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Identification of circulating tumour cells in early stage breast cancer patients using multi marker immunobead RT-PCR.

ABSTRACT: INTRODUCTION: The ability to screen blood of early stage operable breast cancer patients for circulating tumour cells is of potential importance for identifying patients at risk of developing distant relapse. We present the results of a study of the efficacy of the immunobead RT-PCR method in identifying patients with circulating tumour cells. RESULTS: Immunomagnetic enrichment of circulating tumour cells followed by RT-PCR (immunobead RT-PCR) with a panel of five epithelial specific markers (ELF3, EPHB4, EGFR, MGB1 and TACSTD1) was used to screen for circulating tumour cells in the peripheral blood of 56 breast cancer patients. Twenty patients were positive for two or more RT-PCR markers, including seven patients who were node negative by conventional techniques. Significant increases in the frequency of marker positivity was seen in lymph node positive patients, in patients with high grade tumours and in patients with lymphovascular invasion. A strong trend towards improved disease free survival was seen for marker negative patients although it did not reach significance (p = 0.08). CONCLUSION: Multi-marker immunobead RT-PCR analysis of peripheral blood is a robust assay that is capable of detecting circulating tumour cells in early stage breast cancer patients.

SUBMITTER: Raynor MP

PROVIDER: S-EPMC2712470 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Identification of circulating tumour cells in early stage breast cancer patients using multi marker immunobead RT-PCR.

Raynor Michael P MP Stephenson Sally-Anne SA Pittman Kenneth B KB Walsh David C A DC Henderson Michael A MA Dobrovic Alexander A

Journal of hematology & oncology 20090605

<h4>Introduction</h4>The ability to screen blood of early stage operable breast cancer patients for circulating tumour cells is of potential importance for identifying patients at risk of developing distant relapse. We present the results of a study of the efficacy of the immunobead RT-PCR method in identifying patients with circulating tumour cells.<h4>Results</h4>Immunomagnetic enrichment of circulating tumour cells followed by RT-PCR (immunobead RT-PCR) with a panel of five epithelial specifi ...[more]

PMID: 19500345

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Project description:Purpose: There is a quest for novel non-invasive diagnostic markers for the detection of breast cancer. The goal of this study is to identify circulating microRNA signatures using a cohort of Asian Chinese breast cancer patients, and to compare microRNA profiles between tumour and serum samples. Experimental design: MicroRNAs from paired breast cancer tumours, normal tissue and serum samples derived from 32 patients were comprehensively profiled using microarrays (1300 microRNAs against tumour and normal tissues) or LNA RT-PCR panels (742 microRNAs against serum samples). Serum samples from healthy individuals (n=22) were also employed as normal controls. Significant serum microRNAs, identified by logistic regression, were validated in an independent set of serum samples from patients (n=82) and healthy controls (n=53). Results: The 20 most significant microRNAs differentially expressed in breast cancer tumours included miR-21, miR-10b, and miR-145, previously shown to be dysregulated in breast cancer. Interestingly, 16 of the 20 most significant microRNAs differentially expressed in serum samples were novel. MiR-1, miR-92a, miR-133a and miR-133b were identified as the most important diagnostic markers, and were successfully validated; receiver operating characteristic curves derived from combinations of these microRNAs exhibited areas under the curves of 0.944-0.946. Only seven microRNAs were overexpressed in both tumours and serum, suggesting that microRNAs may be released into the serum selectively. Conclusion: The clinical employment of microRNA signatures as a non-invasive diagnostic strategy is promising, but should be further validated for different subtypes of breast cancers. Blood samples were collected in Becton Dickinson (Franklin Lakes, NJ) Vacutainer SST tubes. Serum was harvested by centrifugation at 2200g after allowing blood to clot for 30mins. 32 patient samples and 22 samples from healthy controls were obtained for profiling. Sera samples were stored at -80oC.

Project description:There are currently no highly sensitive and specific minimally invasive biomarkers for detection of early-stage breast cancer. MicroRNAs (miRNAs) are present in the circulation and may be unique biomarkers for early diagnosis of human cancers. The aim of this study was to investigate the differential expression of miRNAs in the serum of breast cancer patients and healthy controls.Global miRNA analysis was performed on serum from 48 patients with ER-positive early-stage breast cancer obtained at diagnosis (24 lymph node-positive and 24 lymph node-negative) and 24 age-matched healthy controls using LNA-based quantitative real-time PCR (qRT-PCR). A signature of miRNAs was subsequently validated in an independent set of 111 serum samples from 60 patients with early-stage breast cancer and 51 healthy controls and further tested for reproducibility in 3 independent data sets from the GEO Database.A multivariable signature consisting of 9 miRNAs (miR-15a, miR-18a, miR-107, miR-133a, miR-139-5p, miR-143, miR-145, miR-365, miR-425) was identified that provided considerable discrimination between breast cancer patients and healthy controls. Further, the ability of the 9 miRNA signature to stratify samples from breast cancer patients and healthy controls was confirmed in the validation set (p = 0.012) with a corresponding AUC = 0.665 in the ROC-curve analysis. No association between miRNA expression and tumor grade, tumor size, menopausal- or lymph node status was observed. The signature was also successfully validated in a previously published independent data set of circulating miRNAs in early-stage breast cancer (p = 0.024).We present herein a 9 miRNA signature capable of discriminating between ER-positive breast cancer and healthy controls. Using a specific algorithm based on the 9 miRNA signature, the risk for future individuals can be predicted. Since microRNAs are highly stable in blood components, this signature might be useful in the development of a blood-based multi-marker test to improve early detection of breast cancer. Such a test could potentially be used as a screening tool to identify individuals who would benefit from further diagnostic assessment.

Dataset Information

Identification of circulating tumour cells in early stage breast cancer patients using multi marker immunobead RT-PCR.

Publications

Identification of circulating tumour cells in early stage breast cancer patients using multi marker immunobead RT-PCR.

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