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Cancer Biology & Therapy 11:2, 284-286; January 15, 2011; © 2011 Landes Bioscience
Tumor marker assessment Points to ponder Yesim Gökmen-Polar1 and Sunil Badve1,2,* Department of Medicine and 2Department of Pathology; Indiana University School of Medicine; Indianapolis, IN USA
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Tumor-associated proteolytic enzymes, proteases, play key roles in tumor invasion and metastasis where they contribute to the degradation of extracellular matrix, and multiple steps of tumor progression including proliferation, migration and angiogenesis.1,2 Among the proteases, the serine protease urokinasetype plasminogen activator (uPA) and its inhibitor (plasminogen activator inhibitor type-1; PAI-1) were recommended by the American Society for Clinical Oncology (ASCO) as prognostic markers in patients with newly diagnosed, node-negative breast cancer, while the level of evidence for cathepsin D (cath-D) was insufficient to recommend it for routine use.3 uPA and PAI-1 belong to the plasminogen activating system, including the receptor for uPA (uPAR) and other inhibitors such as PAI-2. uPA is initially synthesized as an inactive proenzyme (pro-uPA), which can bind uPAR in its active or inactive form. After activation, uPA cleaves plasminogen to its active form, plasmin. Plasmin degrades extracellular matrix (ECM) components and activates matrix metalloproteases (MMPs) leading to proteolysis and degradation of the ECM.4,5 In addition to their role in the degradation of ECM, uPA-uPAR can activate cell signaling pathways such as focal adhesion kinase (FAK), ERK and phosphatidylinositide 3-kinase (PI3K)/Akt and can contribute to cell proliferation, adhesion and migration.5,6 PAI-1 negatively regulates the uPAuPAR complex while the complex can be activated by plasmin, by a feedback loop, and by other enzymes such as cath-D. Cath-D is an aspartic protease involved in different steps of cancer including cell
proliferation, angiogenesis and apoptosis.7 Although overexpression of cath-D is associated with aggressiveness and poor prognosis in breast cancer, its estrogeninduced overexpression is more related to the cell proliferation rather than invasion.7,8 Figure 1 demonstrates a simplified schema of the role of these proteases in tumor progression, cancer invasion and metastasis. In this issue of Cancer Biology & Therapy, Mazouni et al. evaluated the relationship between uPA, PAI-1 and cath-D with respect to estrogen receptor (ER) status by analyzing their expression levels in tumor cytosol fractions using enzymelinked immunosorbent assays (ELISA) in a group of 316 patients with invasive breast cancer who underwent primary surgery (Mazouni et al. this issue).9 In their study, the correlation between these three proteases and their prognostic impact differed when the analysis was performed in the overall population or subsets based on ER expression. The authors categorized ER expression levels into three groups: an ER-negative (ER-) group (defined as less than 15 fmol/mg protein); and an ER-positive group, which was further divided into low (ER+); and high (ER++) based on greater than 75th quartile expression. The levels of uPA, and PAI-1 were significantly higher in ER- patients (p < 0.001), while cath-D levels were similar in ER subsets (p = 0.96). In the overall population, a significant correlation between uPA and PAI-1 and between uPA and cath-D was observed. The association between uPA, PAI-1 and cath-D were significant only in the ER+ (i.e., ER low) population among the ER subsets
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Key words: Breast cancer, estrogen receptor (ER), urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor type-1 (PAI-1), cathepsin D (cath-D), tumor marker, prognosis Submitted: 11/26/10 Accepted: 12/02/10 DOI: 10.4161/cbt.11.2.14370 *Correspondence to: Sunil Badve; Email:
[email protected] Commentary to: Mazouni C, Romain S, Bonnier P, Ouafik L, Martin PM. Prognostic significance of tumor-related proteases as a function of the estrogen receptor status. Cancer Biol Ther 2011; This issue.
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Autophagic Punctum
Figure 1. Schematic description of the role of uPA/PAI-1 in tumor progression.
(Table 3 in Mazouni, et al.). Among these proteases, cath-D (above median) was an independent factor associated with a poor overall survival in the entire population (p < 0.001) and in the ER+ subset (p = 0.02). PAI-1 (upper tertile) was prognostic factor for overall survival (OS) in the ER++ subset and in the entire population (Table 4 in Mazouni et al.). Overexpression of uPA and/or PAI-1 using ELISA assays has been correlated with poor prognosis in early-stage breast cancer.10-16 These markers clearly provide information in addition to classic prognostic markers and have been validated in clinical trials of node-negative breast cancer.16-20 The level of evidence for cathD, on the other hand, was thought to be insufficient to justify routine use.21,22 Cath-D levels are typically determined in breast tumor cytosolic extracts using an ELISA assay. However, the cutoff points used in the studies to classify tumors into positive or negative have been variable. The current study used “median” as a cutoff for cath-D. Cath-D (cut point of 45.2 pmol/mg) was modestly predictive (hazard ratio, 1.39) in both node-negative and node-positive populations by multivariate analysis.21 In another study, using a cut point of 10 pmol/mg, high cath-D expression correlated with a 1.7-fold higher hazard of relapse both in univariate and multivariate analyses.22 Cath-D was not associated with hormone receptor status in one study,23 while a significant association was found between cath-D and both estrogen receptor and progesterone receptor levels in other studies.24,25 In the study by
Kute et al. high levels of cath-D predicted a shorter OS, while PAI-1 and cath-D were independent significant prognostic indicators for disease-free survival (DFS).26 Among the studies that have analyzed the correlation between these three proteases and steroid receptors, and their prognostic significance in breast cancer,23-28 the results have been variable, with one study showing no correlation at all. In this article, Mazouni et al. introduce a more detailed quantitative approach for ER expression levels to determine the relationship and prognostic significance of these proteases in regard to ER status. Most importantly, this study provokes us to rethink about the challenges of validation methods on the ongoing search of standardization in the field of biomarkers. First, the current recommendation of ASCO and College of American Pathologists (ASCO/CAP) guidelines for ER and PR expression is based on immunohistochemistry (IHC) with tumors showing greater than 1% expression being regarded as positive.29 The cutoff values for ligand binding assay and ELISA are poorly defined. Although IHC may not be quantitative (or perhaps even as sensitive) in comparison to an ELISA assay, it permits visualization of the positive cells and exclusion of the nontumor epithelial components. This ability is lost when one uses “grind and bind” techniques. The use of different methods may cause variability in the preanalytic, analytic and postanalytic standardization, which may affect the interpretation of data. More importantly, it significantly limits the ability to
translate the information from different studies. Second, ELISA is the only method that has been shown to be prognostic for the expression of uPA and PAI-1markers.17 Several studies have attempted analysis of these markers using IHC30 or quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR)30-33 with less than satisfactory results. Nucleic acid sequencebased amplification (NASBA) has demonstrated a high concordance between NASBA and uPA/PAI-1 antigen expression determined by ELISA.34 However, none of these methods have reached the clinical utility due to the lack of multicenter validation in the clinic. Third, the ELISA assays, although quantitative, are limited to the use of fresh or frozen tissues. In the case of uPA/PAI1, a minimum of 300 mg of fresh or frozen breast cancer tissue required (ASCO Guidelines3), as large tissue sections from tumors were used in the pooled analysis study17 and in the prospective randomized clinical trial.18 Given the limited availability of frozen tissues in the clinical setting, this raises the question of how best to integrate these quantitative assays to routine practice. Fourth, questions need to be raised as to why uPA was not significant in the current study. Is this a quirk associated with a single institutional dataset or is it due to the use of “upper tertile” as a cutoff or is it because the authors stratified the ER+ cases in high and low categories? It is now well established that breast cancer is a heterogeneous disease and composed of several subtypes.35 Due to this heterogeneity, subtype specific markers are needed to predict outcomes. The availability of a new marker in the domain of Lum A tumors is probably not such a big advance due to existence of assays such as Ki67 and Oncotype Dx®.36,37 In contrast, there is a dire need for new markers in the triple-negative and HER2-positive subgroups. The study by Mazouni et al. highlights the point that the prognostic significance of the markers varies according to the breast cancer subset studied. This raises the following question: Is there any utility in doing prognostic/predictive marker studies in a “breast cancer” population or has the field advanced
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enough to limit them to subpopulations based on either molecular classification or at least on the basis of ER and HER2 expression? Lastly, is five years a long enough time frame to assess utility of a marker in a subset of breast cancer patients with good prognosis? If not, what should be the optimal interval—should this depend on the subtype of breast cancer? In summary, this work raises several important questions in the field of predictive and prognostic markers and the challenges that lie ahead of us. Given the increasingly smaller sizes of tumors and the diminished availability of fresh frozen tissues, there may be a need to develop a strategic plan for performance and analysis of markers in a uniform manner using standard methodologies, and definitions to permit maximal use of this scarce resource. The authors (and their institutions) need to be congratulated for the investing of scarce resources in developing cohorts of fresh frozen tumors with followup data; an extremely valuable resource to identify novel predictive and prognostic markers.
9. Mazouni C, Romain S, Bonnier P, Ouafik L, Martin PM. Prognostic significance of tumor-related proteases as a function of the estrogen receptor status. Cancer Biol Ther 2011; 11: 277-83. 10. Duffy MJ. Urokinase plasminogen activator and its inhibitor, PAI-1, as prognostic markers in breast cancer: from pilot to level 1 evidence studies. Clin Chem 2002; 48:1194-7. 11. Foekens JA, Schmitt M, van Putten WL, Peters HA, Kramer MD, Janicke F, et al. Plasminogen activator inhibitor-1 and prognosis in primary breast cancer. J Clin Oncol 1994; 12:1648-58. 12. Bouchet C, Hacene K, Martin PM, Becette V, Tubiana-Hulin M, Lasry S, et al. Dissemination risk index based on plasminogen activator system components in primary breast cancer. J Clin Oncol 1999; 17:3048-57. 13. Bouchet C, Spyratos F, Martin PM, Hacene K, Gentile A, Oglobine J. Prognostic value of urokinasetype plasminogen activator (uPA) and plasminogen activator inhibitors PAI-1 and PAI-2 in breast carcinomas. Br J Cancer 1994; 69:398-405. 14. Duffy MJ, O’Grady P, Devaney D, O’Siorain L, Fennelly JJ, Lijnen HJ. Urokinase-plasminogen activator, a marker for aggressive breast carcinomas. Preliminary report. Cancer 1988; 62:531-3. 15. Eppenberger U, Kueng W, Schlaeppi JM, Roesel JL, Benz C, Mueller H, et al. Markers of tumor angiogenesis and proteolysis independently define highand low-risk subsets of node-negative breast cancer patients. J Clin Oncol 1998; 16:3129-36. 16. Harbeck N, Schmitt M, Kates RE, Kiechle M, Zemzoum I, Janicke F, et al. Clinical utility of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 determination in primary breast cancer tissue for individualized therapy concepts. Clin Breast Cancer 2002; 3:196-200. 17. Look MP, van Putten WL, Duffy MJ, Harbeck N, Christensen IJ, Thomssen C, et al. Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients. J Natl Cancer Inst 2002; 94:116-28. 18. Janicke F, Prechtl A, Thomssen C, Harbeck N, Meisner C, Untch M, et al. Randomized adjuvant chemotherapy trial in high-risk, lymph node-negative breast cancer patients identified by urokinase-type plasminogen activator and plasminogen activator inhibitor type 1. J Natl Cancer Inst 2001; 93:913-20. 19. Zemzoum I, Kates RE, Ross JS, Dettmar P, Dutta M, Henrichs C, et al. Invasion factors uPA/PAI-1 and HER2 status provide independent and complementary information on patient outcome in node-negative breast cancer. J Clin Oncol 2003; 21:1022-8. 20. Hayes DF, Bast RC, Desch CE, Fritsche H Jr, Kemeny NE, Jessup JM, et al. Tumor marker utility grading system: a framework to evaluate clinical utility of tumor markers. J Natl Cancer Inst 1996; 88:1456-66. 21. Foekens JA, Look MP, Bolt-de Vries J, Meijer-van Gelder ME, van Putten WL, Klijn JG. Cathepsin-D in primary breast cancer: prognostic evaluation involving 2,810 patients. Br J Cancer 1999; 79:300-7. 22. Billgren AM, Tani E, Liedberg A, Skoog L, Rutqvist LE. Prognostic significance of tumor cell proliferation analyzed in fine needle aspirates from primary breast cancer. Breast Cancer Res Treat 2002; 71:161-70. 23. Harbeck N, Alt U, Berger U, Kruger A, Thomssen C, Janicke F, et al. Prognostic impact of proteolytic factors (urokinase-type plasminogen activator, plasminogen activator inhibitor 1, and cathepsins B, D and L) in primary breast cancer reflects effects of adjuvant systemic therapy. Clin Cancer Res 2001; 7:2757-64.
24. Marsigliante S, Biscozzo L, Greco S, Leo G, Storelli C. Relation of cathepsin D level to the estrogen receptor in human breast cancer. Int J Clin Lab Res 1992; 22:52-7. 25. Gion M, Mione R, Dittadi R, Romanelli M, Pappagallo L, Capitanio G, et al. Relationship between cathepsin D and other pathological and biological parameters in 1,752 patients with primary breast cancer. Eur J Cancer 1995; 31:671-7. 26. Kute TE, Grondahl-Hansen J, Shao SM, Long R, Russell G, Brunner N. Low cathepsin D and low plasminogen activator type 1 inhibitor in tumor cytosols defines a group of node negative breast cancer patients with low risk of recurrence. Breast Cancer Res Treat 1998; 47:9-16. 27. Marsigliante S, Biscozzo L, Correale M, Paradiso A, Leo G, Abbate I, et al. Immunoradiometric detection of pS2 and total cathepsin D in primary breast cancer biopsies: their correlation with steroid receptors. Br J Cancer 1994; 69:550-4. 28. Duffy MJ, Reilly D, McDermott E, O’Higgins N, Fennelly JJ, Andreasen PA. Urokinase plasminogen activator as a prognostic marker in different subgroups of patients with breast cancer. Cancer 1994; 74:2276-80. 29. Hammond ME, Hayes DF, Dowsett M, Allred DC, Hagerty KL, Badve S, et al. American Society of Clinical Oncology/College Of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Clin Oncol 2010; 28:2784-95. 30. Schmitt M, Mengele K, Gkazepis A, Napieralski R, Magdolen V, Reuning U, et al. Assessment of urokinase-type plasminogen activator and its inhibitor PAI-1 in breast cancer tissue: historical aspects and future prospects. Breast Care (Basel) 2008; 3:3-10. 31. Biermann JC, Holzscheiter L, Kotzsch M, Luther T, Kiechle-Bahat M, Sweep FC, et al. Quantitative RT-PCR assays for the determination of urokinasetype plasminogen activator and plasminogen activator inhibitor type 1 mRNA in primary tumor tissue of breast cancer patients: comparison to antigen quantification by ELISA. Int J Mol Med 2008; 21:251-9. 32. Leissner P, Verjat T, Bachelot T, Paye M, Krause A, Puisieux A, et al. Prognostic significance of urokinase plasminogen activator and plasminogen activator inhibitor-1 mRNA expression in lymph node- and hormone receptor-positive breast cancer. BMC Cancer 2006; 6:216. 33. Spyratos F, Bouchet C, Tozlu S, Labroquere M, Vignaud S, Becette V, et al. Prognostic value of uPA, PAI-1 and PAI-2 mRNA expression in primary breast cancer. Anticancer Res 2002; 22:2997-3003. 34. Lamy PJ, Verjat T, Servanton AC, Paye M, Leissner P, Mougin B. Urokinase-type plasminogen activator and plasminogen activator inhibitor type-1 mRNA assessment in breast cancer by means of NASBA. Correlation with protein expression. AM J Clin Pathol 2007; 128: 404-413. 35. Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, et al. Molecular portraits of human breast tumours. Nature 2000; 406:747-52. 36. Paik S, Tang G, Shak S, Kim C, Baker J, Kim W, et al. Gene expression and benefit of chemotherapy in women with node-negative, estrogen receptor-positive breast cancer. J Clin Oncol 2006; 24:3726-34. 37. Paik S, Shak S, Tang G, Kim C, Baker J, Cronin M, et al. A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med 2004; 351:2817-26.
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