From the Department of Pharmacology and Pathobiology, Royal Veterinary and Agricultural University, Ridebanevej 9, DK-1870 Frederiksberg C, Copenhagen, Denmark,
Department of Chemical Endocrinology, University Medical Centre Nijmegen, P.O. Box 9101, Geert Groteplein 10, NL-6500 HB Nijmegen, The Netherlands, ¶ Clinical Research Unit, Department of Obstetrics & Gynaecology, Technical University of Munich, Ismaninger Strasse 22, D-81675 München, Germany, and || Rotterdam Cancer Institute (Daniel der Hoed Klinik), Josephine Nefkens Building, Nr. BE 426, Dr. Molewaterplein 50, NL-3015 GE Rotterdam, The Netherlands
A very broad definition of a tumor marker is: a tool that enables the clinician to answer clinically relevant questions regarding a cancer disease (1). However, most researchers in this field would probably prefer the following more specific definition of a tumor marker: a molecule, a process, or a substance that is altered quantitatively or qualitatively in precancerous or cancerous conditions, the alteration being detectable by an assay (2). Alterations can be produced either by the tumor itself or by the surrounding normal tissue as a response to tumor cells (2). Regardless of which definition is preferred, the tumor marker itself can be DNA, mRNA, protein, or processes (apoptosis, angiogenesis, proliferation, etc.) measured quantitatively or qualitatively by an appropriate assay. In addition, the types of specimen in which the tumor marker is detected can be different; tissue, blood (plasma/serum), saliva, urine, etc. are all used. The tumor marker assays can be of very different formats ranging from complex animal models to immunohistochemical test kits. The most commonly used format is probably the immunoassay, which is a well-characterized methodology. However, this field is progressing rapidly, and new and advanced assays such as microarrays and mass spectrometry are becoming established technologies in tumor marker research.
The first known tumor marker was described in 1846, when Henry Bence-Jones reported the precipitation of a protein in acidified urine from patients with multiple myeloma. Detection of the monoclonal immunoglobulin light chain in this disease is still in use, and since then numerous potential tumor markers have been reported on in the literature (1). Examples of such markers in clinical use are: alpha-fetoprotein for tumors of the liver, testis, and other germ cell line tumors, CA125 for ovarian cancer, prostate specific antigen (PSA)1 for prostate cancer, and steroid hormone receptors (estrogen and progesterone receptor) used in management of breast cancer. However, as the field of tumor markers has expanded rapidly over the last two decades with a concomitant increase in published reports, it has become increasingly apparent that a strong need exists for establishment of consensus guidelines for development and use of tumor markers. Such guidelines should be internationally accepted if any of these potential new markers are ever to reach a stage where they will benefit the patients. The guidelines should define the potential specific clinical uses of tumor markers, define specific requirements for the technical development of tumor marker assays, and state specific requirements that are to be fulfilled before clinical implementation of a tumor marker. Suggestions for such guidelines have been made; in 1996, a tumor marker expert panel convened by the American Society of Clinical Oncology proposed a framework to be used for evaluation of tumor marker studies: the tumor marker utility grading system (TMUGS), which also includes a framework for rating published evidence (2). The TMUGS framework is further discussed in "Clinical Testing." However, work in this field is still ongoing, and some important aspects to consider in the process of designing such guidelines will be covered in this review.
The possible clinical uses of tumor markers are manifold, and several categories of markers can be defined. A diagnostic tumor marker is a marker that will aid in detection of malignant disease in an individual. Preferably, the marker should be tissue specific and not be influenced by benign diseases of the particular tissue/organ. Thus, a diagnostic marker should exhibit both high levels of diagnostic sensitivity and specificity (see below) to be of clinical value, especially if the marker is to be used for (mass) screening purposes. A fundamental prerequisite for development of any diagnostic (screening) tumor marker lies in the nature of the disease because this must be relatively prevalent, the biology of the disease must be known, and, naturally, an effective treatment should be at hand (3). Measurement of a prognostic marker gives the clinician a tool for estimating the risk of disease recurrence and/or cancer-related death for an individual patient following the initial surgical removal of the cancer but without administration of adjuvant therapy. In contrast, a predictive tumor marker will foretell how the patient is going to respond to a given therapy. Tumor markers for detection of recurrence or remission are classified as monitoring markers and are used during follow-up of patients who do or do not receive anticancer therapy. Finally, a new and potentially important area includes the use of tumor markers for localizing tumors and for targeting of cytotoxic agents (1).
Thus, the uses of tumor markers are numerous. However, regardless of the type of tumor marker, in order for a marker to be considered for routine implementation it needs to be demonstrated that measurement of the marker ultimately impacts on clinical management of the malignant disease either by improving patient outcome or quality of life or by lowering costs of care (2).
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SCREENING MARKERS |
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The desired or required specificity is also dependent on the prevalence of a particular cancer disease in the study population. For example, by studying a group of individuals with a particularly high risk of disease, e.g. due to family history, an increase in positive predictive value is gained, because a number of the false-positive individuals will be among the nonstudied population. Screening for colon cancer is a good example: If the prevalence of the disease is considered 1:1000 in a population of 50- to 80-year-old individuals, and the test has a specificity of 95%, on average 50 colonoscopies have to be performed in order to detect one colon cancer patient. By preselecting patients at high risk for colon cancer, e.g. one or more family members with prior colon cancer, patients with prior colon adenoma, or patients with inflammatory bowel diseases, the prevalence of colon cancer increases to for example 1:100. If the prevalence increases ten times, one only has to perform one-tenth the number of colonoscopies in order to find one colon cancer. In the present example this means 5 colonoscopies. It should be emphasized that selecting a high-risk population for screening has no impact on the sensitivity of the test. The sensitivity could be increased by, for instance, including an additional screening marker in the test, which is independent of the first marker and thus provides additional information.
A receiver operating curve can graphically illustrate the relationship between specificity and sensitivity. The ideal curve is the one giving the maximum possible area under the curve. Fig. 1 shows a receiver operating curve from one of our studies of plasma concentrations of tissue inhibitor of metalloproteinases-1 (TIMP-1) in healthy individuals and colon cancer patients (4). 1-Specificity is depicted on the x axis and sensitivity shown on the y axis. We added information from measurements of serum concentrations of another protein, carcino-embryonic antigen (CEA) and were able to show that this added significantly to the area under the TIMP-1 curve.
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Lack of patients compliance is a major problem in cancer screening. In order to assure good patient compliance, a screening test should be as noninvasive as possible, and ideally the assay should enable testing either by the patient himself or by the family physician.
Another crucial point that has to be considered for any screening method is that it should primarily detect early stage disease where the chance of cure by subsequent standard treatment is high.
As for any marker study, sufficient evidence for the clinical use of the marker in question has to be provided. For screening markers, this will include randomized controlled studies as well as systematic reviews or meta-analyses of published and hitherto unpublished data.
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PROGNOSTIC MARKERS |
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A good example of a prognostic marker, which was thoroughly developed according to the criteria initially suggested by McGuire et al. (5) (Table I) and which is now being used in clinical practice, is the urokinase-type plasminogen activation system with its components urokinase-type plasminogen activator (uPA) and plasminogen-activator inhibitor type-1 (PAI-1) in breast cancer. Invasion factors uPA and its inhibitor PAI-1 are the first novel tumor biological prognostic factors that have been validated at the highest level of evidence (to be discussed later) with regard to their clinical utility in breast cancer.
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2. Standardized Method
Antigen levels of both factors are determined by standardized, quality assured enzyme-linked immunosorbent assays in extracts of primary tumor tissue (7).
36. Demonstration of Clinical Impact
Numerous international studies have shown that patients with low levels of uPA and PAI-1 have a significantly better survival rate than patients with high levels of either factor (reviewed in Ref. 8).
78. Validation and Therapy Trial
Recently, these data have been validated by a multicenter prospective, randomized therapy trial in node-negative breast cancer ("Chemo N0") (9) and a European Organization for Research and Treatment of Cancer (EORTC) Receptor and Biomarker Group (RBG) pooled analysis comprising more than 8300 breast cancer patients (10).
The particular combination of both factors, uPA/PAI-1 (both low versus either or both factors high), outperforms the single factors as well as other, established prognostic factors with regard to risk group assessment, particularly in node-negative breast cancer.
9. Transfer to Clinical Practice
Node-negative breast cancer patients with low levels of uPA and PAI-1 have a very good prognosis and may thus be candidates for being spared the burden of adjuvant chemotherapy. In contrast, node-negative patients with high uPA/PAI-1 are at substantially increased risk of relapse, comparable to that of patients with three or more involved axillary lymph nodes. First results from the Chemo N0 trial indicated that these high-risk patients benefit from adjuvant chemotherapy. The prospective results have recently been substantiated by a large retrospective analysis indicating that breast cancer patients with high uPA/PAI-1 may derive particular benefit from adjuvant chemotherapy (11). A new therapy trial (NNBC-3, node-negative breast carcinoma) will now investigate the optimal chemotherapy for high-risk node-negative patients with high uPA/PAI-1. Last but not least, uPA and PAI-1 are promising targets for tumor biological therapy, and novel therapeutic approaches are currently being evaluated in preclinical models and early phase clinical trials (12).
Next to uPA/PAI-1, such consistent evaluation of clinical utility for a prognostic factor in breast cancer has only been demonstrated for the proliferation marker thymidine labeling index in Italy (1315). As discussed above for uPA/PAI-1, thorough evaluation of a prognostic marker may be a long process comparable time-wise to that of developing a novel therapeutic agent (Fig. 2).
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Second, prognostic markers that are significantly correlated to a particular course of the disease may not necessarily be clinically useful. The clinical utility of prognostic, and other, markers depends on the fact whether the result of marker determination alters clinical decision making, i.e. whether additional diagnostic tests or treatments will be advised or not or whether closer or less frequent follow-up will be recommended. For example, a marker may significantly split the patient cohort into two groups, yet if the percentage of patients within the high- or low-risk group is too small or too large in order to warrant any clinical consequences, the marker may be clinically useless. Similarly, if patient prognosis in the high- or low-risk group is too poor in order to forgo any additional therapy, the marker may also not be suitable for clinical application. Thus, successful validation of prognostic markers for clinical utility requires an interdisciplinary effort of basic researchers, clinicians, pathologists, and biostatisticians.
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PREDICTIVE MARKERS |
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A more direct way to study the predictive value of a tumor-associated factor is analysis of response-to-therapy in patients with locally advanced or metastatic cancer. In these studies, the end point is the directly measurable effect of treatment on the size of the recurrence(s) or the development of new relapses. This is different from studies performed in the adjuvant setting in which potentially cured cancer patients are evaluated with relapse occurrence as the primary end point, which, as discussed above, in nonrandomized studies not necessarily reflects an association between a marker and efficacy of adjuvant treatment. Furthermore, in contrast to studies performed in the recurrent setting, the type of response (as complete remission, partial remission, stable disease, progressive disease) cannot be studied in adjuvant studies because, by definition, there is no measurable tumor mass and there may be only nonmeasurable occult micrometastases present. Advantages of studying the predictive value of a marker in the metastatic setting are that the follow-up period needed for response assessment may only be less than 1 year and that, because of the yes or no answer, the patient number may be relatively low. Furthermore, tumor samples of nonrandomized patient cohorts can be studied retrospectively because the treatment effect is directly measurable by the size of the recurrence or occurrence of new metastases. In contrast, adjuvant studies need to be randomized, require accrual of hundreds of patients, and require many years of follow-up to give an answer with respect to the predictive value of the marker studied. However, there is one main disadvantage of measuring cell biological factors in the primary tumor, and then later correlating their expression levels to the type of response to systemic therapy that is given for a recurrence up to 10 years after primary therapy. The recurrent tumor, which is used for response assessment, may be phenotypically or genetically different from the primary tumor in which the markers were originally analyzed. Thus, this approach only works for markers that are not known to change their expression between primary lesions and metastases, even while withstanding adjuvant systemic therapy. Moreover, as stated before for the adjuvant setting, proper statistical methods need to be used in order to adjust for confounding factors such as different types of adjuvant therapy, etc.
Nevertheless, for the past 20 years, presence of ER and PgR in primary breast tumors has been the major guide for physicians to treat recurrent breast cancer patients successfully with endocrine therapy. However, the steroid hormone receptor status of the primary tumor does not fully predict which patients will fail or respond to endocrine therapy (Fig. 3). This also holds true with regard to adjuvant endocrine treatment of ER-positive primary breast cancer patients, showing benefit in only 2025% of patients. Therefore, many potential cell biological predictive markers, which may also serve as potential targets for future therapeutic intervention, have been studied (22). The availability of currently employed high-throughput technologies to simultaneously assess the expression levels of tens of thousands of genes will hopefully lead to identification of new and powerful predictive markers, and possibly to the development of new therapeutic strategies.
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MONITORING MARKERS |
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Measuring tumor markers for evaluation of treatment effects is often used as a surrogate end point for treatment efficacy. Such markers are of obvious value, because patients who do not respond to the applied treatment could at a very early time point either be shifted to another treatment or treatment could be stopped and thereby the patient would escape potential toxic side effects of the treatment. In germ cell line tumors, alpha-fetoprotein and human chorionic gonadotropin-beta are both being used to follow treatment efficacy.
In patients with gastrointestinal cancers, CEA and CA19.9 are used to follow the patients after primary therapy in order to detect disease recurrence at an early stage. Similarly, in patients with ovarian cancer, CA125 is a frequently used serum marker for monitoring of recurrent disease. Besides being used for monitoring of treatment efficacy as mentioned above, alpha-fetoprotein and human chorionic gonadotropin-beta are also used to monitor patients with germ cell line tumors in order to detect recurrent disease. It should be emphasized, though, that in order to have clinical value, it has to be demonstrated that early detection of recurrent disease has an impact on patient survival. For example, in breast cancer, no survival benefit has been linked to any tumor marker measurement in regular follow-up after primary therapy. Thus, tumor markers have disappeared form the respective guidelines; yet, they are useful monitoring tools during systemic therapy in those breast cancer patients experiencing recurrence of disease.
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QUALITY ASSURANCE OF BIOMARKERS |
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Standardization of Laboratory Tests
Although immunometric methods are extensively used in clinical research settings, standardization and quality control is often lacking. Still, assay results from unvalidated markers are being made available to clinicians, but guidelines on how the results should be interpreted are often missing. As most of the clinical trials involve multicenter cooperation, special emphasis should be put on the quality and reproducibility of the assays performed in the different laboratories. An excellent example of what can be reached with proper quality assessment and assurance is measurement of steroid hormone receptors in breast cancer tissue extracts. Determination of ER and PgR content of human breast tumors is widely used for prediction of response to endocrine therapy (24) as described above, and also for indicating patient prognosis. Within Europe, a multitude of translational multicenter cancer studies have been coordinated by the EORTC. Within the EORTC, the RBG serves to this consortium to research and advise on common methodologies for biomarker assays and to ensure that appropriate external quality assessment schemes are available. In the past 20 years, large-scale external quality assessment trials for ER and PgR, amounting to 165 participating laboratories from 18 countries, have been carried out organized by the RBG (25, 26). During several workshops, adequate and uniform methodologies for ER and PgR assays performed were defined and applied (2729). By use of calibration vials, the initial between-laboratories coefficients of variation of 45% were substantially reduced to less than 15% (25).
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FINAL TEST RESULTS DEPEND ON TOTAL OF PREANALYTICAL, ANALYTICAL, AND POSTANALYTICAL ASPECTS |
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Analytical Aspects
Prior to producing test results used for clinical application, for each of the methods a (test) laboratory must verify or establish performance specifications of the test. The following analytical specifications have to be assessed: type of standard/reference material, recovery rate, accuracy, precision, sensitivity, specificity, linearity, and interferences.
A standard (reference material) is used to relate the reading of an assay to the quantity of measured analyte. Regarding the standard employed, in immunometric assays one should take care of the stability of the standard, buffer composition, and affinity between the standard and antibody.
It is worth mentioning that analytes extracted from tumor tissue may be different in nature from those present in the peripheral blood circulation of a patient. Therefore, an assay designed for measurement of an analyte in tissue extracts may not always be suitable for assaying the same analyte in plasma or serum. In recovery experiments, a known amount of standard is added to samples with a known amount of biomarker and then recovery of the added marker is calculated. This will provide information on the nature of the analyte versus standard and/or on any interfering process. The accuracy of an assay is the agreement between the best estimate of a quantity and its true value. Still, only for analytes for which a reference method is available is such comparison possible. The precision of an assay is defined by the agreement between replicate measurements. For validation of an assay, at least the intra-sample, intra-assay precision performance should be included. The precision profile is an ideal tool to assess this. Two types of sensitivity are of interest in immunoassays. The limit of detection (analytical sensitivity) is defined by the lowest concentration detected that is significantly different from zero. The limit of quantification (functional sensitivity) is the lowest concentration at which a test result can be reliably measured with a coefficient of variation of <20%. The specificity of immunometric assays strongly depends on antibody characteristics. Polyclonal or monoclonal antibodies or mixtures of both are applied in different test kits. In general, when polyclonal antibodies are used there is increased sensitivity but also an increased risk that one of these antibodies will recognize an epitope on a different antigen, resulting in decreased specificity. In contrast, monoclonal antibodies are directed to a single epitope and have higher specificity. Most assay procedures demand that samples be diluted to within a specified range of protein content prior to the assay, but values multiplied by the dilution factor should give the same results, irrespective of extent of dilution (i.e. parallelism/linearity studies). One has to be aware of, however, that blood from patients treated with immunotherapy may contain antibodies against these therapeutic antibodies, which may interfere in sandwich assays leading to false-positive or false-negative test results. Such heterophilic antibodies can also occur through frequent contact of the patients with animals. Moreover, blood from patients with infectious diseases may contain high amounts of IgM molecules, causing nonspecific reactions in sandwich-type immunoassays. Assays should be designed to reduce these potential interferences.
Postanalytical Aspects
It is of crucial importance to establish specific guidelines for interpretation of assay results if the marker is to be used in the clinical setting, as numerous statistical approaches are available to process the assay read-outs and interpolate them in the standard curves. Also, for multicenter studies, at each of the centers, the same statistical approach to processing of assay data should be used. Reference intervals for the tumor marker are needed for the specification of "high" and "low" levels, according to which patients can be divided into relevant risk groups if the marker is used for prognostic stratification, and also a reference range can help the clinician in identification of patients with cancer disease if the marker is validated for diagnostic purposes. Because of population sampling errors and biological variation, every laboratory should establish its own reference values, if appropriate.
Standardization of Total Protein Assay
In general, the amount of a biological marker detected in a tumor tissue extract is related to the total protein content of the sample. Therefore, it is important to standardize the protein assay as well, as high within- and between-laboratory coefficients of variation of protein assays have been reported (25).
Quality Monitoring
New assays should be thoroughly validated upon first use. Design of an adequate control procedure should start with a definition of quality requirements weighing acceptable error against needed clinical decision levels. Every assay consists of a measurement procedure to determine analyte levels of a biomarker and a control procedure in which, by measurement of control samples, the validity of the measurement of the samples can be checked. Comparison of the analyte values of control samples against predefined limits should always be an integral part of the assay procedure. Control (or reference) preparations should be time and temperature resistant with little or no vial-to-vial variation, homogeneous, similar in buffer/matrix composition to the test material, available at concentrations that cover the physiological range expected in the experimental material, and available in sufficient quantity.
For internal quality control (QC) purposes, the laboratory must include samples of different concentrations of control material (tissue extracts, serum, plasma, bodily fluids, etc.). Repeated measurement of control samples allows determination of the imprecision of the assay system. The long-term trend in assay performance should be checked regularly in order to detect any shift or drift.
For external QC purposes, preparations distributed by a reference laboratory should be included. External QC programs serve to monitor long-term assay performance within a laboratory. Moreover, they provide comparison of assay results between laboratories. This enables the external QC organization to assess systematic errors between laboratories. Systematic differences in test results pave the way for calibration, and successful normalization of data from different laboratories form one of the cornerstones of valid multicenter studies on the potential value of biomarkers. Normalization can only be achieved, however, if a marker is homogeneous in nature, preferentially with only one molecular form present.
It should be noted that in the case of using lyophilized cytosols as external QC samples, no conclusions can be derived with regard to preanalytical issues as the use of external controls only covers reproducibility of the analytical assay procedure and subsequent computation of data. Providing proper instructions is the only feasible way to monitor (between-hospital) variations in sample treatment conditions. Because most clinical trials are carried out on a multicenter basis, the importance of between-laboratory QC cannot be overemphasized. Therefore, all steps in the procedure from taking biopsies to reporting assay results to the clinician, including the preanalytical ones, should be subjected to strict handling. These handlings should be described in standard operating procedures.
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CLINICAL TESTING |
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In 1996, the TMUGS framework was proposed by the American Society of Clinical Oncology (2). In this framework, all available published data for the tumor marker in question forms the basis of an evaluation and subsequent scoring of the marker. However, in recognition of the great variability in quality and therefore in validity of reported tumor marker studies, a system for classification of published data into certain levels of evidence (LOE) was incorporated in the TMUGS framework. Herein, the published papers evaluated are categorized according to evidence levels ranging from V to I depending on study design and size. The lowest level of evidence is LOE V, where the evidence for the tumor marker has been gained from small pilot studies. LOE IV and III are descriptive for retrospective studies of either small (IV) or large size (III); most tumor marker studies are of these two levels. LOE II comes from prospective therapeutic studies, where the primary question is therapeutic; marker testing is a secondary goal. LOE I studies are either high-powered prospective studies with the primary aim of testing the clinical validity of the tumor marker or meta-analyses of (several) studies of lower LOE stages.
With the LOE tool, it is possible to attribute a semiquantitative score to a potential tumor marker based on the published evidence available. The possible marker scores of the TMUGS range from "0" to "+++" (2). A score of "0" signifies that the tumor marker in question is safely concluded to be of no clinical utility based on sufficient data. "NA" (not available) denotes a lack of data for the marker. The scores of "+/-" to "+++" correspond to grades from "investigational with only preliminary data" to "independent information for clinical decision-making". Only markers deemed to be "++" or "+++" should be implemented as standard practice in clinical management of cancerous disease.
Thus, the TMUGS is a framework that covers important aspects to evaluate and take into consideration when working with tumor markers. In particular, it illustrates the need for systematization and rating of evidence when transferring markers from the laboratory into clinical practice.
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CONCLUSIONS |
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Quality assurance is an issue of crucial importance in biomarker research and when implementing biomarkers in the clinic. A major problem associated with evaluating biomarkers in tissues, blood, or bodily fluids is that different procedures (sample collection, sample storage, sample processing) and different assay formats may yield different results. Therefore, assays and procedures have to be standardized and standard operating procedures should be developed for each type of sample and assay format, and the quality of the biomarker assay results should be monitored by continuous between-laboratory proficiency testing of performance. Although for some markers considerable progress has been made in the standardization of methods and assay protocols, efforts should be continued as only the stringent application of quality control systems enables a consistent assessment of the clinical value of biomarkers.
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FOOTNOTES |
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Published, MCP Papers in Press, June 17, 2003, DOI 10.1074/mcp.R300006-MCP200
1 The abbreviations used are: PSA, prostate specific antigen; TMUGS, tumor marker utility grading system; CEA, carcino-embryonic antigen; uPA, urokinase-type plasminogen activator; PAI-1, plasminogen activator inhibitor type-1; EORTC, European Organisation for Research and Treatment of Cancer; RBG, Receptor and Biomarker Group; ER, estrogen receptor; PgR, progesterone receptor; HCG, human chorionic gonadotropin; QC, quality control; LOE, level of evidence.
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Department of Pharmacology and Pathobiology, Royal Veterinary and Agricultural University, Ridebanevej 9, DK-1870 Frederiksberg C, Copenhagen, Denmark. Tel.: +45 35283130; Fax: +45 35353514; E-mail: nbr{at}kvl.dk
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REFERENCES |
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