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Measurements of Proteases or Protease System Components in Blood to Enhance Prediction of Disease Risk or Outcome in Possible Cancer

Departments of Clinical Laboratories, Surgery (Urology), Medicine, and Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY

There is a growing body of evidence that proteases can act as molecular markers for cancer. Indeed, prostate-specific antigen (PSA or KLK3), one of the few molecular markers that is routinely used in all stages of clinical cancer care,¹ is a protease.² Moreover, attempts to improve on the diagnostic and prognostic value of PSA have often involved measuring other kallikrein-like proteases, such as KLK2 (hK2) or KLK11.^3,4 Proteomic and peptidomic analyses have also highlighted the importance of the proteases. For example, in a recent report, Tempst et al⁵ showed that matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF) can be used to identify certain cancer-associated signature combinations of small-sized peptides known to be released from several of the most abundant human serum proteins by cancer-associated exopeptidases. This allows discrimination among malignancies (including prostate cancer) and between the malignant and cancer-free state. The findings of Tempst et al may also relate to the manifestation and regulation of proteolytic activity associated with extracellular matrix degradation and tissue remodeling during invasion and metastasis in malignancy.^6,7 This includes plasmin- and trypsin-facilitated activation of matrix metalloproteases, capable of degrading major basal membrane components, such as collagen type IV, which may be key in allowing invading cancer cells to escape and seed metastases outside the primary organ.⁸

The components of the urokinase-type plasminogen activator system have been subject to particular attention in this context. The urokinase-type plasminogen activator (uPA) is secreted as inactive proenzyme (pro-uPA), which binds to a specific receptor, urokinase plasminogen activator receptor (uPAR), at the cell surface.⁹ Acting as a proximal catalytic template, plasminogen enhances conversion of uPAR-bound pro-uPA to active uPA.¹⁰ In vitro, trypsin, hK2, and other proteases have been found capable of converting pro-uPA to active uPA, while active uPA catalyzes activation of plasminogen to plasmin. Further, uPA is inactivated by formation of stable complexes with various plasminogen activator inhibitors (PAI-1, PAI-2, or PAI-3/PCI), whereas plasmin, uPA, and several other proteases cleave and release uPAR from the cell surface. uPAR is a glycolipid anchored receptor with three homologous domains; the amino terminal (domain I) is needed to bind uPA, while high affinity binding requires intact uPAR(I-III).^11,12 Several proteases (including active uPA and plasmin) liberate uPAR(I) by cleaving uPAR(I-III) between domains I and II, while uPAR(II-III) is retained at the cell surface by the glycolipid anchor.^12,13 uPAR can also be shed from the cell surface due to hydrolysis of the lipid anchor by either phospholipases or proteases.¹⁴ These mechanisms provide a basis for the occurrence of detectable levels of soluble uPAR forms in plasma from healthy individuals.¹⁵

It has previously been shown that uPAR levels are elevated in the blood of patients with non–small-cell lung cancer,¹⁶ colorectal cancer,¹⁷ and breast cancer.¹⁸ Cleaved uPAR(II-III) has been identified in cystic fluid from ovarian cancer patients.¹⁹ Preoperative uPAR levels in plasma or serum are associated with the prognosis of patients with colorectal cancer.²⁰ It has also been proposed that levels of cleaved uPAR forms in blood or tumor tissue more closely reflect the degree of uPA-catalyzed plasminogen activation, and hence, may be more closely associated with patient prognosis than measurements of the sum of all uPAR forms. Applying measurements of the urokinase system components to patients with prostate cancer, Visakorpi et al²¹ showed that amplification of the uPA gene was frequent in metastatic lesions of castrated prostate cancer patients. Cozzi et al²² recently reported on the use of tissue microarrays to show that the expression of both uPA and UPAR is frequently upregulated in high-grade primary tumors, whereas a study by Piironen et al²³ suggested that measurements of certain UPAR forms in the blood may enhance discrimination of men with positive compared to those with negative prostate biopsy findings.

Shariat et al²⁴ report in this issue of the Journal of Clinical Oncology that plasma levels of uPA and uPAR are higher in men with prostate cancer than in healthy controls, and increase with disease progression. Although these results are intriguing, we would do well to remember that research on molecular markers, like that on anticancer agents, passes through several phases. Demonstration of a statistically significant association between a marker and a clinical state, the aim of this article, is the equivalent of a phase I trial of a cytotoxic agent; showing that a biomarker is not independent of outcome, like identifying a dose of an agent that can be taken safely is necessary but clearly insufficient for clinical implementation. An essential next step would be to show that adding data on urokinase levels to currently available clinical data enables more accurate diagnosis or prognosis. For example, does including both PSA and urokinase in a screening test for prostate cancer increase specificity or sensitivity compared with a PSA test alone? Does the addition of measurements of uPAR and uPA levels to clinical stage, tumor grade, and PSA in serum importantly improve the predictive accuracy of the established nomograms commonly used to counsel patients?²⁵ Would more selective measurements of uPAR forms released into blood contribute similar or additional diagnostic improvements?²⁶ Investigators would also need to show that the urokinase measurements improve decision making, as it is quite possible for a new test to have increased accuracy, but for this to make no practical difference in the clinic.

Analysis of molecular markers remains one of the most promising areas of cancer research and proteases are among the most promising markers. There is a clear biologic basis for an association between protease levels and cancer, and preliminary data, such as that provided by Shariat et al,²⁴ are encouraging. The challenge now is to conduct the sort of clinical research that would determine whether the use of proteases such as urokinase as molecular markers would provide tangible benefits to those living with prostate cancer or those who are at increased risk of developing the disease.

AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

Although all authors completed the disclosure declaration, the following author or 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.

Employment: N/A Leadership: N/A Consultant: N/A Stock: Hans Lilja, Arctic Partners Oy Honoraria: N/A Research Funds: N/A Testimony: N/A Other: N/A

AUTHOR CONTRIBUTIONS

Conception and design: Hans Lilja, Andrew Vickers, Peter Scardino

Financial support: Peter Scardino

Administrative support: Hans Lilja, Peter Scardino

Data analysis and interpretation: Hans Lilja, Andrew Vickers, Peter Scardino

Manuscript writing: Hans Lilja, Andrew Vickers, Peter Scardino

Final approval of manuscript: Hans Lilja, Andrew Vickers, Peter Scardino

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