Real‐time quantitative PCR determination of urokinase‐type plasminogen activator receptor (uPAR) expression of isolated micrometastatic cells from bone marrow of breast cancer patients

• Published in: International journal of cancer Vol. 114; no. 2; pp. 291 - 298
• Main Authors: Pierga, Jean‐Yves, Bonneton, Charlyne, Magdelénat, Henri, Vincent‐Salomon, Anne, Nos, Claude, Boudou, Evelyne, Pouillart, Pierre, Thiery, Jean‐Paul, de Cremoux, Patricia
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 20-03-2005; Wiley-Liss
• Subjects: Base Sequence; Biological and medical sciences; Bone Marrow - pathology; breast cancer; Breast Neoplasms - classification; Breast Neoplasms - genetics; Breast Neoplasms - pathology; DNA Primers; Female; Gene Expression Regulation, Neoplastic; Gynecology. Andrology. Obstetrics; Humans; Mammary gland diseases; Medical sciences; micrometastasis; Neoplasm Metastasis - genetics; Neoplasm Staging; Polymerase Chain Reaction - methods; real‐time PCR; Receptors, Cell Surface - genetics; Receptors, Estrogen - analysis; Receptors, Progesterone - analysis; Receptors, Urokinase Plasminogen Activator; Risk Assessment; RNA, Messenger - genetics; Tumors; urokinase‐type plasminogen activator receptor; Human; u-Plasminogen activator; Serine endopeptidases; Enzyme; Breast cancer; Malignant tumor; Metastasis; Gene expression; Real time; Bone marrow disease; Polymerase chain reaction; Mammary gland diseases; Peptidases; Cancerology; Isolated cell; Micrometastasis; Bone marrow; Genetics; Hydrolases; Molecular biology; Quantitative analysis; Biological receptor
• ISSN: 0020-7136; 1097-0215
• DOI: 10.1002/ijc.20698

Disseminated tumor cells (DTC) in bone marrow are independently related to poor outcome in patients with breast cancer. Phenotypic characterization of DTC may be useful to improve evaluation of the metastasizing potential of DTC and also to more accurately target aggressive tumor cells. DTC were screened in bone marrow aspirates from breast cancer patients by immunocytochemistry with an anticytokeratin (anti‐CK) antibody (A45B/B3). Because the cell permeabilization and fixation required for intracellular CK staining is deleterious for mRNA, we used microaspiration to isolate single tumor cells stained with a monoclonal antibody directed against a membrane epitope, epithelial cell adhesion molecule (EpCAM), in CK‐positive cases. Urokinase‐type plasminogen activator receptor (uPAR) was quantified by real‐time quantitative RT‐PCR. The SKBR3 human breast cancer cell line was used to calibrate RT‐PCR. A linear relationship was observed between the cycle threshold (Ct) of uPAR and 18S gene expression and SKBR3 cells spiked (1, 3, 7, 10 and 20) in control patient bone marrow. EpCAM‐positive cells were aspirated in 21 out of 25 bone marrow specimens from breast cancer patients with CK‐positive cells and uPAR mRNA expression was determined in 16 cases. A high level of uPAR mRNA in DTC was detected in 8 out of 16 patients (50%) and was associated with a more aggressive primary tumor phenotype (estrogen receptor [ER]‐negative, progesterone receptor [PR]‐negative or HER2‐positive) (p = 0.01). We demonstrated that real‐time quantitative RT‐PCR was reliably adapted to phenotype analysis of isolated micrometastatic cells. A larger study would be useful to confirm the importance of uPAR to define higher risk subgroups of breast cancer patients with micrometastatic disease. © 2004 Wiley‐Liss, Inc.