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Journal of Clinical Oncology, Vol 24, No 26 (September 10), 2006: pp. 4245-4253 © 2006 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.05.1912 Increased Expression of Urokinase-Type Plasminogen Activator mRNA Determines Adverse Prognosis in ErbB2-Positive Primary Breast CancerFrom the Swiss Institute for Experimental Cancer Research, National Center of Competence in Research, Molecular Oncology; Swiss Institute of Bioinformatics; Epalinges s/Lausanne; OncoScore AG, Riehen; Department of Obstetrics and Gynaecology, University of Basel; Stiftung Tumorbank Basel, Riehen and University of Basel; Zentrum für Tumordiagnostik und Prävention, St Gallen, Switzerland; and the Buck Institute for Age Research, Novato, CA Address reprint requests to Patrick Urban, MD, Stiftung Tumorbank Basel, Lörracherstrasse 50, CH-4125 Riehen, Switzerland; e-mail: patrick.urban{at}unibas.ch
Purpose To evaluate and validate mRNA expression markers capable of identifying patients with ErbB2-positive breast cancer associated with distant metastasis and reduced survival. Patients and Methods Expression of 60 genes involved in breast cancer biology was assessed by quantitative real-time PCR (qrt-PCR) in 317 primary breast cancer patients and correlated with clinical outcome data. Results were validated subsequently using two previously published and publicly available microarray data sets with different patient populations comprising 295 and 286 breast cancer samples, respectively. Results Of the 60 genes measured by qrt-PCR, urokinase-type plasminogen activator (uPA or PLAU) mRNA expression was the most significant marker associated with distant metastasis-free survival (MFS) by univariate Cox analysis in patients with ErbB2-positive tumors and an independent factor in multivariate analysis. Subsequent validation in two microarray data sets confirmed the prognostic value of uPA in ErbB2-positive tumors by both univariate and multivariate analysis. uPA mRNA expression was not significantly associated with MFS in ErbB2-negative tumors. Kaplan-Meier analysis showed in all three study populations that patients with ErbB2-positive/uPA–positive tumors exhibited significantly reduced MFS (hazard ratios [HR], 4.3; 95% CI, 1.6 to 11.8; HR, 2.7; 95% CI, 1.2 to 6.2; and, HR, 2.8; 95% CI, 1.1 to 7.1; all P < .02) as compared with the group with ErbB2-positive/uPA–negative tumors who exhibited similar outcome to those with ErbB2-negative tumors, irrespective of uPA status. Conclusion After evaluation of 898 breast cancer patients, uPA mRNA expression emerged as a powerful prognostic indicator in ErbB2-positive tumors. These results were consistent among three independent study populations assayed by different techniques, including qrt-PCR and two microarray platforms.
The oncogenic receptor tyrosine kinase, ErbB2 (HER2/neu), has received major attention as a breast cancer biomarker and is mechanistically linked with a variety of malignant processes including dysregulated cell growth and proliferation, tumor angiogenesis, tissue invasion, and metastases (reviewed previously1-4). Overexpression of ErbB2 mRNA and protein resulting from the amplified oncogene is observed in 15% to 30% of all newly diagnosed breast cancer patients, in which it is associated with poor prognosis and serves as a predictor of clinical responsiveness to the anti-ErbB2 therapeutic trastuzumab (Herceptin; Genentech, South San Francisco, CA). Based on commonly used breast cancer risk assessment criteria, including the most recent St Gallen international expert consensus criteria, most patients with ErbB2-positive breast cancer are assigned to the highest risk category for metastatic relapse.5 However, not all patients with ErbB2-positive breast cancer develop nodal or distant metastases; moreover, the molecular mechanisms by which ErbB2 overexpression results in clinically more aggressive disease remain poorly understood. Therefore, identification of additional risk markers is needed to elucidate pathways responsible for metastatic relapse as well as to improve the risk classification of patients diagnosed with ErbB2-positive breast cancer. Recently, expression profiling has shown promise in being able to use breast cancer gene expression signatures to predict metastatic relapse.6-8 However, to date these studies have not focused on the identification of outcome predictors for patients newly diagnosed with ErbB2-positive breast cancer. To address this need, 60 candidate genes were selected for expression profiling based on their reported links to malignant cell behavior and breast cancer biology and their representation in all major cancer pathways, including cell proliferation and survival, invasiveness, angiogenic potential, and endocrine dependence (Table 1). All 60 candidates were assessed by quantitative real-time polymerase chain reaction (qrt-PCR) in a set of 317 primary breast cancer biopsies and their expression levels were correlated with clinical outcome data. Five candidate genes were identified whose expression levels showed significant univariate association with distant metastasis-free survival (MFS) in ErbB2-positive tumors. The most significant of these markers, urokinase-type plasminogen activator (uPA or PLAU), was further validated in two independent and previously reported breast cancer study populations for which gene expression (microarray) and patient outcome data are publicly accessible.6,7
Study Patients Fresh frozen tissue from primary breast cancer patients was obtained from the Stiftung Tumorbank Basel (STB, Basel, Switzerland). Exclusion criteria included neoadjuvant therapy, insufficient tumor content (< 50%), and poor RNA quality resulting in 317 eligible samples (STB study). All patients underwent surgery between 1992 and 1996. Quantitative protein levels of hormone receptors, ErbB2 and uPA, were prospectively determined at the time of surgery.9,10 Median patient age at diagnosis was 60 years (range, 27 to 88 years). Fifty-seven patients (18%) developed distant metastasis within a median MFS time of 44 months (range, 8 to 90 months). Forty-six percent of tumors were node-positive, 73% were estrogen-receptor (ER) –positive (> 20 fmol/mg protein by Enzyme Immuno Assay11), and 22% were ErbB2-positive (see Statistical Analysis). Systemic adjuvant hormone therapy was administrated to 43% of patients, chemotherapy to 22% of patients, and combination adjuvant therapy to 16% of patients; virtually all hormonal therapy was in the form of tamoxifen, with chemotherapy primarily consisting of cyclophosphamide, methotrexate, and fluorouracil and 9% of patients receiving anthracyclines. STB is an approved nonprofit laboratory that performs routine tumor biomarker measurements and clinical research under Swiss health regulatory office compliance with specific regard to ethical standards and patient confidentiality. For validation purposes, two additional breast cancer study populations were also analyzed using the publicly accessible microarray and outcome data sets reported by Van de Vijver et al (Amsterdam study; Agilent microarray platform; Agilent Technologies, Palo Alto, CA)6 and Wang et al (Rotterdam study; Affymetrix microarray platform; Affymetrix, Santa Clara, CA).7 Major differences in the three study populations included patient age, nodal status, and adjuvant treatments. MFS was the only outcome parameter commonly available for all three study populations. Comparative patient and tumor characteristics from the STB, Amsterdam, and Rotterdam study populations are summarized in Table 2.
RNA Extraction and Quantitative Real-Time PCR Gene expression measurements by quantitative real-time PCR were performed as reported.12 In brief, RNA was extracted using the RNAeasy Mini Kit (Qiagen, Hilden, Germany), quantified and quality-checked on a Bioanalyzer 2100 (RNA 6000 Nano LabChip-Kit; Agilent Technologies). High quality RNA samples (28S:18S > 1) were reverse transcribed (1µg total RNA, 10 mmol/L DTT, 1µg of hexamer primers, 2 U of MMLV Reverse Transcriptase [Invitrogen, Carlsbad, CA], 40 U of RNasin [Promega, Madison, WI], 0.5 mmol/L each dNTP [Promega], 1x reaction buffer). PCR primers were designed to be cDNA specific and ordered at GeneScan Europe (Freiburg, Germany). PCR was performed in 40 cycles on a ABI Prism 7000 using 2x SYBR Green I Master Mix (Applied Biosystems, Foster City, CA) in a final volume of 25 µL. Relative quantities (  cycle threshold values) were obtained by normalization against ribosomal 18S RNA. The 60 candidate genes measured by quantitative real-time PCR are listed in Table 1.
Statistical Analysis
Candidate Genes Associated With MFS in the ErbB2-Positive STB Breast Cancer Population The prognostic value of all 60 biomarkers detected by quantitative real-time PCR was individually assessed in the 70 STB ErbB2-positive tumors (22%) by univariate Cox regression revealing five candidates (uPA, MMP 3, MMP11, uPAR [PLAUR], and MMP1) significantly associated with MFS (Table 3). All five genes encode proteases and their levels of mRNA expression correlated strongly with one another (Online Only Appendix). Compared to uPA, the effect of MMP11, uPAR, MMP1, and MMP3 was weaker and nonsignificant when taken together with uPA in a multivariate model (data not shown). In contrast, none of the five candidates showed significant prognostic value with respect to MFS by univariate analysis in the 247 STB patients with ErbB2-negative tumors (Table 3). However, uPA, uPAR, and MMP1 were significantly associated with MFS in the overall population. To investigate possible confounding by treatment group, ER status, or proliferation (as defined by the average expression value of BIRC5, TOP2A, TYMS, TK1, and E2F1), stratified Cox analysis was performed; in all of these models uPA retained significant prognostic value in ErbB2-positive but not ErbB2-negative tumors (data not shown).
Prognostic Validation of uPA in Amsterdam and Rotterdam Breast Cancer Populations Amsterdam and Rotterdam breast cancer study sets were dichotomized into ErbB2-positive and ErbB2-negative patients as described (Patients and Methods and Online Only Appendix). Using the established 0.3 and 12.9 mRNA cutoff values to dichotomize the Amsterdam and Rotterdam patients, respectively, 52 of the Amsterdam patients (18%) and 51 of the Rotterdam patients (18%) were classified as ErbB2-positive (Table 2). Within each of these study sets, the prognostic value of uPA was independently assessed against both the ErbB2-positive and ErbB2-negative tumors, with and without stratification by ER status, treatment group (Amsterdam study only), and tumor proliferation. As presented in Table 4, uPA demonstrated significant prognostic association with MFS in ErbB2-positive but not in ErbB2-negative breast cancers. uPA was also significantly associated with MFS in the overall population, although displaying a weaker prognostic effect (Table 4).
Multivariate Cox Analysis The prognostic value of uPA mRNA expression was further assessed by multivariate analysis across all three study populations against nodal status, grade, tumor size, age, hormone receptor status, tumor proliferation, and treatment. Of note, the Rotterdam study patients were all untreated and node-negative, with only information on ER status available. Across all three study populations, uPA retained independent prognostic value and was significantly associated with MFS in ErbB2-positive tumors (Table 5). In addition to uPA, nodal status and chemotherapy retained independent prognostic value. In multivariate analysis, uPA also retained significance as an independent prognostic marker across all breast cancer patients, but not for patients with ErbB2-negative tumors (data not shown). Overall survival analysis demonstrated consistent findings with MFS among the Amsterdam study population, for which this outcome parameter was also available. Multivariate analysis for each of the three data sets can be found in the Online Only Appendix.
Stratification of ErbB2-Positive Tumors by uPA The cutoff value for uPA status was set at the 75th percentile after evaluation of 5-year MFS as a function of uPA mRNA expression in the STB study population (Online Only Appendix). The uPA cutoff was chosen to identify ErbB2-positive tumors with poor prognosis (5-year MFS of less than 50%) and to optimize separation between the groups. The 75th percentile agreed with previously published uPA cutoff values based on protein determination.14,15 This same uPA cutoff value was used to dichotomize ErbB2-positive and ErbB2-negative tumors in all three study populations and for outcome determination by Kaplan-Meier analyses (Fig 1). Within each study set, uPA status proved to be a strong prognostic factor for the development of distant metastasis, but only among ErbB2-positive breast cancer patients (HRs: STB study: 4.3; 95% CI, 1.6 to 11.8; Rotterdam study: 2.8; 95% CI, 1.1 to 7.1]; Amsterdam study: 2.7; 95% CI, 1.2 to 6.2; all P < .02). The prognostic value of uPA overexpression was even more pronounced for overall survival among ErbB2-positive Amsterdam patients (HR, 3.5; 95% CI, 1.5 to 8.6; P < .001, Fig 1). A search for alternative cutoff values for uPA failed to identify any level of uPA mRNA expression significantly associated with MFS among patients with ErbB2-negative breast cancer (Online Only Appendix). These stratification findings were independent of tumor treatment or stage, since multivariate analysis (see Multivariate Cox Analysis section) confirmed the independent prognostic value of uPA in ErbB2-positive but not in ErbB2-negative tumors (Table 5 and the Online Only Appendix).
Clinical and Prognostic Parameters Associated With uPA Stratified ErbB2-Positive Tumors In all three study sets, ER expression was significantly lower in ErbB2-positive as compared with ErbB2-negative tumors. However, no significant difference was found with respect to ER status, nodal status, tumor size, age, or treatment group between the dichotomized ErbB2-positive/uPA–negative and ErbB2-positive/uPA–positive tumors in any of the three study populations (Table 6). In addition, the 70-gene prognostic signature6 was investigated in the Amsterdam study population and found to classify 85% of ErbB2-positive tumors into a poor prognosis group; however, this signature showed no difference with regard to uPA status among patients with ErbB2-positive tumors (Table 6 and the online-only Appendix). Except for the Rotterdam study population, uPA expression was significantly higher in ErbB2-positive compared with ErbB2-negative tumors.
Initial evaluation of mRNA profiles from 60 cancer-related genes identified five protease-related prognostic candidates whose increased expression appeared significantly associated with MFS in ErbB2-positive breast cancer. The most significant of these, uPA, was further validated in two independent breast cancer study populations.6,7 Despite analysis of three distinct study populations by different assay methods to measure uPA mRNA (quantitative real-time PCR v Agilent and Affymetrix microarrays, Table 2), expression levels of this biomarker showed its consistent and independent prognostic significance that was restricted to patients with ErbB2-positive breast cancer. Matrix metalloproteinases (MMP) and the uPA system are known to play important roles in cancer cell invasion and metastasis;16-18 and the initially identified five protease-related prognostic candidates (Table 3) all showed a strong pattern of correlation among the STB study population (Online Only Appendix). uPA and other protein members of the uPA system are well established breastcancer prognostics.10 However, this study showing that uPA mRNA expression is of significant prognostic value only in ErbB2-positive patients is a novel observation. In conjunction with ErbB2 and uPA status, Zemzoum et al19 used another member of the uPA system, plasminogen activator inhibitor-1 (PAI-1), to define a high-risk subgroup of breast cancer patients. Among the STB patients, a strong correlation was observed between uPA and PAI-1 mRNA levels (rs = 0.64). In this same population, PAI-1 was significantly associated with MFS by univariate Cox regression, but showed only borderline significance for ErbB2-positive patients (P = .08), hence it was not included among the five protease-related prognostic candidates (Table 3). Like uPA, PAI-1 was not a significantly associated with MFS in ErbB2-negative tumors. Previously published reports have also demonstrated the predictive value of uPA with respect to adjuvant chemotherapy.20,21 However, the uniform association of uPA mRNA expression with survival observed across three distinct study populations was not likely due to predictive value with regard to chemotherapy because one of the validating study populations (Rotterdam) received no chemotherapy; as well, by multivariate analysis including chemotherapy, uPA remained an independent and significant factor in determining patient outcome in the other two study populations (Table 5). Konecny et al14 and Zemzoum et al19 previously investigated uPA and ErbB2 in breast cancer and reported their independent prognostic value, also illustrating that observed prognostic relationships may be dependent on the method of biomarker assessment (for example, immunohistochemistry v FISH for ErbB2) as well as the choice of the clinical outcome parameter (MFS v disease-free or overall survival). Unlike these earlier reports which evaluated uPA by immunoassays (enzyme-linked immunosorbent assay [ELISA]) on protein level and ErbB2 by protein immunohistochemistry and DNA FISH, this study quantitated expression of uPA and ErbB2 at the mRNA level. ErbB2 mRNA expression has been shown by several groups including our own to correlate well with more standard protein and DNA measures of ErbB2 status.12, 22-24 Although the prognostic value of microarray and quantitative real-time PCR determined uPA mRNA levels were found in this study to be highly comparable, prior analysis of STB patients demonstrated only a moderate correlation between uPA protein content (ELISA) and mRNA levels (rs = 0.46), suggesting some biologic discordance between these two measures of uPA bioactivity. Using an optimized uPA mRNA cutoff value capable of prognostically subsetting ErbB2-positive tumors, 25% of all breast cancer patients in this study scored as uPA–positive, which is consistent with the reported proportion of poor-risk breast cancers overexpressing uPA when measured by protein content.14,15 To avoid overfitting, cutoff optimization was performed only on the STB study population and this optimized value was subsequently validated in the Amsterdam and Rotterdam study populations. Of note, we were unable to find any uPA cutoff showing prognostic significance among the ErbB2-negative breast cancer tumors (Online Only Appendix). Recently, gene expression profiling using microarrays or quantitative real-time PCR have proved useful in predicting breast cancer outcomes.6-8 Interestingly, neither the prognostic gene signatures published by the Amsterdam6 nor the Rotterdam7 groups contained uPA. It was speculated that uPA as a single gene may have limited prognostic value compared with gene signatures and that mRNA expression may not be as informative as protein biomarker expression.25 This study revealed, however, that uPA mRNA level was highly informative and a prognostically valuable risk indicator when used for ErbB2-positive breast cancers. Given the finding that uPA mRNA expression had no apparent prognostic value among ErbB2-negative breast cancers, it is possible that the overall prognostic importance of uPA (and other genes) has been underestimated in past breast cancer studies not stratified by ErbB2 status. Interestingly, the 70-gene prognostic signature published by the Amsterdam group6 classified almost all ErbB2-positive patients into the poor prognosis group. In terms of survival there was no difference between the poor prognosis/ErbB2-positive and poor prognosis/ErbB2-negative tumors (Online Only Appendix). In contrast to uPA and other proteases, the 70-gene prognostic signature appears to largely discriminate a low-risk group from among patients with ErbB2-negative tumors. The existence of a molecular mechanism linking uPA upregulation with ErbB2 overexpression is supported by numerous reports that ErbB2 activation increases the transcriptional expression of uPA and various members of the MMP family.4,26-28 Consistent with these reports, we observed significantly higher uPA expression levels in ErbB2-positive relative to ErbB2-negative tumors; and similar findings have been reported for other cancers.29-31 However, the mechanistic interactions linking ErbB2 and uPA overexpression with increased breast cancer metastatic potential are likely more complex, also because ErbB2 overexpression occurs only within tumor epithelium while uPA and other proteases are primarily overexpressed in tumor stroma.32,33 Stromal production of uPA (and other proteases) must be considered when microdissection techniques are used to obtain breast cancer RNA for expression profiling. The functional interaction between overexpressed ErbB2 and uPA also deserves greater study at both cellular and clinical levels since it is unclear why ErbB2-negative/uPA–positive and ErbB2-positive/uPA–negative breast cancers are no more clinically aggressive than ErbB2-negative/uPA–negative breast cancers. Apparently the proteolytic activity of uPA alone is insufficient to determine the metastatic outcome, and perhaps requires the cell proliferation and survival advantages provided by activation of ErbB2. Similarly, ErbB2 activation may only predispose to a metastatic outcome in conjunction with cellular expression of the uPA and other proteases. In conclusion, this study indicates that overexpression of uPA mRNA levels in ErbB2-positive breast cancers determines the aggressive and highly metastatic clinical behavior previously attributed only to activation of the ErbB2 receptor tyrosine kinase. ErbB2 status is determined on all newly diagnosed breast cancers. While the prognostic and predictive potential of uPA ELISA-based measurements is supported by the highest level of clinical evidence, such uPA assays have proven impractical for widespread clinical application. Future studies showing that the uPA quantitative real-time PCR assay as employed in this study may also be adapted to small paraffin-archived breast cancer samples will undoubtedly lead to more rapid and widespread clinical measurement of uPA. With expression profiling now integrated into several ongoing multinational breast cancer adjuvant trials, attention to breast tumor uPA mRNA levels, measured alone or as part of gene expression signature, will further enhance the prognostic and predictive value of this important protease biomarker.
Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
Dollar Amount Codes (A) < $10,000 (B) $10,000-99,999 (C)
We thank pathologists for providing tumor tissues in particular R. Caduff, MD, H. Moch, MD, M. Mihatsch, MD, and G. Sauter, MD, for the FISH assessment of ErbB-2. We are indebted to all clinicians for their collaboration in collecting clinical data in particular H. Dieterich, MD, D. Fink, MD, and K. Lüscher, MD. We are grateful to Thierry Sengstag, PhD, for implementing mixture model statistics, to Sabine Ehret for technical assistance, and to Ulrike Weissenstein, PhD, and Chrisitine Wullschleger for data management.
Supported by the Stiftung Tumorbank Basel, the Swiss National Science Foundation (Grant No. 3100-059819.99/1), and the National Center of Competence in Research Molecular Oncology—a research instrument of the Swiss National Science Foundation. Quantitative real-time polymerase chain reaction analysis was supported by OncoScore AG. P.U. and V.V. contributed equally to this article. Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
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