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

<p>Abstract</p> <p>Background</p> <p>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. Molecular characterization of CTCs is difficult to address since they are very rare and the amount of available sample is very limited.</p> <p>Methods</p> <p>We quantified by RT-qPCR <it>CK-19, MAGE-A3, HER-2, TWIST1, hTERT α+β+</it>, and <it>mammaglobin </it>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.</p> <p>Results</p> <p>RT-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, <it>CK-19 </it>was detected in 42.4%, <it>HER-2 </it>in 13.6%, <it>MAGE-A3 </it>in 21.2%, <it>hMAM </it>in 13.6%, <it>TWIST-1 </it>in 42.4%, and <it>hTERT α+β+ </it>in 10.2%. In 26 patients with verified metastasis, <it>CK-19 </it>was detected in 53.8%, <it>HER-2 </it>in 19.2%, <it>MAGE-A3 </it>in 15.4%, <it>hMAM </it>in 30.8%, <it>TWIST-1 </it>in 38.5% and <it>hTERT </it>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.</p> <p>Conclusions</p> <p>Molecular 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.</p

Molecular detection of micrometastatic disease in breast cancer

Circulating tumor cells represent an important biological link in the prevalence of breast cancer, between primary tumor and metastatic disease. CTCs have already been established as strong predictors of prognosis in patients with metastatic breast cancer. The expression of epithelial cancer marker CK-19 in 179 samples of patients with early breast cancer and we found out that the patients that were positive for mRNA of CK-19 were associates with disease free interval (P=0,0140) and overall survival (P=0,0510). Quantitative real-time PCR was developed for the transcription factor TWIST-1 and the gene HER-2. CTCs were detected concerning the expression of TWIST-1 in 12,5% of patients with early breast cancer, in 5.5% concerning the expression of HER-2 and in 11,1% in patients with verified metastasis. Quantitative multiplex real-time PCR was developed for the genes CK-19, MAGE A3, HER-2, PBGD. CTC’s were detected in patients with early breast cancer 42,2% for CK-19, 18,8% for MAGE A3, 12,5% for HER-2 and in patients with verified metastasis, 51,8% for CK-19, 14,8% for MAGE A3, 18,5% for HER-2.Τα κυκλοφορούντα καρκινικά κύτταρα αντιπροσωπεύουν έναν σημαντικό βιολογικό σύνδεσμο στη διάδοση του καρκίνου, από το στάδιο πρωτογενή όγκο μέχρι την εμφάνιση μεταστάσεων. Τα CTCs έχουν καθιερωθεί ήδη ως ισχυροί ενδεικτικοί παράγοντες στην πρόγνωση ασθενών με μεταστατικό καρκίνο μαστού. Μελετήθηκε η έκφραση του επιθηλιακού καρκινικού δείκτη CK-19 σε 179 ασθενείς με πρώϊμο καρκίνου μαστού και βρέθηκε ότι οι ασθενείς που ήταν θετικοί στο mRNA της CK-19 είχαν μειωμένο ελεύθερο νόσου διάστημα (P=0,0140) και μικρότερη επιβίωση (P=0,0510). Αναπτύχθηκαν μεθοδολογίες ποσοτικής real-time PCR για τον μεταγραφικό παράγοντα TWIST-1 και το HER-2. Ανιχνεύτηκαν CTCs ως προς την έκφραση του TWIST-1 σε 12,5% ασθενών με πρώϊμο καρκίνο μαστού, ενώ ως προς την έκφραση του HER-2. 5,5% βρέθηκαν θετικοί και 1 1.1% σε ασθενείς με μεταστατικό καρκίνο μαστού. Αναπτύχθηκε πολλαπλή ποσοτική real-time PCR για τα γονίδια CK-19, HER-2, MAGE A3, PBGD. Ανιχνεύτηκαν CTC’s σε ασθενείς με πρώϊμο καρκίνο μαστού 42,2% για τη CK-19, 18,8% για το MAGE A3, 12,5% για το HER-2 και σε μεταστατικούς ασθενείς, 51,8% για τη CK-19, 14,8% για το MAGE A3, 18,5% για το HER-2

Detection and Molecular Characterization of Circulating Tumour Cells: Challenges for the Clinical Setting

Over the last decade, liquid biopsy has gained much attention as a powerful tool in personalized medicine since it enables monitoring cancer evolution and follow-up of cancer patients in real time. Through minimally invasive procedures, liquid biopsy provides important information through the analysis of circulating tumour cells (CTCs) and circulating tumour-derived material, such as circulating tumour DNA (ctDNA), circulating miRNAs (cfmiRNAs) and extracellular vehicles (EVs). CTC analysis has already had an important impact on the prognosis, detection of minimal residual disease (MRD), treatment selection and monitoring of cancer patients. Numerous clinical trials nowadays include a liquid biopsy arm. CTC analysis is now an exponentially expanding field in almost all types of solid cancers. Functional studies, mainly based on CTC-derived cell-lines and CTC-derived explants (CDx), provide important insights into the metastatic process. The purpose of this review is to summarize the latest findings on the clinical significance of CTCs for the management of cancer patients, covering the last four years. This review focuses on providing a comprehensive overview of CTC analysis in breast, prostate and non-small-cell lung cancer. The unique potential of CTC single-cell analysis for understanding metastasis biology, and the importance of quality control and standardization of methodologies used in this field, is also discussed

RNA-Based CTC Analysis Provides Prognostic Information in Metastatic Breast Cancer

In metastatic breast cancer (MBC) the molecular characterization of circulating tumor cells (CTCs) provides a unique tool to understand metastasis-biology and therapy-resistance. We evaluated the prognostic significance of gene expression in EpCAM(+) CTCs in 46 MBC patients based on a long follow-up. We selected a panel consisting of stem cell markers (CD24, CD44, ALDH1), the mesenchymal marker TWIST1, receptors (ESR1, PGR, HER2, EGFR) and the epithelial marker CK-19. Singleplex RT-qPCR was used for TWIST1 and CK-19 and multiplex RT-qPCR for stem cell markers and receptors. A group of 19 healthy donors (HD) was used as control. Univariate (p = 0.001) and multivariate analysis (p = 0.002) revealed the prognostic value of combined gene expression of CK-19(+), CD44high/CD24low, ALDH1high/CD24low and HER2 over-expression for overall survival (OS). The Kaplan–Meier estimates of OS were significantly different in patients positive for CK-19 (p = 0.028), CD44high/CD24low (p = 0.002), ALDH1high/CD24low (p = 0.007) and HER2-positive (p = 0.022). Our results indicate that combined gene expression analysis in EpCAM(+) CTCs provides prognostic information in MBC

Prognostic Significance of TWIST1, CD24, CD44, and ALDH1 Transcript Quantification in EpCAM-Positive Circulating Tumor Cells from Early Stage Breast Cancer Patients

(1) Background: The aim of the study was to evaluate the prognostic significance of EMT-associated (TWIST1) and stem-cell (SC) transcript (CD24, CD44, ALDH1) quantification in EpCAM+ circulating tumor cells (CTCs) of early breast cancer patients. (2) Methods: 100 early stage breast cancer patients and 19 healthy donors were enrolled in the study. CD24, CD44, and ALDH1 transcripts of EpCAM(+) cells were quantified using a novel highly sensitive and specific quadraplex RT-qPCR, while TWIST1 transcripts were quantified by single RT-qPCR. All patients were followed up for more than 5 years. (3) Results: A significant positive correlation between overexpression of TWIST1 and CD24(-flow)/CD44(high) profile was found. Kaplan-Meier analysis revealed that the ER/PR-negative (HR-) patients and those patients with more than 3 positive lymph nodes that overexpressed TWIST1 in EpCAM(+) cells had a significant lower DFI (log rank test; p &lt; 0.001, p &lt; 0.001) and OS (log rank test; p = 0.006, p &lt; 0.001). Univariate and multivariate analysis also revealed the prognostic value of TWIST1 overexpression and CD24(-low)/CD44high and CD24(-flow)/ALDH1(high) profile for both DFI and OS. (4) Conclusions: Detection of TWIST1 overexpression and stem-cell (CD24, CD44, ALDH1) transcripts in EpCAM(+) CTCs provides prognostic information in early stage breast cancer patients

Molecular characterization of circulating tumor cells in breast cancer by liquid bead array hybridization assay

BACKGROUND: Molecular characterization of circulating tumor cells (CTCs) is crucial to identify novel diagnostic and therapeutic targets for individualized therapies. We developed a multiplexed PCR-coupled liquid bead array to detect the expression of multiple genes in CTCs. METHODS: mRNA isolated from immunomagnetically enriched CTCs was subjected to multiplex PCR for KRT19 (keratin 19; also known as CK19), ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian); also known as HER2], SCGB2A2 (secretoglobin, family 2A, member 2; also known as MGB1, mammaglobin A), MAGEA3 (melanoma antigen family A, 3), TWIST-1 [twist homolog 1 (Drosophila)], and HMBS (hydroxymethylbilane synthase; also known as PBGD). Biotinylated amplicons were hybridized against fluorescent microspheres carrying gene-specific capture probes and incubated with streptavidin-phycoerythrin. Wequantified the captured labeled amplicons and decoded the beads by Luminex flow cytometry. The assay was validated for limit of detection, specificity, and comparison with reverse-transcription quantitative PCR (RT-qPCR), and its clinical performance was evaluated in 64 patients with operable breast cancer, 20 patients with metastasis, and 17 healthy individuals. RESULTS: The assay was specific for each gene in complex multiplexed formats and could detect the expression of each gene at the level of a single SK-BR-3 cell. The assay produced results comparable to those for RT-qPCR for each gene. None of the genes tested was detected in the CTC fraction of healthy donors. We detected KRT19, ERBB2, MAGEA3, SCGB2A2, and TWIST1 in 26.6%, 12.5%, 18.7%, 10.9%, and 31.2% of operable breast cancer patients, respectively, and detected the corresponding genes in 65%, 20%, 30%, 20%, and 20% of patients with verified metastasis, respectively. CONCLUSIONS: The expression of 6 genes in CTCs can be measured simultaneously and reliably, thereby saving precious sample and reducing the costs and time of analysis. © 2010 American Association for Clinical Chemistry