| Backgroud and ObjectiveEarly detection of breast cancer can reduce the morbidity and mortality of breast cancer patients . Breast cancer mortality occurs in a substantial portion of breast cancer patients and is increasingly linked to early metastases, which are often occult at the time of primary diagnosis. Because undetected micrometastases can contribute to the failure of primary treatment, their identification in patients with early breast cancer relapse may have a substantial impact on prognosis and treatment choice for these patients. It is very convenient for collecting peripheral blood samples to detect occult circulating peripheral tumor cells, which is more favourable for clinical monitoring consecutively. Thus, improved direct identification of these occult metastases in blood offers a critical opportunity to optimize management of breast cancer patients.Microsphere-based suspension array technologies, such as the Luminex xMAP system, offer a new platform for high-throughput nucleic acid detection which is being used in a variety of applications. Some benefits of suspension array technology include rapid data acquisition, excellent sensitivity and specificity, and multiplexed analysis capability. As compared to planar microarrays, suspension arrays have the benefits of ease of use, low cost, statistical superiority, faster hybridization kinetics and more flexibility in array preparation.So we want to develop a Suspension array assay for the detection of marker genes expression for human circulating breast cancer cells ,in order to analyze the independent prognostic predictors of breast cancer metastasis.Methods1. The Primer 5.0 software is used to design the primers and probes sequences for marker genes for human circulating breast cancer cells.2. In order to prepare Luminex hybridization liquid, the specific probes was used to coat Luminex beads.3. The human MCF7 cells is used as a model to develop a Suspension array assay for the detection of marker genes expression for human circulating breast cancer cells.4. Then our suspension array is used to examine the multigene expression in PBMC of 73 breast cancer patients,including the breast cancer relapse patients.5. COX proportional hazard modle was used to find the independent prognostic predictors of breast cancer relapse.ResultsWe develop successfully a suspension array assay for the detection of marker genes expression for human circulating breast cancer cells (including hMAM,HER2,CK19,SBEM,EPG2,hTERT,β-HCG and B305D) ,and the detection limit of this assay is to detect 50 MCF7 tumor cells per milliliter peripheral blood. Resolution of the mix of amplified products was achieved by PCR product hybridization to corresponding probe sequences, attached to unique sets of fluorescent beads. The assay was optimized to allow for maximum sensitivity in a multiplexed format. And there were no false-positive results or false-negative results being detected. Gene expression analysis of cells collected from blood of 73 women with breast cancer was performed with the multigene RT-PCR and suspension array assay. hMAM expression was detected in 58%, HER2 in 58%, CK19 in 53%, SBEM in 52%, EPG2 in 31%, hTERT in 26%,β-HCG in 22% and B305D in 15% of the blood samples, respectively. But detection in peripheral blood of any of the eight marker genes included in the multigene assay is not significantly associated with increasing age, tumor size and tumor type.And we discover that hMAM, SBEM, HER2 and ER could be considered as the independent prognostic predictors of breast cancer relapse by using Cox PH model, and the Relative hazard(HR) of hMAM, SBEM, HER2 and ER is 2.66,2.88,2.63 and 0.43, respectively.ConclusionThe sensitivity and specificity of the suspension array assay for the detection of marker genes expression for human circulating breast cancer cells are rather high, and the characterization of circulating tumor cells based on the detection of breast-specific marker genes with a suspension array assay has good potential for early detection of early detection of breast cancer relapse. And the expression of hMAM,SBEM and HER2 in peripheral blood can be considered as the independent prognostic predictors of breast cancer metastasis.
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