Abstract. Urokinase plasminogen activator (uPA) and its main inhibitor, plasminogen activator inhibitor type-1. (PAI-1) determined in tumor tissue by means of ...
Anatomic Pathology / UPA AND PAI-1 MRNA EXPRESSION IN BREAST TUMORS
Urokinase-type Plasminogen Activator and Plasminogen Activator Inhibitor Type-1 mRNA Assessment in Breast Cancer by Means of NASBA Correlation With Protein Expression Pierre-Jean Lamy, PharmD, PhD,1* Thibault Verjat, PhD,2* Anne-Claire Servanton, MS,1 Malik Paye, MS,2 Philippe Leissner, PhD,2 and Bruno Mougin, PharmD, PhD2 Key Words: Breast cancer; Urokinase plasminogen activator; Plasminogen activator inhibitor 1; Molecular diagnostics; Prognostic factors; Tumor marker; Nucleic acid sequence–based amplification; NASBA; ELISA; Quantitative PCR DOI: 10.1309/K4JAF2NMD5EJU67Y
Abstract Urokinase plasminogen activator (uPA) and its main inhibitor, plasminogen activator inhibitor type-1 (PAI-1) determined in tumor tissue by means of enzymelinked immunosorbent assay (ELISA) can discriminate patients with primary breast cancer at high risk vs low risk for recurrence. The aim of this study was to analyze uPA and PAI-1 messenger RNA (mRNA) expression by means of quantitative nucleic acid sequence–based amplification (NASBA) on 77 primary breast tumor samples and to correlate this expression with the uPA and PAI-1 protein content. We observed that the 2 markers were significantly overexpressed (uPA, P < .0001; PAI-1, P = .0042) in mRNA in the ELISA+ group. The receiver operating characteristic (ROC) curves demonstrated high concordance between NASBA and ELISA (area under the ROC curve of 0.84 and 0.70 for uPA and PAI-1, respectively) and showed that uPA and PAI-1 status could be predicted by using the molecular assay with sensitivity and specificity values of 80.8% and 82.4% and sensitivity and specificity values of 66.7% and 74.0%, respectively.
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Am J Clin Pathol 2007;128:404-413 DOI: 10.1309/K4JAF2NMD5EJU67Y
Malignant tumors can degrade the extracellular matrix, and this breakdown is crucial for cancer invasion and metastasis. These processes involve a complex interplay between tumor cells and nonmalignant stromal cells. It is now well known that proteolytic enzymes such as matrix metalloproteinases, cysteine proteases, and serine proteases have an important role in cancer metastasis.1 One important component of the proteolytic system is the urokinase-type plasminogen activator system, which consists of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), and uPA inhibitors 1 and 2 (PAI-1 and PAI-2).2 uPA is a 50-kd serine protease synthesized by fibroblasts, monocytes, neutrophils, epithelial cells, and tumor cells involved in tissue-remodeling processes. uPA-mediated proteolysis is regulated at several levels, particularly by PAI-1, which belongs to the serpin superfamily. Their exact roles in invasion and metastasis are not fully understood, and questions remain about cellular distribution of the uPA system molecular components. Besides proteolysis, new mechanisms whereby the uPA system could promote cancer progression have been described. Studies have shown that uPA expression is up-regulated by some growth factors3 and maintains an elevated basal level of activated ERK that could inhibit apoptosis.4 The paradoxical role of PAI-1 could be explained by its ability to interact with uPAR, integrins, and vitronectin, thereby controlling cell migration. It is now well known that high levels of uPA and PAI-1 are associated with a poor prognosis in breast cancer, in particular, node-negative breast cancer.5-9 The prognostic value of these markers has been validated by means of a European Organization for Research and Treatment of Cancer Receptor and Biomarker Group meta-analysis comprising more than 8,300 patients with breast cancer.10 Thus, uPA and PAI-1 were © American Society for Clinical Pathology
Anatomic Pathology / ORIGINAL ARTICLE
the first novel tumor biologic factors to be validated at the highest level of evidence (level 1) with regard to their clinical usefulness in breast cancer. These markers can now be considered for routine assessment of prognosis in patients with newly diagnosed breast cancer. The status of uPA and PAI-1 has most commonly been determined by means of an immunometric assay (enzyme-linked immunosorbent assay [ELISA]) in primary tumor tissue extracts. Several standardized ELISA methods, such as the Femtelle assay (American Diagnostica, Stanford, CT), that are robust enough for clinical routine determination are commercially available. Most of the prognostic studies referring to uPA and PAI-1 protein levels that have been published have used the ELISA method.5,7,8,11-20 Real-time polymerase chain reaction (PCR) assays allow convenient quantification of target messenger RNA (mRNA) transcripts and quantification of target-derived nucleic acids in clinical specimens such as breast tumors.21 The study of uPA and PAI-1 expression in mRNA by means of reverse-transcription (RT)–quantitative PCR (qPCR) analysis, easily applied to very small samples, could constitute an alternative to ELISA, a method limited by the need for milligram quantities of frozen tissue. Significant correlation between mRNA quantified by RT-qPCR and protein has been observed for uPA and PAI-1.22-24 Nucleic acid sequence–based amplification (NASBA) is an efficient and specific method for amplifying mRNA with detection performed in real time using molecular beacons.25 A NASBA-based assay simultaneously measuring the mRNA levels of estrogen receptor α (ERα), progesterone receptor (PR), and the HER-2 gene in primary breast tumor specimens has been reported by our group.26 To complete this menu with markers of interest in breast cancer according to the recommendations of the European Group on Tumor Markers,27 we have developed, for the first time, 2 duplex real-time NASBA assays to study the level of mRNA coding for uPA and PAI-1 with cyclophilin B (PPIB) as the normalizing gene, whose expression has been shown to be stable in breast cancer cell lines and tumors.28 We report the results of a study comparing the uPA and PAI-1 mRNA expression levels assessed by means of real-time NASBA with the cognate protein content measured by means of the reference ELISA Femtelle method with a panel of cell lines and 77 breast tumor samples.
Materials and Methods Tumor Samples Tumor samples from 77 patients with primary breast cancer were collected at the Centre Regional de Lutte contre le Cancer, Montpellier, France. Clinicopathologic characteristics are summarized in ❚Table 1❚. Tumor samples obtained during
❚Table 1❚ Patient and Tumor Clinicopathologic Characteristics Characteristic
No. of Cases
Age (y)
