Worldwide, more than two million women are diagnosed with breast, cervical, endometrial or ovarian cancer each year. These cancers contribute to 45% of total female malignancies and approximately 880000 cancer related deaths annually [
1]. Although several improvements have been made in early diagnosis during the past few decades, many patients still die of visceral metastasis, which is the main cause for tumor-related death. In these patients, the hematogenous spread of malignant cells remains undetected at the time of initial therapy. Since T. R. Ashworth first reported circulating tumor cells (CTC) in the blood of cancer patients in 1869 [
2], the presence of CTC has been described for several solid tumors, such as colorectal, lung, kidney, squamous oesophageal, liver, prostate and pancreatic cancer [
3]. Among cancers specific to women, the majority of CTC based research has been performed in breast cancer patients (reviewed in [
3‐
6]), whereas few data exist for CTC in ovarian [
7,
8], cervical [
9], and endometrial cancer [
10,
11] patients. Recent studies have demonstrated the prognostic role of CTC [
12‐
14]; and the presence of tumor cells in the peripheral blood was considered to be established as an additional staging parameter [
15]. Hence, many efforts have been made to develop reliable procedures for the sensitive and specific detection of CTC, either at the protein level (antibody-based cell staining) or at the mRNA level (reverse transcription PCR). While the first approach is the gold standard technique for the detection of tumor cells in the bone marrow of breast cancer patients, the latter has been proven to be more sensitive and amenable to high-throughput analysis [
6]. Nevertheless, the detection of CTC is often hampered by the heterogeneity of the primary tumor and by the loss of epithelial antigens as occurs during epithelial to mesenchymal transition [
3]. It has been shown that normal-like breast cancer cells characterized by aggressive behaviour and worse treatment options are not recognized by the CellSearch circulating tumor cell test (Veridex LLC, San Diego, CA), which uses EpCAM for cell isolation [
16]. This test is the only diagnostic test that is currently approved by the US Food and Drug Administration for the automated detection and enumeration of circulating tumor cells [
17].
EpCAM (epithelial cell adhesion molecule) is not a perfect marker for CTC detection due to the high variation in its gene expression between tumor subtypes and its illegitimate transcription from leukocytes [
18],. Likewise, the analysis of
hMAM (human mammaglobin A), the most widely studied marker after
CK19 (cytokeratin 19) in breast cancer patients, gene expression identifies patients with nearly 100% specificity at the same sensitivity as
CK19 (1 tumor cell in 10
6 peripheral blood mononuclear cells) [
19,
20]. Nevertheless, mammaglobin A gene expression is highly variable in female cancers and is detected in the blood of approximately 10 to 30% of breast cancer patients [
21]. Hence, there is a high scientific and clinical need for the identification of new markers for the detection of CTC.
In this study, we aimed to identify new gene markers for the RT-qPCR based detection of CTC in the blood of female cancer patients following a step-down strategy utilizing a whole genome analysis with oligonucleotide microarrays (Applied Biosystems) and TaqMan® Low Density Array (TLDA) based RT-qPCR using microfluidic technology. Based on the results of these experiments, a panel of six candidate gene markers was selected for future routine diagnosis of circulating tumor cells.