Detection of mammaglobin expressing cells in blood of breast cancer patients
Introduction
A tumor arises as a consequence of multiple genomic alterations in individual cells. Subsequent tumor metastasis is an active multi-step process including neovascularization/angiogenesis of the tumor, local invasion of the extracellular matrix, into lymphatics or vasculature, dissemination of cells in the circulation and extravasation into secondary organ tissue [1]. Since even small tumors show dissemination of cells their disclosure could be a tool to improve early cancer detection. Furthermore after surgical removal of the primary tumor, disseminated cells remain in the lymphatic/systemic circulation. This, by conventional clinical measures undetected cell dissemination is ultimately responsible for the later recurrence of disease [2]. Consequently the presence of disseminated cancer cells is of relevance for cancer progression and their molecular characterization will improve prognostication, therapy selection and monitoring of disease [3], [4].
Numerous attempts have been made to characterize minimal residual cancer cells (MRCC) at the mRNA level. Reverse transcription-polymerase chain reaction (RT-PCR) provides the necessary analytical sensitivity to detect up to one cancer cell in 107 normal cells [5]. For the detection of MRCC in blood of patients with breast cancer a specific marker mRNA is still missing. Monitoring of the expression of epidermal growth factor receptor (EGFR) [6], erbB-2 [7], prostate-specific antigen (PSA) [8] or maspin [9] as well as of CEA, CK19, CK20, β-HCG and progesterone receptor [4] is hampered in general by insufficient sensitivity and/or specificity.
In 1996, the cDNA for a novel gene termed human mammaglobin (hMAM) was isolated [10]. The hMAM gene is localized to chromosome 11q13 [11]. Mammaglobin encodes a glycoprotein and is distantly related to a family of epithelial secretory proteins. The amino acid sequence of hMAM exhibits homology to the uteroglobin gene family, but its cellular function is not known yet. Watson et al. [10] reported that the expression of hMAM is restricted to the adult mammary gland and to mammary tumor cell lines. In an analysis of breast tumor biopsies, hMAM mRNA levels were increased in 23% of the cases. Zach et al. [12] reported in a study of peripheral blood of breast cancer patients hMAM mRNA was frequently up-regulated, too. Mammaglobin expression among breast tumors did not appear to correlate with histological type, tumor grade, tumor stage or hormone receptor status. Expression of mammaglobin was undetectable in human uterus, lung, ovary and placenta. This finding supports the hypothesis that the mammaglobin expressing cells are a subclass of tumor cells expressing and metastasizing only in few specific tissues. Furthermore some authors [13], [14], [15] reported that lymph nodes from patients with metastatic breast cancer contained detectable mammaglobin mRNA whereas hMAM expression in uninvolved lymph nodes was not detectable.
We have evaluated a novel quantitative RT-PCR assay based on TaqMan fluorescence methodology to quantify the hMAM expression in peripheral blood of breast cancer patients as well as in several cell lines derived from various types of cancer. We determined the sensitivity and specificity of mammaglobin mRNA expression to evaluate the advantage of mammaglobin as tumor indicating parameter.
Section snippets
Cell lines
The cell lines were cultured in RPMI 1640 (LIFE Technologies, Germany) culture medium (90%), containing 10% fetal bovine serum without antibiotics at 37°C with 5% CO2 in atmosphere. The culture medium was replaced once a week. Cultured cells were harvested according to American Type Culture Collection (ATCC) guide lines. Cells were pelleted (400×g, 10 min), washed two times with culture medium and resuspended in 10 ml phosphate–buffered saline (PBS) to define the amount of living cells by
Specificity of hMAM expression in cell lines of various origin
As a first step we investigated the hMAM mRNA expression of cell lines derived from various cancer types. The quantification of the starting copy number in an unknown sample was performed by preparing a standard curve using serial dilutions of a known amount of standard cDNA of the cell line EFM-192. Standards were used to calculate the amount of mRNA denoted as cell equivalents (CEQ). The ratios of cell equivalents of the hMAM and the house keeping gene GAPDH is to give the relative parameter
Discussion
RT-PCR allows the specific detection of tumor cells at the mRNA level in secondary sites like peripheral blood [5], [18] or bone marrow [19] with an analytical sensitivity of one tumor cell measurable in a background of 107 normal cells without the need of tumor cell enrichment or purification prior to analysis. In contrast to other tumors, specific marker genes for the detection of disseminated cancer cells in blood of breast cancer patients are not yet available. Most tumor or epithelium
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