The development of distant metastases is the primary cause of death in patients with breast cancer. Although the presence of involved lymph nodes is a negative prognostic factor, it remains impossible to reliably identify those patients who will eventually relapse with metastatic disease only by assessing the lymph node status. Advances in the development of immunocytochemical and molecular assays now enable detection of metastatic cells even at a single cell stage and thus carry the promise to positively identify those patients with early microscopic distant dissemination.
To detect dissemniated epithelial cells (DEC) in blood and bone marrow (BM) of patients with breast cancer, we have developed a sensitive real time reverse transcriptase polymerase chain reaction (RT-PCR). The amplification of specific mRNA markers relies on the screening for tumour associated and/or organ specific markers: genes that are expressed by disseminated tumour cells but are absent on the surrounding normal cells. Cytokeratins are widely evaluated as targets for detection of DEC. Mammaglobin, a member of the uteroglobulin gene family, is reported to be exclusively expressed in mammary epithelium, making it also a potentially useful RT-PCR marker. To detect DEC in blood and bone marrow of patients with breast cancer we quantify cytokeratin-19 (CK-19) and human mammaglobin (MAM) expression using a quantitative real time RT-PCR method. All PCR reactions are performed on the ABI Prism 7700 Sequence Detection System (Applied Biosystems) using the fluorescent Taqman methodology. The sensitivity and specificity of the RT-PCR method is compared to immunocytochemistry (ICC), which is regarded as the standard method to detect DEC in blood and bone marrow. Immunostaining to detect cytokeratin-positive cells in the bone marrow of patients with breast cancer is done with the Epimet-kit (Micromet AG, Germany) on an automatic immunostainer (Autostainer, Dako).
Large prospective studies have
demonstrated the clinical importance of DEC in BM representing an
independent predictive and prognostic factor for distant relaps and
overall survival in nonmetastatic patients with breast cancer.
Although bone marrow aspiration is an acknowledged clinical method,
it remains cumbersome, especially if repetitive examinations are
necessary. Peripheral blood is the second best organ to evaluate a
patient for the presence of disseminated epithelial cancer cells,
although tumour cells in blood and tumour cells in bone marrow do
not necessarily have the same metastatic potential. Also repetitive
sampling of peripheral blood is more feasible.
The aim of our study is:
1. to prove a stochastic correlation between the molecular method
and the ICC-based quantification of DEC in bone marrow of patients
with breast cancer;
2. to compare results from blood and bone marrow concerning the
utility for detection of DEC.
Furthermore, clinico-pathological parameters, e.g. TNM stage,
hormone receptor status, differentiation grade, menopausal status,
and others are recorded and associations with presence or absence
of DEC in blood and bone marrow are studied. The prognostic
importance of DEC in blood and bone marrow will be investigated.
The study protocol is approved by the ethical committees of the
Faculty of Medicine, University of Antwerp, and of the General
Hospital Sint-Augustinus. All patients and volunteers sign a
written informed consent.
Relevant publications:
- Benoy IH, Elst H, Van Dam P, Scharpe S, Van Marck E, Vermeulen PB, Dirix LY. Detection of circulating tumour cells in blood by quantitative real-time RT-PCR: effect of pre-analytical time. Clin Chem Lab Med. 2006;44(9):1082-7.
- Benoy IH, Elst H, Philips M, Wuyts H, Van Dam P, Scharpe S, Van Marck E, Vermeulen PB, Dirix LY. Prognostic significance of disseminated tumor cells as detected by quantitative real-time reverse-transcriptase polymerase chain reaction in patients with breast cancer. Clin Breast Cancer. 2006 Jun;7(2):146-52.
- Benoy IH, Elst H, Philips M, Wuyts H, Van Dam P, Scharpe S, Van Marck E,Vermeulen PB, Dirix LY. Real-time RT-PCR detection of disseminated tumour cells in bone marrow has superior prognostic significance in comparison with circulating tumour cells in patients with breast cancer. Br J Cancer. 2006 Mar 13;94(5):672-680.
- Benoy I, Salgado R, Elst H, Van Dam P, Weyler J, Van Marck E, Scharpe S, Vermeulen P and Dirix L. Relative microvessel area of the primary tumour, and not lymph node status, predicts the presence of bone marrow micrometastases detected by reverse transcriptase polymerase chain reaction in patients with clinically non-metastatic breast cancer. Breast Cancer Res. Treat. 2005;7(2):R210-9. Epub 2005 Jan 10.
