M€ uller and Pantel, 2009;Strati et al, 2011;Powell et al, 2012). These findings advocate for the usage of CTCs as 'liquid biopsy' to refine therapeutic regimens.…”