Molecular detection of breast cancer cells in the peripheral blood of advanced-stage breast cancer patients using multimarker real-time reverse transcription-polymerase chain reaction and a novel porous barrier density gradient centrifugation technology.
OBJECTIVE
The goal of this study was to develop a molecular diagnostic assay to detect circulating breast cancer cells in the peripheral blood for the purpose of staging breast cancer. Our aim was to make available an assay that was not limited by the low concentration of circulating breast cancer cells and the background gene expression that is typically found in peripheral blood.


METHODS
In this study, we investigated the ability of two new technologies to significantly enhance the quantification of gene expression in the peripheral blood: enrichment by a novel porous barrier density gradient centrifugation technology; and multimarker real-time reverse transcription-PCR (RT-PCR).


RESULTS
Using fluorescence-labeled breast cancer cells and flow cytometry, we show that processing peripheral blood by porous barrier density gradient centrifugation results in a 300-fold enrichment of breast cancer cells. Real-time RT-PCR analysis confirmed a concomitant reduction in background expression of the CK19 and MUC1 genes after enrichment. In a pilot study, porous barrier density gradient centrifugation and multimarker real-time RT-PCR enabled our laboratory to detect breast cancer-associated gene overexpression in 13 of 20 (65%) stage IV breast cancer patients. Nine of these 14 patients overexpressed three or more markers.


CONCLUSIONS
These results confirm the promise of such a molecular diagnostic assay and suggest that additional studies are needed to precisely define the clinical relevance.
Department of Surgery, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Megan K
MK
Baker
Kaidi
K
Mikhitarian
Walid
W
Osta
Kathi
K
Callahan
Rana
R
Hoda
Frank
F
Brescia
Rayna
R
Kneuper-Hall
Michael
M
Mitas
David J
DJ
Cole
William E
WE
Gillanders
D014408:Biomarkers, Tumor; D001943:Breast Neoplasms / Q000097:blood* / Q000175:diagnosis* / Q000235:genetics*; D045744:Cell Line, Tumor; D002499:Centrifugation, Density Gradient; D018076:DNA, Complementary / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D009360:Neoplastic Cells, Circulating; D010865:Pilot Projects; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D014408:Biomarkers, Tumor; D018076:DNA, Complementary; D012333:RNA, Messenger