Detection of Tumor Cell-Specific mRNA in the Peripheral Blood of Patients with Breast Cancer-Evaluation of Several Markers with Real-Time Reverse Transcription-PCR

It is widely known that cells from epithelial tumors, e. g., breast
cancer, detach from their primary tissue and enter blood
circulation. We show that the presence of circulating tumor cells
(CTCs) in samples of patients with primary and metastatic breast
cancer can be detected with an array of selected tumor-marker-genes
by reverse transcription real-time PCR. The focus of the presented
work is on detecting differences in gene expression between healthy
individuals and adjuvant and metastatic breast cancer patients, not
an accurate quantification of these differences. Therefore, total
RNA was isolated from blood samples of healthy donors and patients
with primary or metastatic breast cancer after enrichment of
mononuclear cells by density gradient centrifugation. After reverse
transcription real-time PCR was carried out with a set of marker
genes (BCSP, CK8, Her2, MGL, CK18, CK19). B2M and GAPDH were used
as reference genes. Blood samples from patients with metastatic
disease revealed increased cytokine gene levels in comparison to
normal blood samples. Detection of a single gene was not sufficient
to detect CTCs by reverse transcription real-time PCR. Markers used
here were selected based on a recent study detecting cancer cells
on different protein levels. The combination of such a marker array
leads to higher and more specific discovery rates, predominantly in
metastatic patients. Identification of CTCs by PCR methods may lead
to better diagnosis and prognosis and could help to choose an
adequate therapy.