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.
Highlights
Circulating 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
We have recently shown that the detection of CTCs post-chemotherapy in breast cancer patients is associated with involvement of more than three axillary lymph nodes with significantly increased clinical relapses and disease-related deaths [15]
By using a multi-marker assay in CTCs in early breast cancer, we have shown that CK-19, Mammaglobin, and HER-2 positive CTCs are associated with shorter disease free survival [11]
Summary
Circulating 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. Metastasis is a multi-stage process [1] that selects for Circulating Tumor Cells (CTCs) that can infiltrate, survive in and colonize distant organs [2]. Recent advances in this field are supportive for the early dissemination model of metastasis, through the observation that Disseminated Tumour Cells (DTCs) isolated from bone marrow or lymph nodes display disparate changes on all levels of genomic resolution as compared to primary tumor cells [3]. Our group has previously shown that the detection of CTCs in peripheral blood of early breast cancer patients before and after chemotherapy through the epithelial molecular marker Cytokeratin-19 (CK-19) is of prognostic significance [8,9,10,11,12]. Enumeration and molecular characterization of CTCs can be used as a liquid biopsy for repeated follow up examinations in a variety of human cancers [16,17,18] and may play a major role in helping to guide targeted therapy [16,17,18,19,20]
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