|Home | About | Journals | Submit | Contact Us | Français|
The new Latin word fibrinolysis describes a process whereby a fibrin clot – the product of blood coagulation involving transformation of the liquid plasma protein fibrinogen into solidified fibrin – is lysed by proteolytic enzymes such as plasmin . The serine protease plasmin is generated from the plasma protein plasminogen by the action of plasminogen activators, e.g. the tissue-type plasminogen activator (tPA) and the urokinase-type plasminogen activator (uPA) [2, 3]. The proteolytic activity of plasmin is inactivated by alpha-2-antiplasmin or alpha-2-macroglobulin, that of tPA or uPA essentially by two specific inhibitors, plasminogen activator inhibitor type-1 (PAI-1) and type-2 (PAI-2) . These observations are not new; Astrup described plasminogen activator activity, fibrinolysin, in 1950, which at that time was a term for any of various proteolytic enzymes, especially plasmin, capable of digesting fibrin in the bloodstream [5, 6]. It was only a few years earlier, in 1948, that MacFarlane and Biggs had reviewed previous work on the proteolytic factors of serum since different terms had been applied to identical substances, which had led to confusion . The authors suggested that such names as serum trypsin, serum protease, serum tryptase, fibrinolysin, thrombolysin and others should be abandoned in favour of a more specific designation, and proposed adhering to a nomenclature suggested by Christensen and MacLeod in 1945 . Here, the proteolytic enzyme of plasma is named plasmin, its precursor plasminogen, and its inhibitor antiplasmin. The term fibrinolysin was not recommended since this term was also applied to the streptococcal filtrate known to activate plasminogen in vivo. This filtrate factor became known as streptokinase.
Soon after, in 1951, Lewis and Ferguson  reported compounds capable of activating plasminogen in blood; in the same year, Williams  demonstrated the presence in urine of a substance (urokinase, uPA) able to activate plasminogen, which was later isolated from urine by Ploug et al. . uPA is present in normal and malignant tissues and plasma as well, a fact which was recognised for ovarian cancer tissues in 1976 by Astedt and Holmberg  and a few years later (1982) for plasma by Wun et al.  and Tissot et al. . The concentration of uPA in plasma amounts to about 3.5 ng/ml, which is low compared to the relatively high concentration of 200-300 ng/ml in urine. Regarding the second major plasminogen activator, tPA, the prefix ‘tissue-type’ of tPA refers to the original observation in the 1940s that tPA is present in tissues and tissue extracts . The concentration of tPA in plasma amounts to 5–10 ng/ml, but varies strongly under different physiological and pathological conditions.
Although it was already known before 1970 that plasminogen activator activity may be increased in tumour tissues over non-neoplastic tissue, for some time, interest was turned away from the possible role of plasminogen activators in cancer progression [15,16,17,18,19,20], particularly because the techniques used then did not distinguish between the two types of plasminogen activators, uPA and tPA [21, 22]. In the years following Astedt and Holmberg's observation that uPA is released by human ovarian cancer cells, several other authors reported elevated uPA concentrations in tumour tissues compared to non-neoplastic tissues [23,24,25,26]. These observations prompted several investigators to restart detailed analyses of plasminogen activators, especially uPA, in tumour tissue and blood samples from cancer patients. Due to refined analytical tools and instruments, the structure of uPA, its proteolytic activation and role in the pathophysiology of tumour stroma degradation and tumour spread was investigated. This was also enhanced by the fact that, in 1985, a cell surface receptor for uPA (uPAR; CD87) was detected and it also became clear that the proteolytic activity of uPA and tPA in thrombolysis and fibrinolysis is counterbalanced by inhibitors of tPA and uPA and that uPAR is a focal adhesion point for localised uPA-mediated proteolysis in the physiological and malignant state [27, 28].
In 1966, Brakman et al.  described the presence of a plasminogen activator inhibitor in a group of patients with an impaired plasma fibrinolytic system, but it took another 18 years before the inhibitor PAI-1 was isolated . The PAI-1 concentration in plasma is about 20 ng/ml. The other inhibitor, PAI-2, was first detected in human placental tissue  and was therefore named placenta-type plasminogen activator inhibitor. Later it became clear that PAI-2 is also present in various types of white blood cells and in tumour tissue . The PAI-2 concentrations in plasma are usually low, but can be high (above 35 ng/ml) in pregnant women.
The fundamental role of the uPA/PAI-1 system in tumour invasion and metastasis has first been derived from correlations between pathophysiological phenomena and tumour tissue-associated proteolytic activity. In essence, the finding was that plasminogen activators are linked to degradation and remodeling of normal and cancer tissue and the surrounding extracellular matrix [2, 21, 22]. Concerning the clinical relevance of uPA, in 1985 O'Grady et al.  in a first comprehensive report determined total plasminogen activator proteolytic activity, uPA activity, and tPA activity in benign breast tumours and primary breast cancer. Benign tumours contained predominantly tPA activity whereas uPA activity was significantly higher in the malignant tumours compared with the benign ones. Then, in 1988, Duffy et al.  reported that patients with primary breast carcinomas containing high levels of uPA proteolytic activity had a significantly shorter disease-free interval than patients with low levels of activity. This finding led the authors to conclude that uPA may serve as a new prognostic marker in breast cancer. In the following years, Jänicke et al. [35, 36] presented evidence of a high correlation between elevated uPA antigen levels in the primary tumour and poor outcome of breast cancer patients, followed by a report of Jänicke et al. in 1991  announcing that the same applies for the inhibitor of uPA, PAI-1, but not for tPA. Since then many independent reports applying antigen measurements (enzyme-linked immunosorbent assay (ELISA)) to quantify uPA and PAI-1 antigen content in cancer tissue extracts have been published describing such a correlation (table (table1),1), not only for breast cancer but also for other solid malignant tumours [2, 38, 39]. These findings were important as such cancer biomarkers were urgently needed for the individualisation of oncologic therapy. Surveying the current literature (National Center for Biotechnology Information (NCBI) MedLine), it is evident that the majority of investigations has been conducted in breast cancer. In the years 1984 to July 2008, 762 articles (141 reviews) were listed in MedLine regarding the clinical relevance of uPA in cancer; for PAI-1, 536 articles (85 reviews). Regarding breast cancer, within that period of time, 336 articles (75 reviews) were listed in MedLine for uPA and 237 articles (48 reviews) for PAI-1. The peak of publications was between 2000 and 2003 (fig. (fig.11).
Given the now known biological importance of uPA and PAI-1 in tissue remodeling, angiogenesis, cell migration, and proliferation [40,41,42] – not only in the physiological but also in the pathophysiological state – and the clinical relevance of these cancer biomarkers for cancer prognosis and prediction of therapy response, several approaches have been developed targeting the uPA/PAI-1 system in cancer to reduce tumour invasion and metastasis. Such preclinical strategies include the use of antisense oligonucleotides or small interfering RNAs (siRNAs) to silence the uPA gene, antibodies to uPA or uPAR as well as recombinant or synthetic uPA or uPAR analogues to prevent binding of naturally occurring uPA (and uPA:PAI-1 complexes) to uPAR, and naturally and synthetic serine protease inhibitors blocking uPA enzymatic activity to reduce tumour cell proliferation, invasion, and metastasis [2, 43,44,45,46].
Drug candidates that emerged from synthetic uPA inhibitors shown to be effective in experimental tumour-bearing animals  are now in clinical phase I/II testing involving late-stage cancer patients afflicted with tumours of the breast, pancreas, ovary, or the gastrointestinal tract. Goldstein (this issue) reports the promising results of a phase Ib trial with the serine protease inhibitor WX-UK1 in patients with solid tumours, including breast cancer patients, in combination with the chemotherapeutic agent capecitabine. Also, WX-671 (MESUPRON®, an oral pro-drug of WX-UK1) was studied in a phase Ib trial with patients with head and neck cancer (Lang et al., this issue). In July 2008, Wilex AG, Munich, Germany, successfully completed recruitment of a randomised clinical phase II trial of advanced pancreatic cancer patients. Patients were treated with MESUPRON in combination with the chemotherapeutic agent gemcitabine (Gemzar®; Eli Lilly and Company, USA). MESUPRON is currently being tested clinically (phase II) in breast cancer patients in combination with the chemotherapeutic agent capecitabine.
Over the years, substantial efforts have been made in breast cancer to subdivide patient populations into groups that behave differently, so that therapy can be applied more efficiently. Still, since these efforts are based on clinical outcomes related to clinical cancer size and the presence or absence of pathologically involved lymph nodes, subgroups with different biological behaviours cannot be defined correctly . Nonetheless, in the last decade, basic and clinical scientists have studied a plethora of novel cancer biomarkers at the gene and protein level [48, 49]. For breast cancer, several hundreds of such markers have been reported, yet only a handful have actually gained widespread clinical use, including the steroid hormone receptors estrogen receptor (ER) and progesterone receptor (PR), the oncogene HER2, and the tumour invasion factors uPA and PAI-1 [38, 50]. This lack of acceptance, due to controversial test results, at least in part comes from the biological diversity of the breast cancer disease, poorly designed or non-validated clinical studies, non-validated tools and test systems, poor statistics, and/or low quality of the tumour material tested.
The lack of contradictory evidence on the prognostic impact of uPA and PAI-1 is quite unique for any cancer biomarker and is remarkable considering the variety of demographic conditions covered by studies in Europe and abroad . Table Table11 depicts key references in which studies are described showing the prognostic impact of uPA and/or PAI-1 in primary breast cancer. It is worth mentioning that uPA and PAI-1 have reached the highest level of evidence (LOE-1) according to the tumour marker utility grading system  by fulfilling the criteria of a prospective therapy trial (Chemo-N0) to test the clinical utility of the two cancer biomarkers  and by a meta-analysis (pooled analysis) encompassing 8377 patients and published databases from 18 different study centres . Finally, in November 2007, determining the uPA/PAI-1 content in a breast cancer patient's primary tumour tissue was incorporated into the breast cancer treatment guidelines of the American Society of Clinical Oncology (ASCO) to provide for the appropriate adjuvant systemic treatment .
It is worth mentioning that no significant correlation was found between plasma and tumour tissue levels of uPA and PAI-1, indicating that determination of these factors in plasma does not reflect their concentration in tumour tissue. Therefore, measurement of uPA and PAI-1 in blood cannot be recommended for assessing prognosis in breast cancer patients .
We would like to stress that so far, clinically relevant, validated data regarding uPA and PAI-1 in breast cancer have been obtained only by measuring these two cancer biomarkers by ELISA, either in tumour tissue cytosolic fractions or in the detergent-released tumour tissue fraction [54, 55]. Such ELISAs are commercially available (e.g. FEMTELLE®; American Diagnostica Inc., Stamford, CT, USA) and robust enough for clinical routine use. The quality of the test kits is assessed and assured by the European Organization for Research and Treatment of Cancer (EORTC) PathoBiology Group (Brussels, Belgium). Use of the non-ionic detergent Triton X-100 for tumour tissue extraction is recommended since this method of extraction yields considerably more release of uPA antigen than uPA freed into the cytosol fraction. No such difference is observed for PAI-1 . The test can be applied to primary tumour biopsies, core needle biopsies, and cryostat sections. Standard operating protocols for tumour tissue disintegration and uPA/PAI-1 test implementation are published and described explicitly [56, 57]. Therefore, when evaluating other ways of determining uPA and PAI-1 in breast cancer tissue, the ELISA should be considered as the gold standard.
The use of fixed, archived paraffin-embedded tissue specimens, enabling more widely available determination of uPA and PAI-1, e.g. by applying specific antibodies in immunohistochemistry, is hampered by the fact that both uPA and PAI-1 antigens are presented by tumour cells and surrounding stroma cells and that these biomarkers are released into the tissue as well, making reliable scoring rather difficult. Still, a first comparison of uPA values obtained by ELISA and by immunohistochemical score was already published in 1990 by Jänicke et al. . A statistically significant increase in the uPA values determined by ELISA was noted with increasing staining intensity in immunohistochemistry. Such a correlation was also published for PAI-1 by Reilly et al. . Various antibodies to uPA and PAI-1 generated in animals have been established and tested by different groups; an overview of published work describing distribution of the uPA/PAI-1 antigens in breast cancer tumour tissue specimens is presented in table table2.2. Work is in progress utilising novel approaches to scan i mmunohistochemically stained breast cancer tumour tissue specimens by use of a high-resolution virtual microscope (figs. (figs.22 and and3)3) combined with automatic image analysis systems. The aim of such studies is to provide an alternative to determination of uPA/PAI-1 in tumour tissue extracts by ELISA to allow worldwide quantification of these cancer biomarkers in routinely available breast cancer specimens, including preoperative core needle biopsies.
Since assessment of uPA and PAI-1 expression status in breast cancer tumour tissues by ELISA requires fresh or fresh-frozen tissue, alternative methods of cancer biomarker analysis using formalin-fixed biopsy material have been investigated. One option is assessment of uPA and PAI-1 marker expression at the transcriptional level. Thus, recently, highly sensitive quantitative reverse transcription-poly-merase chain reaction (RT-PCR) assays requiring only small amounts of mRNA and using formalin-fixed tissue specimens as the test material were established by Biermann et al. . Interestingly, for uPA and PAI-1, when assessing breast cancer cell lines, a significant correlation of transcript and antigen (protein) levels was noted; this, however, did not apply to breast cancer tissue samples, confirming earlier studies in which also no significant correlation of mRNA level with antigen expression was found. Spyratos et al. , by assessing breast cancer tissue specimens, found only borderline correlation of uPA antigen with mRNA expression but a significant correlation for PAI-1. Although these results point to the fact that for clinical decision-making the uPA/PAI-1 ELISAs cannot be replaced at present by uPA/PAI-1 mRNA determination, transcription level determination of these cancer biomarkers may have prognostic impact in certain patient subpopulations, for instance by predicting nodal status, malignant transformation, distant metastasis, disease recurrence, or disease-free survival. Significant differences in the topographical distribution of transcription and protein expression levels of uPA and PAI-1 were noted by Castello et al.  when comparing uPA/PAI-1 expression levels assessed by in situ hybridisation to those obtained by immunohistochemical analyses. Lamy et al.  used a very novel approach, nucleic acid sequence-based amplification (NASBA), and showed high concordance between NASBA and uPA/PAI-1 antigen expression determined by ELISA.
Although still a matter of debate, especially for PAI-1, transcription can also be influenced by genetic factors leading to nucleotide polymorphisms (e.g. 4G/5G) of the PAI-1 gene [63, 64]. In contrast to these studies, Sternlicht et al. , screening more than 2500 tumour tissue samples of breast cancer patients, did not find such an association of PAI-1 polymorphism with mRNA levels and frequency differences between tumour and control collectives, and also no association with annual mortality rates between the different allele subsets. Thus, until now, allele assessment of the 4G/5G PAI-1 polymorphism shows no consistent association with clinical factors.
Epigenetics represents an additional level of gene transcription control . Methylation of cytosine residues in so-called CpG dinucleotide repeats in specific gene promoter regions can influence transcriptional activity, associating methylated CpGs with transcriptional silencing of the respective gene. This mechanism is facilitated by a complex machinery of enzymes, including DNA methyl transferases (DNMTs), demethylases, methylated DNA binding proteins (MBDs) and histone-modifying enzymes, linking DNA methylation with transcriptional repressive chromatin status . Since epigenetic markers are DNA based, assessment of DNA methylation markers can easily be carried out in formalin-fixed, paraffin-embedded biopsies using DNA array technology, sequencing, or PCR-based assays. Xing et al.  and Gao et al.  showed an association of uPA and PAI-1 promoter methylation status with respective mRNA expression in breast cancer cell lines and an association with invasive capacity, which could be modulated by methylating (S-adenosylmethionine, SAM), demethylating (5-azacytidine, decitabine), or histone deacetylase-in-hibiting drugs (Trichostatin, TSA). In breast cancer tissues, Pakneshan et al.  found a strong correlation of uPA DNA methylation status with its respective mRNA levels and increased demethylation of the promoter region with increasing tumour grading. New approaches for silencing of uPA or PAI-1 transcription involve RNA interference (short hairpin RNA (shRNA), antisense RNA) as demonstrated by Meyret-Figuieres et al. , Arens et al. , and Ishii et al. . Future breast cancer studies may thus, in addition to uPA/PAI-1 antigen and mRNA measurements, consider assessment of nucleotide modifications and epigenetic variations of the uPA/PAI-1 genes to provide additional clinical information eventually leading to improved management of the breast cancer disease.
The cancer biomarkers uPA and PAI-1 are linked to tumour invasion and metastasis in patients afflicted with solid malignant tumours, such as breast cancer. The prognostic and predictive value of these proteolytic factors was shown in numerous validated retrospective and prospective studies, including a multicentre clinical trial (Chemo-N0). Thus, uPA and PAI-1 were awarded the highest level of evidence, LOE-1, based on the ASCO tumour marker utility grading system. So far, most of the clinically relevant data have been collected by quantitatively determining the uPA and PAI-1 antigens contents in primary breast cancer tumour tissue extracts by certified ELISA tests. Although these tests are highly validated and quality-assured, alternative techniques not requiring fresh-frozen tissue are currently being explored. So far, none of the alternative ways of assessment at the gene or protein level has yielded satisfactory results, but research in this direction is encouraged. In particular, improved immunohistochemistry formats and quantitative assessment of epigenetic modifications of uPA and PAI-1 may provide new tools and vistas to determine these important cancer biomarkers even in small tissue samples such as core needle biopsies or single cells. Comparison of the clinical i mpact of uPA and PAI-1 in breast cancer to breast cancer mRNA signatures, such as the Amsterdam 70-gene signature (Mamma-print®), the Rotterdam 76-gene signature, the Oncotype DX®, or the H/I signature, have not yet been published [73, 74].