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WO2011047688A1 - Valeur pronostique et prédictive des formes intactes et clivées du récepteur de l'activateur de la plasminogène urokinase dans le cancer de la prostate - Google Patents

Valeur pronostique et prédictive des formes intactes et clivées du récepteur de l'activateur de la plasminogène urokinase dans le cancer de la prostate Download PDF

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WO2011047688A1
WO2011047688A1 PCT/DK2010/050280 DK2010050280W WO2011047688A1 WO 2011047688 A1 WO2011047688 A1 WO 2011047688A1 DK 2010050280 W DK2010050280 W DK 2010050280W WO 2011047688 A1 WO2011047688 A1 WO 2011047688A1
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Prior art keywords
upar
level
lll
prostate cancer
forms
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Ib Jarle Christensen
Klaus Brasso
Gunilla Højer HANSEN
Charlotte Almasi
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Rigshospitalet
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Rigshospitalet
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/972—Plasminogen activators
    • G01N2333/9723—Urokinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the present invention relates to the methods of classifying the severity, selecting a treatment for prostate cancer in a subject and/or monitoring the progression of the disease before, during and after administering a treatment, wherein a predetermined level of one or more uPAR forms above a reference level indicates the need for administering a treatment.
  • the present invention further relates to a kit and a device that may be used in the method of the present invention comprising means for measuring the level of one or more uPAR forms in a sample; and means for comparing the measured level of one or more uPAR forms with at least one reference level of one or more uPAR forms.
  • PC Prostate cancer
  • PSA prostate-specific antigen
  • More than 90% of PC cases are diagnosed as either localized or locally advanced disease, where the 5-year survival rate approaches 100%.
  • the patients with metastatic PC at diagnosis face a poor prognosis with 5-year relative survival rate of only 33.5% (1995-2001 figures) and a median survival of 2 - 3 years (3-5).
  • ADT metastatic PC androgen-deprivation therapy
  • ADT may be managed with either castration - surgical or medical- with or without supplementary anti-androgen therapy.
  • OS overall survival
  • EOD bone metastasis
  • WHO PS World Health Organization performance status
  • weight loss age at diagnosis, presence of co-morbidity
  • levels of hemoglobin, lactate dehydrogenase, bone-specific alkaline phosphatase and other markers relating to bone remodeling also play a role, although they appear to be less important than EOD (8, 1 1 ).
  • a single marker or method that would facilitate selecting between treatments of varying efficacy and/or monitoring the progression or determining the stage of a prostate cancer prior to, during and following administration of a given treatment would greatly improve the ease with which these selection and monitoration processes occur today.
  • markers are known to be associated with specific cancer types; examples hereof include HER2 which is a marker of certain forms of breast cancer and EGFR which has been linked to lung cancer and glioblastoma multiforme.
  • Administering the best possible treatment for each individual patient with prostate cancer would improve the efficacy of any treatment whether it involves administration of medicaments, surgery, or other and independent of whether the treatment given is prophylactic, curative or ameliorative.
  • a classification of the individuals suffering from a prostate cancer according to survival prognosis and time to progression would be of assistance in determining the best possible treatment, improving the effect of an administered treatment, improving the survival rate, lower relapse risks, and heighten the quality of life following the outbreak of prostate cancer.
  • monitoring the treatment administered to any individual patient depending upon the progression and/or state of their disease would be of assistance in
  • the urokinase plasminogen activator (uPA) system is an extracellular protease system, active in both normal and pathological tissue remodeling processes, including cancer invasion (14, 15).
  • uPA is bound to its receptor, uPAR, and by proteolytic cleavage uPA converts plasminogen into active plasmin, which is involved in degradation of the extracellular matrix (ECM).
  • ECM extracellular matrix
  • uPAR is a glycosyl-phosphatidylinositol anchored 55-60 kD membrane glycoprotein consisting of three homologous domains, denoted I, II and III. Intact uPAR (uPAR l-lll) is required for high affinity binding of uPA (16, 17).
  • uPA and plasmin generated by uPA can cleave intact uPAR (uPAR(l-lll)) liberating domain I (uPAR(l))(18).
  • the remaining domain, uPAR(ll-lll) will be left on the cell surface, but can like intact uPAR(l-lll) be shed from the cells.
  • uPAR(l-lll), uPAR(l) and uPAR(ll-lll) are therefore all found in blood, and particularly the levels of the two latter forms may reflect the overall activity of the uPA system in the body (47).
  • uPAR has previously been measured by ELISA methods that detect the total amount of uPAR immunoreactive proteins, and high uPAR levels were found to be associated with short OS when measured in tumor tissue extracts from patients with lung, breast, gastric and colorectal cancer (21 -24) and in blood from patients with colorectal and breast cancer (25, 26). In patients with localized PC, high preoperative serum uPAR was shown to correlate with early biochemical progression (27).
  • uPAR(l) The NH 2 -terminal domain, uPAR(l), is required for uPA binding to uPAR(l-lll), so cleavage abolishes the binding potential of uPAR.
  • uPAR(l-lll) lacking at least the lipid moiety of the glycolipid anchor cannot be cleaved by physiologically relevant concentrations of uPA (48)..
  • In vitro proteases other than uPA have been shown to cleave both uPAR(l-lll) and uPAR(l-lll), but their functionality in vivo remains to be demonstrated (47).
  • uPA is the only protease shown to cleave uPAR(l-lll) in vivo (20).
  • uPA is the only protease capable of in vivo uPAR(l-lll) cleavage
  • all the uPAR(ll-lll) found in body fluids is the result of shedding of cell surface uPAR(ll-lll) and all uPAR(l) found results from uPA catalyzed uPAR(l-lll) cleavage.
  • Components of the uPA system including uPAR are, like components of other matrix degrading systems, in many types of cancer mainly or exclusively expressed by stromal cells such as fibroblasts, macrophages and neutrophils and this is also the case in prostate cancer (44).
  • hormones, including estrogens often exert their effect in the normal prostate gland as well as in prostate cancer by acting on stromal cells rather than epithelial cells (45, 46).
  • uPAR(l) levels were found to be significantly associated to overall survival (OS), being a stronger prognostic parameter than the total amount of uPAR as measured by ELISA (49). It has been shown, that serum levels of uPAR(l) and uPAR(ll-lll) were higher in PC than in benign prostate hyperplasia (BPH). There was only a weak correlation between uPAR forms and PSA, and a combination of PSA and uPAR forms significantly increased the detection of PC (30).
  • TR- FIAs time-resolved fluoroimmunoassays
  • a main aspect of the present invention relates to a method for pre-diagnosing, and/or classifying the severity based on aggressiveness and/or determining a therapy and/or monitoring a therapeutic treatment of a prostate cancer in a subject, said method comprising:
  • a specific aspect regards the above method for classifying the severity and/or determining a therapy and/or monitoring a therapeutic treatment of a prostate cancer in a subject, preferably of metastatic prostate cancer.
  • the one or more reference levels of one or more uPAR forms may for instance be a set of reference levels of one or more uPAR forms obtained by measuring the one or more uPAR forms levels in samples from healthy individuals and/or individuals with untreated prostate cancer: a first reference level being the median value of one or more uPAR forms in samples from healthy individuals, a second reference level being the 95 th percentile of one or more uPAR forms in samples from healthy individuals, a third reference level being the 99 th percentile in samples from healthy individuals, a fourth reference level being the median value of one or more uPAR forms in individuals with previously untreated prostate cancer, a fifth reference level being the 95 th percentile of one or more uPAR forms in samples from individuals with previously untreated prostate cancer, a sixth reference level being the 99 th percentile in individuals with previously untreated prostate cancer, a seventh reference level being a factor at least above 1 .1 of the median value of one or more uPAR forms in healthy individuals, an eight reference level being a factor at least
  • the one or more reference levels may for instance be a set of reference levels of uPAR (l-lll) obtained by measuring the uPAR(l-lll) levels in samples from healthy individuals and/or individuals with untreated prostate cancer: a first reference level being the median value of uPAR(l-ll l) in samples from healthy individuals, a second reference level being the 95 th percentile of uPAR(l-lll) in samples from healthy individuals, a third reference level being the 97.5 th percentile of uPAR(l-lll) in samples from healthy individuals in individuals a fourth reference level being the median value of uPAR(l-lll) in individuals with previously untreated prostate cancer, a fifth reference level being the 95 th percentile of uPAR(l-lll) in samples from individuals with previously untreated prostate cancer, a sixth reference level being the 99 th percentile of uPAR(l-lll) in individuals with previously untreated prostate cancer, a seventh reference level being a factor at least above 1
  • the one or more reference levels may for instance be a set of reference levels of uPAR (l-lll)+(ll-lll) comprises a set of reference levels of uPAR (I- lll)+(ll-lll) obtained by measuring the uPAR (l-lll)+(ll-lll) levels in samples from from healthy individuals and/or individuals with untreated prostate cancer: a first reference level being the median value of uPAR (l-lll)+(ll-lll) in samples from healthy individuals, a second reference level being the 95 th percentile of uPAR (l-lll)+(ll-lll) in samples from healthy individuals, a third reference level being the 97.5 th percentile of uPAR (l-lll)+(ll- III) in samples from healthy individuals in individuals a fourth reference level being the median value uPAR (l-lll)+(ll-lll) in individuals with previously untreated prostate cancer, a fifth reference level being the 95 th percentile of
  • the one or more reference levels may for instance be a set of reference levels of uPAR (l-lll) comprises a set of reference levels of uPAR (l-lll) obtained by measuring the uPAR (l-lll) levels in samples from from healthy individuals and/or individuals with untreated prostate cancer: a first reference level being the median value of uPAR (l-lll) in samples from healthy individuals, a second reference level being the 95 th percentile of uPAR (l-lll) in samples from healthy individuals, a third reference level being the 97.5 th percentile of uPAR (l-lll)+(ll-lll) in samples from healthy individuals in individuals a fourth reference level being the median value uPAR ((l-lll) in individuals with previously untreated prostate cancer, a fifth reference level being the 95 th percentile of uPAR (l-lll) in samples from individuals with previously untreated prostate cancer, a sixth reference level being the 99 th percentile of uPAR (l-l)
  • one embodiment of the first aspect of the present invention regards a method for classifying the severity of a prostate cancer based on the aggressiveness of said prostate cancer in a subject, said method comprising
  • a second embodiment of the present invention regards a method for determining a therapy for a prostate cancer in a subject, said method comprising:
  • a third embodiment regards a method for monitoring therapeutic treatment of a prostate cancer in a subject, said subject being treated for the specific disease, said method comprising
  • the level of one or more uPAR forms with respect to the one or more reference levels indicates the development of said prostate cancer during or after the specific treatment regime and therefore the prognosis. More specifically the prognosis of survival, such as e.g. the prognosis of survival expressed in months.
  • the reference value is, in an alternative embodiment, a reference value obtained from the individual itself and thus a set of embodiments relate to this aspect:
  • An embodiment of the present invention regards a method for pre-diagnosing and/or classifying the severity based on the aggressiveness of said prostate cancer and/or determining a therapy and/or monitoring a therapeutic treatment of a prostate cancer in a subject, said method comprising:
  • a level of one or more uPAR forms in the sample above the reference level indicates the presence of a prostate cancer and/or the severity of said cancer is deduced from said comparison and/or deducing the progress and/or state of said cancer by said comparison, and/or based thereon determining a therapy to be initiated, continued, terminated or replaced.
  • a specific embodiment regards the monitoring of the prostate cancer during androgen- deprivation therapy, ADT, managed with total androgen blockade (TAB), estrogen, a chemotherapeutic agent, radiotherapy or biologies.
  • a more specific embodiment regards the monitoring of the prostate cancer during treatment with a chemotherapeutic agent.
  • the monitoring is during treatment with androgen-deprivation therapy, ADT, managed with total androgen blockade (TAB), or administration of estrogen.
  • Another specific embodiment regards the use of one or more uPAR forms as a biomarker for the pre-diagnosis of a prostate cancer and/or for classifying the severity of a prostate cancer based on the aggressiveness of said prostate cancer and/or for determining a therapy for a prostate cancer and/or for monitoring a therapeutic treatment of a prostate cancer in a subject.
  • the prostate cancer is a metastatic prostate cancer.
  • Yet another specific embodiment regards the use of one or more uPAR forms as a biomarker for determining a specific therapeutic treatment of said prostate cancer in a subject.
  • the prostate cancer is a metastatic prostate cancer.
  • the present invention as described herein further relates to a device for the pre- diagnosis of the presence of a prostate cancer, and/or for classifying the severity of a prostate cancer based on the aggressiveness of said prostate cancer and/or for determining a therapy for a prostate cancer and/or for monitoring a therapeutic treatment
  • the device comprises means for measuring the level of one or more uPAR forms in a sample; and means for comparing the measured level of one or more uPAR forms with at least one reference level of one or more uPAR forms.
  • the prostate cancer is a metastatic prostate cancer.
  • the present invention as described herein relates to a kit of parts comprising
  • ii) means for comparing the measured level of one or more uPAR forms with one or more reference levels of one or more uPAR forms
  • Another and equally important embodiment regards a method for improving the prognosis of survival in individuals with prostate cancer, said method comprising
  • Figure 1 A-F Kaplan-Meier estimates of survival probabilities for the entire study population (A,D,G), the subpopulation of patients treated by TAB ( ⁇ , ⁇ , ⁇ ) and the subpopulation treated by PEP (C,F,I) for the indicated uPAR forms.
  • Patients are stratified in tertiles according to their serum levels of the uPAR forms. The numbers of patients at risk at 0, 24, 48 and 72 months are shown below the axis and the number of deaths to the left. P-values are calculated by the log rank test for trend.
  • Figure 2 Kaplan-Meier plots for overall survival of the subgroup of prostate cancer patients with pre-treatment uPAR(l-lll)+ uPAR (ll-lll) levels above the median (1 1 1 .9 fmol/ml).
  • the two curves represent patients treated with either intramuscular injections of polyestradiol phosphate (PEP) or total androgen blockade (TAB), respectively.
  • PEP polyestradiol phosphate
  • TAB total androgen blockade
  • FIG. 3A and 3B Dipstick embodiments seen from above. Dipstick support material (1 .) with assay field (2.) for use with the biological sample and one control or standard field (3. in Figure 3A) or multiple control or standard fields (4a. to 4.e. in Figure 3B). Standards of a single (for 3.) or various (one concentration for each field in increasing or decreasing order, e.g.) concentrations of one or more uPAR forms may be applied to the control or standard fields to enable reading a positive / negative result with the stick portrayed in fig. 3A or assessing an approximate concentration of one or more uPAR forms in the biological sample compared to which of the control fields in Fig. 3B the sample / assay field resembles the most post testing. Definitions
  • Ameliorate As used herein, is intended to mean to improve or make better; in association with a disease state a lessening in the severity or progression of a disease state, including remission or cure thereof, alternatively the perceived lessening of severity such as lessening of associated pain.
  • Androgen-deprivation therapy As used herein is intended to mean any Androgen-deprivation therapy or androgen suppression therapy including surgical castration (orchiectomy) and/or chemical castration. ADT reduces the levels of the male hormones, androgens, in the body.
  • the main androgens are testosterone and dihydrotestosterone (DHT). Androgens are produced mainly in the testicles and stimulate prostate cancer cells to grow.
  • DHT dihydrotestosterone
  • Antibody As used herein, is intended to mean Immunoglobulin molecules and active portions or fragments of immunoglobulin molecules such as Fab and F(ab').sub.2 which are capable of binding an epitopic determinant of one or more uPAR forms. Antibodies are for example intact immunoglobulin molecules or fragments thereof retaining the immunologic activity.
  • the term "antigen”, as used herein, is intended to mean an immunogenic full-length or cleaved form of an uPAR molecule.
  • Biological sample As used herein, is intended to mean a sample obtained from a subject or individual. Examples include fluid samples such as blood samples, biopsies or other. It is necessary to validate the assays used in the present invention for each biological sample and also for the types of glass or plastic used as well as taking into account for how long said samples are kept prior to analysis.
  • Biomarker As used herein, is intended to mean a molecular indicator of a specific biological property, such as a pathological or physiological state.
  • uPAR(ll-lll) Cleaved uPAR containing domain II and III
  • CAB Combined androgen blockade
  • ELISA Enzyme-linked immunosorbent assay
  • a specific antibody is affixed to a surface, and the antigen is washed over the surface so that it binds to the catching (fixed) antibody .Then another specific antibody is washed over the surface and binds to the antigen.
  • This antibody could be linked to an enzyme, or a third antibody specific for the second antibody and linked to an enzyme and in the final step a substance is added that the enzyme can convert to a detectable signal.
  • Extent of osseous dissemination is a way to quantify the bone involvement in patients with metastatic PC. Internationally accepted methods include the Soloway score, grouping patients in accordance to the relative bone involvement.
  • Intact uPAR (uPAR(l-lll)): As used herein, is intended to mean the intact, uncleaved form of uPAR.
  • mAb As used herein, is intended to mean a monoclonal antibody.
  • a mAb is an antibody produced by a hybridoma cell. Methods of making monoclonal antibody- synthesizing hybridoma cells are well known to those skilled in the art, e.g, by the fusion of an antibody producing B lymphocyte with an immortalized myeloma cell line.
  • uPAR forms As used herein, is intended to mean any uPAR form including but not limited to intact uPAR (l-lll), cleaved uPAR containing domain (ll-lll), uPAR (I), suPAR, suPAR (l-lll) and suPAR (ll-lll).
  • PSA Prostate-specific antigen
  • kallikrein III also known as kallikrein III, seminin, semenogelase, ⁇ -seminoprotein and P-30 antigen
  • PSA is often elevated in the presence of prostate cancer and in other prostate disorders.
  • a blood test to measure PSA levels is a test currently available for the early detection of prostate cancer.
  • Severity "Severe stage”, “severity”, “less severe” and “more severe”, as used herein, are intended to mean a graduation of severity according to for example prognosis for being cured, prognosis for survival, or according to different predetermined stages of diseases. Such stages may be according to various symptoms, and/or traditionally measureable levels of biomarkers, physical functions etc. When focusing on the development of a disease in one and same subject, then a more severe stage refers to a worsening of the disease, whereas a less severe stage than previously determined refers to a bettering of the disease, e.g. due to a satisfactory treatment regime.
  • Severity based on the aggressiveness of prostate cancer is intended to mean how aggressive a prostate cancer is metastatically.
  • Soluble uPAR As used herein, is intended to mean the soluble form of the uPA receptor (uPAR).
  • Subject and/or individual is intended to mean a single member of a species, herein preferably a mammalian species.
  • the term "mammal”, as used herein, is intended to include both humans and non-humans.
  • the term "patient” as used herein, is intended to mean any individual suffering from a prostate cancer.
  • determining a therapy and/or therapeutic treatment cover in principle any treatment that a person skilled in the art would administer to a subject.
  • the subject may be a subject for which the level of one or more uPAR forms has been determined and compared to that of one or more reference levels.
  • the terms cover the most optimal therapy and/or treatment.
  • the treatment that is best suited for the individual patient in terms of any of the following: ameliorating discomfort, alleviating symptoms, curing the disease or at least attempting same, providing the best possible quality of life and so forth for the subject.
  • the terms “best possible”, “most optimal”, and so forth in regards to a therapy and/or therapeutic treatment are used interchangeably herein.
  • the therapies and or therapeutic treatments to be administered, continued, terminated, altered or replaced may be any kind of therapy such as, but not limited to the administration of medicaments and/or surgery, and may be prophylactic, curative or ameliorative.
  • a therapy and/or therapeutic treatment may be initiated if none is ongoing, or may be continued if it is already taking place.
  • a therapy and/or therapeutic treatment may be terminated if it is found unsuitable or if it requires replacing by an alternative method of therapy and or therapeutic treatment.
  • altering a treatment is understood that the treatment is changed for example the dosage is increased or decreased, the concentrations of the drugs are increased or decreased, the administration / dosage regiment is increased or decreased and so on.
  • Total androgen blockade As used herein is intended to mean any type of prostate cancer hormone therapy which combines an anti-androgen with either chemical castration or surgical castration.
  • uPA receptor As used herein is intended to mean the urokinase receptor (uPA receptor, uPAR or CD87).
  • uPAR domain I As used herein is intended to mean domain I liberated from uPAR.
  • Urokinase plasminogen activator a serine protease. The primary
  • plasminogen which is an inactive zymogen form of the serine protease plasmin. Activation of plasmin triggers a proteolysis cascade which, depending on the physiological environment participate in thrombolysis or extracellular matrix degradation.
  • WHO PS World Health Organization performance status
  • the level of one or more uPAR forms can be used as a biomarker for determining a therapy for and/or monitoring a therapeutic treatment of a prostate cancer in a subject, said determination being based on the severity of a specific prostate cancer as judged by the level of one or more uPAR forms by comparison with one or more reference levels of one or more uPAR forms.
  • the invention regards metastatic prostate cancer.
  • the present inventors have furthermore found that the level of one or more uPAR forms can be used as a marker for keeping track of the development of a prostate cancer, i.e.
  • the level of one or more uPAR forms can be used not only to determine which treatment to administer based on a determination of the severity of a prostate cancer in a subject as judged by the level of one or more uPAR forms, but also to determine which treatment to continue with as determined by monitoring of the severity of a prostate cancer as judged by the level of one or more uPAR forms in a subject.
  • the level of one or more uPAR forms can be used as a biomarker for determining which treatment may be used in order to improve overall survival of an individual with prostate cancer.
  • the present invention discloses that the level of one or more uPAR forms can be used as a biomarker giving an indication of the presence of a prostate cancer. Accordingly, by the method according to the present invention the level of one or more uPAR forms can be used to diagnose the presence of a prostate cancer, preferably in conjunction with another biomarker including but not limited to prostate-specific antigen (PSA), YKL-40 and the aminoterminal propeptide of type III procollagen (P-III-NP). It is further envisaged that the method according to the present invention, may be used as a pre-diagnostic tool in connection with a companion diagnostic test in personalized medicine.
  • PSA prostate-specific antigen
  • YKL-40 the aminoterminal propeptide of type III procollagen
  • determining a therapy and/or therapeutic treatment cover in principle any treatment that a person skilled in the art would administer to a subject for which the level of one or more uPAR forms has been determined and compared to that of one or more reference levels.
  • the terms cover the most optimal therapy and/or treatment.
  • the treatment that is best suited for the individual patient in terms of any of the following: ameliorating discomfort, alleviating symptoms, curing the disease, providing the best possible quality of life and so forth for the subject.
  • best possible “most optimal”, and so forth in regards to a therapy and/or therapeutic treatment are used interchangeably herein.
  • the therapies and or therapeutic treatments to be administered, continued, terminated, altered or replaced may be any kind of therapy such as, but not limited to the administration of medicaments and/or surgery, and may be prophylactic, curative or ameliorative.
  • a therapy and/or therapeutic treatment may be initiated if none is ongoing, or may be continued if it is already taking place.
  • a therapy and/or therapeutic treatment may be terminated if it is found unsuitable or if it requires replacing by an alternative method of therapy and or therapeutic treatment.
  • altering a treatment is understood that the treatment is changed for example the dosage is increased or decreased, the concentrations of the drugs are increased or decreased, the administration / dosage regiment is increased or decreased and so on.
  • an aspect of the present invention relates to a method for pre-diagnosing and/or classifying the severity based on the aggressiveness and/or determining a therapy and/or monitoring a therapeutic treatment of a prostate cancer in a subject, said method comprising:
  • determining the level of one or more uPAR forms in a sample obtained from the subject i) determining the level of one or more uPAR forms in a sample obtained from the subject; and ii) comparing said level of one or more uPAR forms with a reference level of one or more uPAR forms;
  • said prostate cancer is metastatic prostate cancer.
  • an embodiment of this aspect of the invention relates to a method for determining a therapy for a prostate cancer in a subject, said method comprising:
  • said prostate cancer is metastatic prostate cancer.
  • another embodiment of the first aspect relates to a method for monitoring therapeutic treatment of a prostate cancer in a subject, said subject being treated for the specific disease, said method comprising
  • the level of one or more uPAR forms with respect to the one or more reference levels indicates the development of said prostate cancer during or after the specific treatment regime and therefore the prognosis. More specifically the prognosis of survival, such as e.g. the prognosis of survival expressed in months.
  • prostate cancer is metastatic prostate cancer.
  • Yet another aspect of the invention relates to a method for classifying the severity based on the aggressiveness of said prostate cancer and/or determining a therapy and/or monitoring a therapeutic treatment of a prostate cancer in a subject, said method comprising:
  • reference level of one or more uPAR forms said reference level being a previously determined level of one or more uPAR forms from the same subject;
  • a level of one or more uPAR forms in the sample above the reference level indicates the presence of a prostate cancer and/or the severity based on the aggressiveness of said cancer is deduced from said comparison and/or deducing the progress and/or state of said cancer by said comparison, and/or based thereon determining a therapy to be initiated, continued, terminated or replaced.
  • said prostate cancer is metastatic prostate cancer.
  • an embodiment of this aspect relates to a method for classifying the severity based on aggressiveness of a prostate cancer in a subject, said method comprising:
  • a level of one or more uPAR forms in the sample being increased by at least a factor 1 .1 compared to the reference level of one or more uPAR forms indicates that the prostate cancer has evolved to a more severe stage of the prostate cancer; and wherein a level of one or more uPAR forms in the sample being decreased by at least a factor 1 .1 compared to the reference level of one or more uPAR forms indicates that the prostate cancer has evolved to a less severe stage of the prostate cancer.
  • prostate cancer is metastatic prostate cancer.
  • another embodiment of this particular aspect relates to a method for determining a therapy for a prostate cancer in a subject, said method comprising: i) determining the level of one or more uPAR forms in a sample obtained from the subject;
  • reference levels of one or more uPAR forms said reference levels being one or more previously determined levels of one or more uPAR forms from the same subject wherein the level of one or more uPAR forms with respect to the reference levels indicates which treatment should be initiated;
  • a level of one or more uPAR forms in the sample being increased to at least a factor 1 .1 compared to the reference level of one or more uPAR forms indicates that the prostate cancer has evolved to a more severe stage of the prostate cancer, and thus requires a therapy of parenteral estrogen to be initiated.
  • said prostate cancer is metastatic prostate cancer.
  • said prostate cancer is metastatic prostate cancer.
  • a third aspect of the present invention relates to the use of one or more uPAR forms as a biomarker for classifying the severity based on the aggressiveness of said prostate cancer and/or determining a therapy. Further details for this aspect of the present invention will be apparent from the text describing the above mentioned methods of the invention. Accordingly, any features mentioned in relation to the methods of the invention apply mutatis mutandis to the use of one or more uPAR forms as a biomarker for classifying the severity of a prostate cancer.
  • said prostate cancer is metastatic prostate cancer.
  • Another and equally important embodiment regards a method for improving the prognosis of survival in individuals with prostate cancer, said method comprising i) determining the level of one or more uPAR forms in a sample obtained from the subject;
  • the prognosis of survival such as e.g. the prognosis of survival expressed in months.
  • Prostate cancer is a disease in which cancer develops in the prostate, a gland in the male reproductive system. It occurs when cells of the prostate mutate and begin to multiply uncontrollably. These cells may metastasize (spread) from the prostate to other parts of the body, particularly the bones and lymph nodes. Prostate cancer may cause pain, difficulty in urinating, problems during sexual intercourse, erectile dysfunction. PC incidence varies widely across the world, with the highest rates in Western Europe and USA, whereas clinical prostate cancer is less frequent in Asia. Prostate cancer develops most frequently in men over the age of fifty and is one of the most prevalent types of cancer in men.
  • prostate cancer may be indicated by symptoms, physical examination, prostate specific antigen (PSA), or biopsy. There is concern about the accuracy of the PSA test and its usefulness in screening. Suspected prostate cancer is typically confirmed by taking a biopsy of the prostate and examining it under a microscope. Further tests, such as CT scans and bone scans, may be performed to determine whether prostate cancer has spread.
  • PSA prostate specific antigen
  • the object of the present invention is to provide a means, by measuring the levels of one or more uPAR forms of the individual suffering from a prostate cancer and relating the measured levels to one or more reference levels, to aid in the pre-diagnosis of the disease and/or to help classify the severity of the disease in terms of the disease's aggressiveness and ultimately assist in the best possible choice of initial and follow up treatment by monitoration of the levels of one or more uPAR forms of the individual.
  • Treatment options for prostate cancer include surgery, radiation therapy, hormonal therapy, ADT, TAB, chemotherapy, proton therapy, cryosurgery, high intensity focused ultrasound (HIFU).
  • the age and underlying health of the man, the extent of metastasis, appearance under the microscope, and response of the cancer to initial treatment are important prognostic factors.
  • the decision whether or not to treat localized prostate cancer (a tumor that is contained within the prostate) with curative intent is a patient trade-off between the expected beneficial and harmful effects in terms of patient survival and quality of life.
  • TNM system abbreviated from Tumor/Nodes/Metastases. Its components include the size of the tumor, presence of lymphnode metastasis and the presence of any other metastases.
  • TNM pelvic organs
  • Stage I disease is cancer that is found incidentally in a small part of the sample when prostate tissue was removed for other reasons, such as benign prostatic hypertrophy,.
  • Stage II more of the prostate is involved and a lump can be felt within the gland.
  • Stage III the tumor has spread through the prostatic capsule and the lump can be felt on the surface of the gland.
  • Stage IV disease the tumor has invaded nearby structures. Grading is based on cellular content and tissue architecture from biopsies (Gleason) which provides an estimate of the destructive potential and ultimate prognosis of the disease.
  • TX cannot evaluate the primary tumor
  • T1 tumor present, but not detectable clinically or with imaging
  • T1 a tumor was incidentally found in less than 5% of prostate tissue resected (for other reasons)
  • T1 b tumor was incidentally found in greater than 5% of prostate tissue resected
  • T1 c tumor was found in a needle biopsy performed due to an elevated serum PSA
  • T2 the tumor can be felt (palpated) on examination, but has not spread outside the prostate
  • T2a the tumor is in half or less than half of one of the prostate gland's two lobes
  • T2b the tumor is in more than half of one lobe, but not both T2c: the tumor is in both lobes
  • T3 the tumor has spread through the prostatic capsule (if it is only part-way through, it is still T2)
  • T3a the tumor has spread through the capsule on one or both sides
  • T3b the tumor has invaded one or both seminal vesicles
  • T4 the tumor has invaded other nearby structures
  • T2c a tumor which is palpable in both lobes of the prostate. Tumors which are found to be bilateral on biopsy only but which are not palpable bilaterally should not be staged as T2c. Evaluation of the regional lymph nodes (' ⁇ ')
  • NX cannot evaluate the regional lymph nodes
  • M1 a the cancer has spread to lymph nodes beyond the regional ones
  • M1 b the cancer has spread to bone
  • M1 c the cancer has spread to other sites (regardless of bone involvement)
  • the grade of the cancer (how different the tissue is from normal tissue) is evaluated separately from the stage; however, for prostate cancer, grade information is used in conjunction with TNM status to group cases into four overall stages.
  • G1 the tumor closely resembles normal tissue (Gleason 2-4)
  • G2 the tumor somewhat resembles normal tissue (Gleason 5-6)
  • G3 ⁇ 1 the tumor resembles normal tissue barely or not at all (Gleason 7-10)
  • this system of describing tumors as “well-”, “moderately-”, and “poorly-” differentiated based on Gleason score of 2-4, 5-6, and 7-10, respectively persists in SEER and other databases but is generally outdated.
  • pathologists rarely assign a tumor a grade less than 3, particularly in biopsy tissue.
  • a more contemporary consideration of Gleason grade is:
  • Gleason 3+3 tumor is low grade (favorable prognosis)
  • Gleason 3+4 / 3+5 tumor is mostly low grade with some high grade
  • Gleason 4+3 / 5+3 tumor is mostly high grade with some low grade
  • the tumor, lymph node, metastasis, and grade status can be combined into four stages of worsening severity.
  • the Urokinase receptor also known as uPA receptor or uPAR or CD87 (Cluster of Differentiation 87), is three domain glycoprotein tethered to the cell membrane with a glycosylphosphatidylinositol (GPI) anchor.
  • GPI glycosylphosphatidylinositol
  • the complete amino acid (SEQ ID NO: 1 ) of human uPAR is found in GenBank under Accession number: Q03405. Human full- length unprocessed, uncleaved uPAR contains a single polypeptide chain of 335 amino acids. The mature protein has an apparent molecular weight of 55-60 kD.
  • the three homologous domains are denoted I, II and III.
  • uPAR (l-lll) comprise residues 1 -283 (SEQ ID NO: 2)
  • uPAR (ll-lll) comprise residues either 84-283 or 90-283 (SEQ ID NOs: 3 and 4)
  • uPAR (I) comprise residues either 1 -83 or 1 -89 (SEQ ID NOs: 5 and 6).
  • the urokinase plasminogen activator (uPA) system is an extracellular protease system, active in both normal and pathological tissue remodellingremodeling processes, including cancer invasion (14, 15).
  • uPA urokinase plasminogen activator
  • ECM extracellular matrix
  • uPAR is a glycosyl-phosphatidylinositol anchored 55-60 kD membrane glycoprotein consisting of three homologous domains, denoted I, II and III. Intact uPAR is required for high affinity binding of uPA (16, 17).
  • catalytically active uPA can cleave intact uPAR (uPAR(l-lll), (SEQ ID NO:2)) liberating domain I (uPAR(l), SEQ ID NO: 4)).
  • the remaining uPAR(ll-lll, SEQ ID NO: 3) will be left on the cell surface, but can like intact uPAR(l-lll) be shed from the cells.
  • uPAR(l-lll), uPAR(l) and uPAR(ll-lll) are therefore all found in blood, and particularly the levels of the two latter forms may reflect the overall activity of the uPA system in the body (47).
  • uPA is the only protease shown to cleave uPAR(l-lll) in vivo (20).
  • the NH 2 -terminal domain, uPAR(l) is required for uPA binding to uPAR(l-lll), so cleavage abolish the binding potential of uPAR(l-lll).
  • uPAR(l-lll) lacking at least the lipid moiety of the glycolipid anchor cannot be cleaved by physiologically relevant concentrations of uPA.
  • uPAR has previously been measured by ELISA methods that detect the total amount of uPAR
  • immunoreactive proteins, and high uPAR levels were found to be associated with short OS when measured in tumor tissue extracts from patients with lung, breast, gastric and colorectal cancer (21 -24) and in blood from patients with colorectal and breast cancer (25, 26).
  • high preoperative serum uPAR was shown to correlate with early biochemical progression (27).
  • uPA is the only protease capable of in vivo uPAR(l-lll) cleavage, then all the suPAR(ll-lll) found in body fluids is the result of shedding of cell surface uPAR(ll-lll) and all uPAR(l) found results from uPA catalyzed uPAR(l-lll) cleavage. Detection of one or more forms of uPAR
  • Peptides and proteins of the invention include functional derivatives of one or more of the uPAR forms.
  • functional derivative is meant the “fragments,” “variants,” “analogs,” or “chemical derivatives” of a molecule.
  • a “variant” of such molecule refers to a naturally occurring molecule substantially similar to either the entire molecule, or a fragment thereof.
  • An “analog” of a molecule refers to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.
  • a molecule is said to be “substantially similar” to another molecule if the sequence of amino acids in both molecules is substantially the same. Substantially similar amino acid molecules will possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if one of the molecules contains additional amino acid residues not found in the other, or if the sequence of amino acid residues is not identical.
  • a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half-life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th Ed., Mack Publishing Co., Easton, Pa., 1980. Minor modifications of the primary amino acid sequence of one or more uPAR forms may result in proteins and peptides that have substantially similar activity as compared to the peptides of one or more uPAR forms described herein.
  • modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the peptides produced by these modifications are included herein as long as the biological activity of the one or more uPAR forms still exists. Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its biological activity. This can lead to the development of a smaller active molecule which would have broader utility. For example, one can remove amino or carboxy terminal amino acids which may not be required for the enzyme to exert the desired catalytic or antigenic activity.
  • the immunoassay procedure used must be quantitative so that levels of the one or more uPAR forms in an individual with disease may be distinguished from normal levels which may be present in healthy humans and/or be distinguished from subjects suffering from benign prostatic disease and/or subjects suffering from malignant prostatic disease and/or background levels measured in the individual.
  • the label will provide a detectible signal indicative of binding of antibody to the antigen of the one or more uPAR forms.
  • the antibody or antigen may be labeled with any label known in the art to provide a detectible signal, including radioisotopes, enzymes, fluorescent molecules, chemiluminescent molecules, bioluminescent molecules and colloidal gold.
  • fluoroimmunoassay FIA
  • time-resolved fluoroimmunoassay TR-FIA time-resolved fluoroimmunoassay
  • radioimmunoassay RIA
  • ELISA enzyme-linked immunoassay
  • TR-FIA time-resolved TR-FIA
  • a fluorescent label will, therefore, be the preferred label.
  • the level of the one or more uPAR forms is determined using an immunoassay.
  • the immunoassay is a quantitative immunoassay.
  • the immunoassay is a quantitative time- resolved TR-FIA immunoassay.
  • the immunoassay uses two monoclonal antibodies that can bind the said uPAR form simultaneously to measure one or more uPAR forms.
  • the immunoassay uses a polyclonal antibody to measure of the one or more uPAR forms.
  • a detectable label selected from the group consisting of radioisotopes, enzymes, fluorescent molecules, chemiluminescent molecules, bioluminescent molecules and colloidal metals may be used to measure the one or more uPAR forms.
  • RIA procedures use radioisotopes as labels, provide high levels of sensitivity and reproducibility, and are amenable to automation for rapid processing of large numbers of samples. However, all RIA procedures require a separation step, since the parameter measured (nuclear decay) cannot be controlled by changing assay conditions or components.
  • isotopes are costly, have relatively short shelf lives, require expensive and complex equipment, and extensive safety measures for their handling and disposal must be followed.
  • TR-FIA is a separate group of FIA because its principles can be adapted to both heterogenous and homogenous assay format.
  • TR-FIA chelates of lanthanides and palladium ions are used as labels.
  • Highly sensitive TR-FIA methods were recently developed that allowed analysis of different uPAR forms (28, 30, 51 ).
  • Any suitable lanthanide may be used, such as but not limited to Samarium, Europium, Terbium, Dysprosium, Holmium, may be used as a label. Most preferred label is Europium.
  • plasma levels of the one or more uPAR forms may be determined in duplicates by a two-site, sandwich-type TR-FIA.
  • a TR-FIA may be used, more preferably two TR-FIAs may be used and even more preferably three TR-FIAs may be used.
  • TR-FIA 1 may be used for quantification of uPAR
  • TR-FIA 2 may be used for quantification of uPAR (l-lll) and uPAR (ll-lll)
  • TR-FIA 3 and/or TR-FIA 4 may be used for quantification of uPAR (I).
  • the sensitivity (detection limit) of the assay of the invention is about 0.3 pmol/L for TR- FIA 1 and TR-FIA 2 and about 1 .9 pmol/L for TR-FIA 3 (28, 30) and about 0.65 pmol/L for TR-FIA 4 (51 ).
  • the limits of quantification of the assays used in invention are about ⁇ 3 pmol/L citrate plasma diluted 1 :10 and serum diluted 1 :10 for all three TR-FIAs.
  • the reference level may be one or more reference levels that for instance each reflects an increased severity of a prostate cancer, or the reference level may for instance be one or more reference levels obtained by previous measurements of samples from the same subject.
  • Reference levels may in general be obtained by measuring levels of one or more uPAR forms in a group of individuals and determining an average or median level of the one or more uPAR forms from these measurements as is known to those skilled in the art.
  • the group of individuals who form the basis for the calculation of the reference level may be a group of healthy individuals or may be a group of patients with untreated prostate cancer or may be a group of patients suffering from benign prostatic disease or may be a group of patients suffering from malignant prostatic disease.
  • the group of individuals forming the basis for the calculation of the reference level is a group of patients with untreated prostate cancer.
  • the group of individuals who form the basis for the calculation of the reference level may be a group of men.
  • Reference levels may also be obtained from the same individual as the sample level of the one or more uPAR forms of that is to be compared with the reference level.
  • the one or more reference levels may for example be levels of the one or more uPAR forms measured in one or more samples obtained prior to diagnosis of the prostate cancer (pre-illness), prior to the
  • any of the indicated groupings of individuals for the determination of a reference level of the one or more uPAR forms may furthermore be combined.
  • the one or more reference levels of the one or more uPAR forms is provided by measuring the levels of the one or more uPAR forms in samples from healthy individuals or from patients with untreated prostate cancer.
  • the one or more reference levels is one or more previously determined levels of the one or more uPAR forms from the same subject. Based on these measurements the person skilled in the art may determine a therapy and/or a therapeutic treatment.
  • the reference level is the serum level of uPAR (l-lll) in healthy individuals in a range from about 10 to about 30 pmol/L (5th percentile range), or a plasma level of less than about 48 pmol/L (95th percentile.
  • the reference level is an uPAR (l-lll) plasma level in a range from about 20 to about 50 pmol/L, such as preferably from about 25 to about 50 pmol/L.
  • the median level is an uPAR (l-lll) plasma level of about 35 pmol/L.
  • the reference level is the serum level of uPAR (l-lll, ll-lll) in healthy individuals in a range from about 70 to about 140 pmol/L (5th percentile range), or a plasma level of less than about 108 pmol/L (95th percentile.
  • the reference level is an uPAR (l-lll, ll-lll) plasma level in a range from about 60 to about 130 pmol/L, such as preferably from about 70 to about 120 pmol/L.
  • the median level is an uPAR (l-lll, ll-lll) plasma level of about 80 pmol/L.
  • one of the one or more reference levels of the one or more uPAR forms is an average or median level obtained by measuring the levels of the one or more uPAR forms in samples from healthy individuals.
  • the median level is used.
  • one of the one or more reference levels of the one or more uPAR forms is an average or median level obtained by measuring the levels of the one or more uPAR forms in samples from patients with previously untreated prostate cancer.
  • the median level is used.
  • the average level is the serum level of uPAR (l-lll) in patients with previously untreated prostate cancer at about 27 pmol/L (5th percentile range), or a plasma level of less than about 1 16 pmol/L (95th percentile range).
  • the reference level is an uPAR (l-lll) plasma level in a range from about 18 to about 1 10 pmol/L, such as preferably from about 25 to about 60 pmol/L.
  • the median level is an uPAR (I- III) plasma level of about 45 pmol/L.
  • the reference level is the serum level of uPAR (l-lll, ll-lll) in healthy individuals at about 70 pmol/L (5th percentile range), or a plasma level of less than about 180 pmol/L (95th percentile.
  • the reference level is an uPAR (l-lll, ll-lll) plasma level in a range from about 60 to about 270 pmol/L, such as preferably from about 80 to about 140 pmol/L.
  • the median level is an uPAR (l-lll, ll-lll) plasma level of about 1 10 pmol/L.
  • a cut-off value is a value the typically divides a number of individuals into two groups: those that have a level of the one or more uPAR forms above a specific cut-off value, and those that have a level of the one or more uPAR forms below the specified cut-off value.
  • the cut-off value may be any value that represents a physiological level of the one or more uPAR forms as measured in any type of biological sample, or as chosen by a person skilled in the art.
  • the cut-off value is the median level of one or more uPAR forms in patients with previously untreated prostate cancer.
  • the reference level of the one or more uPAR forms is a cut-off value corresponding to the 95 th percentile of uPAR (l-lll) or uPAR (l-lll)(ll-lll) in serum in patients with previously untreated prostate cancer, such as for example a plasma value 70 pmol/L of uPAR (l-lll) or 210 pmol/Lof uPAR (l-lll)(ll- III) and more preferably it is a cut-off value corresponding to the 99 th percentile of plasma value of one or more uPAR forms in patients with previously untreated prostate cancer.
  • the cut-off value may be used as a yes or no indicator of whether an individual is within a certain category, in relation to the present invention this corresponds to how the individual will respond to a certain treatment.
  • An individual with a level of one or more uPAR form above the cut-off / median value will respond better to treatment with PEP (polyestradiol phosphate) than with TAB (Total androgen blockage).
  • one of the one or more reference levels of the one or more uPAR forms is a cut-off value corresponding to the 80 th percentile of the one or more uPAR forms as determined in patients with previously untreated prostate cancer.
  • one of the one or more reference levels of the one or more uPAR forms is a cut-off value corresponding to the 85 th percentile of the one or more uPAR forms as determined in patients with previously untreated prostate cancer.
  • one of the one or more reference levels of the one or more uPAR forms is a cut-off value corresponding to the 90 th percentile of the one or more uPAR forms as determined in patients with previously untreated prostate cancer.
  • one of the one or more reference levels of the one or more uPAR forms is a cut-off value corresponding to the 95 th percentile of the one or more uPAR forms as determined in patients with previously untreated prostate cancer.
  • the best possible treatment is a treatment tailored to each individual, and to the stage/severity of a disease or a disorder in said individual.
  • the present invention provides a method of classifying the severity of a prostate cancer, so as each individual may be classified according to e.g. a prognosis of survival.
  • the invention further provides a method of classifying the severity of a prostate cancer, where a prostate cancer may be followed by monitoring the development of the disease or the disorder to determine whether the diseases or disorder evolve towards a more or a less severe stage of the prostate cancer.
  • the classification and monitoration is based on the measurement of the levels of one or more uPAR forms in biological samples taken from the individuals to be classified / monitored and comparing the found levels with that of one or more reference levels.
  • both the ameliorative and the curative effect of the administered treatment will improve, the survival rate of the patients as whole improve, the relapse risks will be lowered, and the quality of life will be heightened. Furthermore, there will be a financial benefit in that the amount of drugs administered may be adjusted acutely. Also, the ability to monitor this group of individuals and determined the development in disease severity will be of assistance in choosing the most effective immediate and follow-up treatment, and be of guidance when counseling on for example required lifestyle changes.
  • the classification of individuals based on their levels of one or more uPAR forms may be performed according to the results described in the Examples. As can be seen from these there is a relationship between increased levels of one or more uPAR forms and increased hazard of death. High hazard ratios indicate increased risk of death and are calculated as known to those skilled in the art. Accordingly, when classifying the severity of a prostate cancer according to the methods of the present invention, the severity of the prostate cancer may be deduced from the correlation with the hazard of death, i.e. the higher the level of one or more uPAR forms, the higher the hazard and therefore the higher the severity of the prostate cancer.
  • An increased level of one or more uPAR forms is indicative of an increased risk of death/short OS as can be seen in the Examples.
  • Different forms of uPAR preferably uPAR (l-lll) and/or uPAR (l-lll)+ uPAR (ll-lll) are thus a biomarkers that allows a prognosis of survival for the individual for which the level of one or more uPAR forms has been determined.
  • the prognosis may be correlated with other signs of health status known to those skilled in the clinical arts.
  • the level of one or more uPAR forms preferably of uPAR (l-lll) and/or uPAR (l-lll)+ uPAR (ll-lll)
  • a prognosis of death or reduced OS may be issued.
  • the pre-treatment serum levels of uPAR (l-lll), uPAR (l-lll)+ uPAR (ll-lll) and uPAR (I) are elevated compared to the median levels of uPAR (l-lll), uPAR (l-lll)+ uPAR (ll-lll) and uPAR (I). These elevated serum levels are associated with short OS as can be seen in the Examples.
  • a high level of one or more uPAR forms is also indicative of which treatment the patient will respond the best to as can be seen in the Examples.
  • Different forms of uPAR preferably uPAR (l-lll) and/or uPAR (l-lll)+(ll-lll) are thus a biomarkers that allows a determination of a treatment for the individual for which the level of one or more uPAR forms has been determined. See herein below for details about the different treatment forms.
  • individuals may be grouped according to their levels of one or more uPAR forms.
  • the levels of one or more uPAR forms may be used for determining a therapy and/or a therapeutic treatment.
  • ADT Androqen-deprivation therapy
  • Any type of ADT is intended to be in the scope of the present invention, particularly orchiectomy (surgical castration) and chemical ADT using any of the methods described herein below.
  • Chemical ADT includes treatment with any drug selected from the group consisting of Luteinizing hormone-releasing hormone (LHRH) analogs/ LHRH agonists, Luteinizing hormone-releasing hormone (LHRH) antagonists, anti- androgens and other androgen-suppressing drugs including but not limited to estrogen. These drugs lower testosterone levels just as well as orchiectomy by lowering the levels of androgens.
  • LHRH Luteinizing hormone-releasing hormone
  • LHRH analogs include but are not limited to leuprolide, goserelin, and triptorelin.
  • Luteinizing hormone-releasing hormone (LHRH) antagonists include but are not limited to Abarelix/Firmagon, degarelix.
  • Anti-androgens block the body's ability to use any androgens.
  • Anti-androgens include but are not limited to flutamide, bicalutamide, and nilutamide.
  • Other androgen-suppressing drugs include but are not limited to estrogens and Ketoconazole.
  • Estrogens are a group of steroid compounds/hormones, named for their importance in the estrous cycle, and functioning as the primary female sex hormone.
  • the group of estrogens includes but is not limited to any estriol, any estradiol such as polyestradiol phosphate (PEP) and any estrone which are all produced from androgens.
  • the estrogens of the present invention may be administered by any route including but limited to parenteral, intramuscular, oral, pulmonary, topical, subcutaneous and nasal administration. In a preferred embodiment the estrogens are administered parenterally. Other types of treatment may also be relevant within the scope of the present invention. These include but are not limited to any angiogenesis inhibitor such as thalidomide, methods utilizing siRNA or antisense, any prostate cancer vaccine, membrane antigens as well as astrazan and archivetin. Combination therapy
  • Anti-androgen treatment may be combined with orchiectomy or LHRH analogs as first- line hormone therapy. This is called combined androgen blockade (CAB). There is still some debate as to whether CAB is more effective in this setting than using orchiectomy or an LHRH analog alone.
  • CAB combined androgen blockade
  • Total androgen blockade is a type of prostate cancer hormone therapy which combines an anti-androgen with either chemical castration or surgical castration. Any type of TAB may be used as a treatment within the present invention. In a preferred embodiment of the present invention TAB/CAB may be used interchangeably and may be done by either bilateral orchiectomy or by administration of triptorelin in combination with antiandrogene flutamide.
  • a high level of one or more uPAR forms is indicative of which treatment the patient will respond the best to as can be seen in the Examples.
  • Different forms of uPAR preferably uPAR (l-lll) and/or uPAR (l-lll)+ uPAR (ll-lll) are thus a biomarkers that allows a determination of a treatment for the individual for which the level of one or more uPAR forms has been determined.
  • the pre-treatment serum levels of uPAR (l-lll), uPAR (l-lll)+ uPAR (ll-lll) and uPAR (I) are elevated compared to the median levels of uPAR (l-lll), uPAR (l-lll)+ uPAR (ll-lll) and uPAR (I). These elevated serum levels are associated with short OS as can be seen in the
  • Short OS is especially pronounced for patients with elevated pre-treatment serum levels of uPAR (l-lll), uPAR (l-lll)+ uPAR (ll-lll) and uPAR (I) when said patients are treated with TAB instead of PEP.
  • patients with elevated pre-treatment serum levels of uPAR (l-lll), uPAR (l-lll)+ uPAR (ll-lll) and uPAR (I) may be treated with PEP.
  • the present invention relates to the monitoring of individuals based on the prognosis of their survival as measured from their levels of one or more uPAR forms.
  • one or more uPAR forms as a prognosis of overall survival in individuals suffering from prostate cancer facilitates administration of the most optimal treatment for each individual.
  • the administration of an effective treatment improves both the ameliorative and curative effect of the administered treatment as well as the survival chances of the individuals, and lessens relapse risks.
  • one or more uPAR forms can be used for monitoring the sufficiency of medical treatment of patients with any prostate cancer and thus improve the curative, ameliorate and general quality of life for an individual (subject) suffering from a prostate cancer.
  • the administration of the most effective treatment is also an issue when assessing the cost/benefits of the given treatment.
  • the method for monitoring therapeutic treatment of a prostate cancer in a subject may be based on the following method which comprises:
  • the level of one or more uPAR forms with respect to the one or more reference levels indicates the development of said prostate cancer during or after the specific treatment regime and therefore the prognosis. More specifically the prognosis of survival, such as e.g. the prognosis of survival expressed in months.
  • said prostate cancer is metastatic prostate cancer.
  • the reference level may be a reference level based on previous determinations of the levels of one or more uPAR forms in the individual such as the method comprising:
  • reference level of one or more uPAR forms said reference level being a previously determined level of one or more uPAR forms from the same subject;
  • a level of one or more uPAR forms in the sample above the reference level indicates the presence of a prostate cancer and/or the severity of said cancer is deduced from said comparison and/or deducing the progress and/or state of said cancer by said comparison, and/or based thereon determining a therapy to be initiated, continued, terminated or replaced.
  • said method comprises:
  • determining the level of one or more uPAR forms in a sample obtained from the subject ii) comparing the level of one or more uPAR forms with one or more reference levels of one or more uPAR forms; said reference levels being one or more previously determined levels of one or more uPAR forms from the same subject wherein the level of one or more uPAR forms with respect to the reference levels indicates the progress and/or state of said prostate cancer, and therefore the degree of efficacy of the ongoing therapeutic treatment;
  • a level of one or more uPAR forms in the sample being increased by at least a factor 1 .1 compared to the reference level of one or more uPAR forms indicates that the prostate cancer has evolved to a more severe stage of the prostate cancer, and thus requires a therapy of parenteral estrogen (PEP) to be initiated; and
  • PEP parenteral estrogen
  • a level of one or more uPAR forms in the sample being decreased by at least a factor 1 .1 compared to the reference level of one or more uPAR forms indicates that the prostate cancer has evolved to a less severe stage of the prostate cancer and thus requires a therapy of total andogen blockade (TAB) to be initiated.
  • TAB total andogen blockade
  • Prostate cancers are commonly treated with one or more of surgery, chemotherapy or (biological) therapy. It is an important aspect of the present invention to monitor the status of an individual suffering from a prostate cancer during treatment of the disease.
  • Chemotherapeutic and biological agents which may be employed in the treatment of cancer include, but are not limited to: 5-Fluouracil (5-FU), Capecitabine (Xeloda R), Tegafur-uracil / Ufteral, S1 , Irinotecan, Oxaliplatin, Gemcitabin, Mitomycin, Taxoterre, Taxol, Carboplatin, Epirubicin, Doxorubicin, Erlotinib (Tarceva R), Sunitinib (Sutent R) and Sorfenib (Nexavar R); VEGF-inhibitors: Bevacizumab (Avastin R), Kinase inhibitors of the VEGF receptor, PDGF-Receptor KIT (CD1 17) RET, CSF-1 R, flt3, Sunitinib (Sutent R), monoclonal antibodies that inhibit the EGF receptor such as Cetuximab (Erbitux R) and Panitumumab (Ve
  • M-Tor inhibitors Sirolimus, Everolimus (Cartisan R) and Temsirolimus (Torisel), Compounds that target the Raf/Mek/Erk signal pathway such as Sorafenib (Nexavar R).
  • suitable treatments include but are not limited to any angiogenesis inhibitor such as thalidomide, methods utilizing siRNA or antisense, any prostate cancer vaccine, membrane antigens as well as astrazan and exptin.
  • the prostate cancer may be treated with any ADT agent from the group including, but not limited to Luteinizing hormone-releasing hormone (LHRH) analogs/ LHRH agonists, including but not limited to leuprolide, goserelin, and triptorelin, Luteinizing hormone-releasing hormone (LHRH) antagonists including but not limited to Abarelix, degarelix, Anti-androgens including but not limited to flutamide, bicalutamide, and nilutamide, estrogens and Ketoconazole.
  • LHRH Luteinizing hormone-releasing hormone
  • LHRH Luteinizing hormone-releasing hormone
  • the prostate cancer may be treated with any type of TAB or any estrogen.
  • the previously untreated prostate cancer may be treated with any estrogens, preferably with PEP (polyestradiol phosphate).
  • the therapeutic treatment is an ADT agent such as, but not limited to a treatment involving the administration of at least one of the following compounds: Luteinizing hormone-releasing hormone (LHRH) analogs/ LHRH agonists, including but not limited to leuprolide, goserelin, and triptorelin.
  • LHRH Luteinizing hormone-releasing hormone
  • Luteinizing hormone-releasing hormone (LHRH) antagonists including but not limited to Abarelix, degarelix, Anti- androgens including but not limited to flutamide, bicalutamide, and nilutamide, estrogens and Ketoconazole. More preferably the treatment may be TAB or estrogen, preferably PEP.
  • an increased level of one or more uPAR forms is indicative of which treatment the patient will respond the best to as can be seen in the Examples.
  • Different forms of uPAR preferably uPAR (l-lll) and/or uPAR (l-lll)+ uPAR (ll-lll) are thus biomarkers that allows a determination of a treatment for the individual for which the level of one or more uPAR forms has been determined.
  • the previously untreated prostate cancer may be treated with any estrogen, preferably with PEP.
  • a person skilled in the art Based upon the classification of the subject according to level of one or more uPAR forms as measured and compared to at least one reference level of one or more uPAR forms a person skilled in the art is better equipped than ever before to determine the best possible treatment of the specific prostate cancer. Thus it is possible for the person skilled in the art based on the method herein disclosed to initiate, continue, terminate, alter or replace a therapy or therapeutic treatment in a subject suffering from the cancer.
  • One or more uPAR forms as a marker of prostate cancer
  • the levels of one or more uPAR forms are increased in subjects suffering from prostate cancers and thus the levels of one or more uPAR forms of an individual may assist in the pre-diagnosis of the presence of a prostate cancer in a subject.
  • the levels of one or more uPAR forms may be part of a series of test a person skilled in the art applies in order to give the diagnosis.
  • One embodiment of the invention relates to a method for pre-diagnosing, classifying the severity based on the aggressiveness of said prostate cancer and/or determining a therapy and/or monitoring a therapeutic treatment of a prostate cancer in a subject, said method comprising:
  • said prostate cancer is metastatic prostate cancer.
  • Another and equally important embodiment regards a method for improving the prognosis of survival in individuals with prostate cancer, said method comprising i) determining the level of one or more uPAR forms in a sample obtained from the subject;
  • the prognosis of survival such as e.g. the prognosis of survival expressed in months.
  • whether the reference level is obtained from a group of healthy individuals a group of people suffering from untreated prostate cancer, benign prostatic disease or malignant prostatic disease or obtained as a previous measurement from the same individual regards a method for diagnosing the presence of a prostate cancer wherein a level of uPAR(l-lll) in the sample being increased by at least a factor of 1 .1 or more compared to the uPAR (I- 111) reference level indicates the presence of a prostate cancer and/or that the prostate cancer has evolved to a more severe stage of the cancer based on the aggressiveness of said prostate cancer, more preferably increased to at least a factor of 1 .2, such as e.g.
  • l-l I preference level indicates the presence of a prostate cancer and/or the severity of said cancer has evolved to a more severe stage of said cancer and optionally requires a therapy of parenteral estrogen to be initiated.
  • whether the reference level is obtained from a group of healthy individuals , a group of people suffering from untreated prostate cancer, benign prostatic disease or malignant prostatic disease or obtained as a previous measurement from the same individual regards a method for diagnosing the presence of a prostate cancer wherein a level of uPAR(l-lll)+ uPAR(ll-lll) in the sample being increased by at least a factor of 1 .1 or more compared to the uPAR (l-lll)+(ll-lll) reference level indicates the presence of a prostate cancer and/or that the prostate cancer has evolved to a more severe stage of the cancer based on the aggressiveness of said prostate cancer, more preferably increased to at least a factor of 1 .2, such as e.g.
  • compared to the uPAR (l-lll)+(ll-lll) reference level indicates the presence of a prostate cancer and/or the severity of said cancer has evolved to a more severe stage of said cancer and optionally requires a therapy of parenteral estrogen to be initiated.
  • the reference level may be obtained from a group of healthy individuals , a group of people suffering from untreated prostate cancer, benign prostatic disease or malignant prostatic disease or obtained as a previous measurement from the same individual, and regards a method for diagnosing the presence of a prostate cancer wherein a level of uPAR(l-lll)+ uPAR(ll-lll) in the sample is increased by at least 5 %, such as al least 10%, for example at least 15%, such as at least 20%, such as at least 25%, for example at least 30%, such as at least 35% compared to the uPAR (I- 111) reference level indicates that the prostate cancer requires a therapy of parenteral estrogen to be initiated.
  • the level of uPAR(l-lll)+ uPAR(ll-lll) in the sample is increased by at least 35%.
  • the reference level may be obtained from a group of healthy individuals , a group of people suffering from untreated prostate cancer, benign prostatic disease or malignant prostatic disease or obtained as a previous measurement from the same individual, and regards a method for diagnosing the presence of a prostate cancer wherein a level of uPAR (l-lll)+(ll-lll) in the sample being increased by at least 5 %, such as al least 10%, for example at least 15%, such as at least 20%, for example at least 25% compared to the uPAR (l-lll)+(ll-lll) reference level indicates that the prostate cancer requires a therapy of parenteral estrogen to be initiated.
  • the level of uPAR(l- lll)+ uPAR(ll-lll) in the sample is increased by at least 25%.
  • One particular embodiment relates to a method for classifying the severity based on the aggressiveness of said prostate cancer and/or determining a therapy and/or monitoring a therapeutic treatment of a prostate cancer in a subject, said method comprising:
  • determining the level of one or more uPAR forms in a sample obtained from the subject i) determining the level of one or more uPAR forms in a sample obtained from the subject; and ii) comparing said level of one or more uPAR forms with a reference level of one or more uPAR forms, said reference level being a previously determined level of one or more uPAR forms from the same subject; wherein a level of one or more uPAR forms in the sample above the reference level indicates the presence of a prostate cancer and/or the severity of said cancer is deduced from said comparison and/or deducing the progress and/or state of said cancer by said comparison, and/or based thereon determining a therapy to be initiated, continued, terminated or replaced.
  • said prostate cancer is metastatic prostate cancer.
  • a level of one or more uPAR forms in the sample being increased by at least a factor of 1 .1 or more compared to the uPAR (l-lll)+(ll-lll) reference level indicates the presence of a prostate cancer and/or that the prostate cancer has evolved to a more severe stage of the cancer based on the aggressiveness of said prostate cancer, more preferably increased to at least a factor of 1 .2, such as e.g.
  • At least a factor of 1 .3, or at least a factor of 1 .4 compared to the reference level of one or more uPAR forms indicates the presence of a prostate cancer and/or the severity of said cancer has evolved to a more severe stage of said cancer and optionally requires a therapy of high efficacy to be initiated and/or requires a therapy with higher efficacy than the ongoing therapy to be initiated.
  • the one or more uPAR forms are independent biomarker for classifying the severity of a prostate cancer and may be used accordingly.
  • the one or more uPAR forms may also be used in combination with other known biomarkers such as any one or more of the biomarkers from the following non-limiting group: prostate-specific antigen (PSA), YKL-40 and the aminoterminal propeptide of type III procollagen (P-III-NP).
  • PSA prostate-specific antigen
  • YKL-40 the aminoterminal propeptide of type III procollagen
  • P-III-NP aminoterminal propeptide of type III procollagen
  • the device comprises means for assaying prostate-specific antigen (PSA).
  • the present invention provides means for pre-diagnosing, classifying the severity and/or determining a therapy and/or monitoring a therapeutic treatment of a prostate cancer in a subject according to their levels of one or more uPAR forms in combination with levels of other biomarkers these being selected from the non- limiting group consisting of: prostate-specific antigen (PSA), the aminoterminal propeptide of type III procollagen (P-III-NP) and YKL-40.
  • PSA prostate-specific antigen
  • P-III-NP aminoterminal propeptide of type III procollagen
  • YKL-40 YKL-40
  • the device comprises means for assaying prostate-specific antigen (PSA).
  • the above mentioned embodiments may be comprised in a kit of parts together with any required medical and or sampling equipment and instructions for use of the equipment and how to perform the assay of choice.
  • a biological sample is a sample obtained from a subject.
  • a biological sample may be a sample selected from the group consisting of tissue, blood, serum, plasma samples, urine, cerebrospinal fluid, synovial fluid, ascites, and saliva.
  • samples of blood, serum or plasma more preferably the biological sample is serum or plasma.
  • the values as described herein can be applied for both plasma and serum samples. It is necessary to validate the assays used in the present invention for each biological sample and also for the types of glass or plastic used as well as taking into account for how long said samples are kept prior to analysis.
  • the subjects herein referred to are single members of a species, herein preferably a mammalian species. Any mammalian species is an object of the present invention, although any of the following species are of particular relevance: mouse, rat, guinea pig, hamster, rabbit, cat, dog, pig, cow, horse, sheep, monkey, and human. Most preferably the subject of the present invention is a human.
  • the subjects may in the present text also be referred to as patients or individuals.
  • a fourth aspect of the present invention relates to a device for diagnosing, classifying the severity and/or determining a therapy and/or monitoring a therapeutic treatment of a prostate cancer in a subject, wherein the device comprises means for measuring the level of one or more uPAR forms in a sample; and means for comparing the measured level of one or more uPAR forms with at least one reference level of one or more uPAR forms.
  • the means for measuring the level of one or more uPAR forms in a sample may for example be a test system that applies any of the above mentioned assay systems, such as an immunoassay.
  • An immunoassay is preferred for the present device, preferably a TR-FIA.
  • the preferred uPAR forms are uPAR (l-lll), uPAR (l-lll)+uPAR (II- III) and uPAR(l).
  • a device according to the present invention may for example comprise a rapid, qualitative and/or quantitative test system mounted on a solid support for the determination of the levels of one or more uPAR forms in biological samples.
  • the solid support can be used in any phase in performing any of the above assays, particularly immunoassays, including dipsticks, membranes, absorptive pads, beads, microtiter wells, test tubes, and the like.
  • test devices which may be conveniently used by the testing personnel or the patient for self-testing, having minimal or no previous training.
  • Such preferred test devices include dipsticks and membrane assay systems. The preparation and use of such conventional test systems is well described in the patent, medical, and scientific literature. If a stick is used, the antibody to one or more uPAR forms is bound to one end of the stick such that the end with the antibody can be dipped into or onto the biological samples.
  • the samples can be applied onto the antibody-coated dipstick or membrane by pipette, dropper, tweezers or the like, or be squirted directly from the body and onto the stick.
  • the device is a dipstick.
  • any biological sample that is or may be converted to a fluid is preferred.
  • biological samples that are obtainable from a body as a fluid are preferred; examples hereof include, and are not limited to: blood, serum, plasma, urine, cerebrospinal fluid, synovial fluid, ascites, semen, and saliva. More preferably serum and plasma samples It is necessary to validate the assays used in the present invention for each biological sample and also for the types of glass or plastic used as well as taking into account for how long said samples are kept prior to analysis.
  • the antibody against one or more uPAR forms can be of any isotype, such as IgA, IgG or IgM, Fab fragments, or the like.
  • the antibody may be a monoclonal or polyclonal and produced by methods as generally described in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, incorporated herein by reference. See also section on immunoassays.
  • the antibody can be applied to the solid support by direct or indirect means. Indirect bonding allows maximum exposure of the binding sites of one or more uPAR forms to the assay solutions since the sites are not themselves used for binding to the support.
  • the solid support is preferably non-specif ically blocked after binding the monoclonal antibodies to one or more uPAR forms to the solid support.
  • Non-specific blocking of surrounding areas can be with whole or derivatized bovine serum albumin, or albumin from other animals, whole animal serum, casein, non-fat milk, and the like.
  • the sample is applied onto the solid support with bound mAb specific to one or more uPAR forms such that the one or more uPAR forms will be bound to the solid support through said mAbs. Excess and unbound components of the sample are removed and the solid support is preferably washed so the antibody-antigen complexes are retained on the solid support.
  • the solid support may be washed with a washing solution which may contain a detergent such as Tween-20, Tween-80.
  • the second mAb may be labelled, preferably with a visible label.
  • the labels may be soluble and may include dyed immunoglobulin binding substances, simple dyes or dye polymers, dyed latex beads, dye-containing liposomes, or metallic, organic, inorganic, or dye solids.
  • the labels may be bound to the mAbs to one or more uPAR forms by a variety of means that are well known in the art. In some embodiments of the present invention, the labels may be enzymes that can be coupled to a signal producing system.
  • visible labels examples include alkaline phosphatase, beta- galactosidase, horseradish peroxidase, and biotin.
  • enzyme-chromogen or enzyme-substrate-chromogen combinations are known and used for enzyme-linked assays.
  • the solid support is washed again to remove unbound labeled antibody and the labeled antibody is visualized and quantitated.
  • the accumulation of label will generally be assessed visually. This visual detection may allow for detection of different colors, e.g., red color, yellow color, brown color, or green color, depending on label used.
  • Accumulated label may also be detected by optical detection devices such as reflectance analyzers, video image analyzers and the like.
  • the visible intensity of accumulated label could correlate with the concentration of one or more uPAR forms in the sample.
  • the correlation between the visible intensity of accumulated label and the amount of one or more uPAR forms may be made by comparison of the visible intensity to a set of reference standards.
  • the standards Preferably, the standards have been assayed in the same way as the unknown sample, and more preferably alongside the sample, either on the same or on a different solid support.
  • the concentration of standards to be used can range from 0.016-10 ⁇ g/L purified calibrators; in the range of 0.5-325 pmol/L of uPAR(l-lll), in the range of 0.8-476 pmol/L of uPAR(ll-lll) and in the range of 1 .5- 961 pmol/L of uPAR(l).
  • the device such as the herein described dipstick or other solid support based test system, may thus be used in aid of determining the approximate level of one or more uPAR forms in a biological sample by comparison to one or more standards / control fields.
  • concentration of one or more uPAR forms can be ascertained to be within a range between two of the concentrations of one or more uPAR forms applied to the standard / control fields of the device.
  • concentration of one or more uPAR forms can be judged to be above or below a cut-off value of one or more uPAR forms, the chosen concentration for the cut-off value being applied to the control field of the dipstick.
  • the device may be used as a yes no test, to compare a level of one or more uPAR forms in a sample with one reference level, i.e. to see whether the level of one or more uPAR forms of the sample is above or below the reference level.
  • the device comprises a single reference level, representing a cut-off value.
  • the reference level may as any of the reference levels described herein above in the section termed "reference levels”.
  • each of the steps can be carried out in the same vessel, such as a test tube, if it is cleaned and washed after each of the steps, a fast and convenient on-site assay is best performed according to the invention by using three separate vessels for each of the steps, one for the sample, one for washing, and one for developing the detectable label.
  • the level of one or more uPAR forms of a biological sample for use in the classification according to a reference level of one or more uPAR forms of the individual from which the biological sample originated is measured by use of a dipstick, (see Figure 3A and 3B).
  • the device further comprises means for assaying additional biomarkers than one or more uPAR forms, such as any one or more of the biomarkers from the following non-limiting group: prostate-specific antigen (PSA), the aminoterminal propeptide of type III procollagen (P-III-NP) and YKL-40.
  • PSA prostate-specific antigen
  • P-III-NP aminoterminal propeptide of type III procollagen
  • YKL-40 YKL-40
  • the device comprises means for assaying prostate- specific antigen (PSA).
  • kits All the materials and reagents required for assaying one or more uPAR forms according to the present invention can be assembled together in a kit, such kit includes at least elements in aid of assessing the level of one or more uPAR forms in a biological sample obtained from an individual, and the instruction on how to do so.
  • Said elements may be a method of detecting the levels of one or more uPAR forms such as an immunoassay, or parts required to perform an immunoassay specific for detection of one or more uPAR forms.
  • the kit of parts may further comprise equipment for obtaining one or more biological samples, such equipment may for example be syringes, vials or other.
  • the kit of parts may be packed for single use or for repeated usage, and the elements therein may be disposable such as to be disposed of after a single use or may be of a quality that allows repeated usage.
  • a specific embodiment of the present invention relates to a kit of parts comprising i) means for measuring the level of one or more uPAR forms in a sample; ii) means for comparing the measured level of one or more uPAR forms with one or more reference levels of one or more uPAR forms; and iii) optionally instructions on how to age adjust the reference level of one or more uPAR forms, according to the age of the subject providing the sample.
  • the kit further comprises means for assaying additional biomarkers than one or more uPAR forms, such as any one or more of the biomarkers from the following non-limiting group: prostate-specific antigen (PSA), ,the aminoterminal propeptide of type III procollagen (P-III-NP) and YKL-40.
  • PSA prostate-specific antigen
  • the device comprises means for assaying prostate-specific antigen (PSA).
  • the kit comprises at least one device as described herein above.
  • Means for measuring the level of one or more uPAR forms in a sample may include one or more solutions containing a known concentration of one or more uPAR forms, a washing solution, a solution of a chromogen which changes color or shade by the action of the enzyme directly or indirectly through action on a substrate, an antibody to one or more uPAR forms conjugated to a label such that it could be detected, pipettes for the transfer of said solutions, test tubes for said solutions, and a solid support, in particular adapted to be inserted into the test tubes, carrying on the surface thereof a polyclonal antibody to of one or more uPAR forms.
  • the kit may also contain one or more solid support having an antibody to of one or more uPAR forms for use in assaying one or more samples simultaneously or individually, and the necessary reagent required to develop the label. Included in means for comparing the measured level of one or more uPAR forms with at least one reference level of one or more uPAR forms may be standards of one or more uPAR forms that can be assayed fresh along with the unknown sample.
  • Such kits will comprise distinct containers for each individual reagent.
  • the reagents may be supplied from storage bottles or one or more of the test tubes may be prefilled with the reagents or controls.
  • the components of the kit may also be provided in dried or lyophilized forms.
  • reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means.
  • kits of the present invention also will typically include a means for containing the reagents such as vials or tubes in close confinement for commercial sale such as, e.g. injection or blow-molded plastic containers into which the desired vials are retained.
  • the kits will also comprise a set of instructions on how to perform the assay.
  • kit according to the present invention may furthermore comprise a device according to the invention as described above here in the section termed "device”.
  • One embodiment of the invention relates to a method as described herein for classifying the severity of a prostate cancer based on the aggressiveness of said prostate cancer in a subject, said method comprising
  • Another embodiment relates to a method as described herein wherein the one or more reference levels is one or more previously determined levels of one or more uPAR forms from the said same subject.
  • Another embodiment relates to a method, as described herein wherein the one or more uPAR forms is uPAR (I).
  • Another embodiment relates to a method, as described herein wherein one of the one or more reference levels of uPAR (I) is a median plasma level of 27 pmol/L in individuals with previously untreated prostate cancer.
  • the one or more reference levels of one or more uPAR forms comprises a set of reference levels of one or more uPAR forms obtained by measuring the one or more uPAR forms levels in samples from healthy individuals and/or individuals with untreated prostate cancer: a first reference level being the median value of one or more uPAR forms in samples from healthy individuals, a second reference level being the 95 th percentile of one or more uPAR forms in samples from healthy individuals, a third reference level being the 99 th percentile in samples from healthy individuals of one or more uPAR forms in healthy individuals, a fourth reference level being the median value of one or more uPAR forms in individuals with previously untreated prostate cancer, a fifth reference level being the 95 th percentile of one or more uPAR forms in samples from individuals with previously untreated prostate cancer, a sixth reference level being the 99 th percentile in individuals with previously untreated prostate cancer, a seventh reference level being a factor at least above 1 .1 of the median value of
  • the one or more reference levels of uPAR(l-lll) forms comprises a set of reference levels of uPAR (l-lll) obtained by measuring the uPAR(l-lll) levels in samples from healthy individuals and/or individuals with untreated prostate cancer: a first reference level being the median value of uPAR(l-lll) in samples from healthy individuals, a second reference level being the 95 th percentile of uPAR(l-lll) in samples from healthy individuals, a third reference level being the 97.5 th percentile of uPAR(l-lll) in samples from healthy individuals in individuals a fourth reference level being the median value of one or more uPAR forms in individuals with previously untreated prostate cancer, a fifth reference level being the 95 th percentile of uPAR(l-lll) in samples from individuals with previously untreated prostate cancer, a sixth reference level being the 99 th percentile of uPAR(l-lll) in individuals with previously untreated prostate cancer
  • Another embodiment relates to a method as described herein wherein the median value of uPAR (l-lll) in individuals with previously untreated prostate cancer is a plasma level of above 45 pmol/L, and the 95 th percentile of uPAR (l-lll) is a plasma level of 80 pmol/l.
  • Another embodiment relates to a method as described herein wherein the median value of uPAR (l-lll) in healthy individuals is a plasma level of 35 pmol/L, and the 95 th percentile of uPAR (l-lll) is a plasma level of 48 pmol/l.
  • the one or more reference levels of uPAR (l-lll)+(ll-lll) comprises a set of reference levels of uPAR (I- lll)+(ll-lll) obtained by measuring the uPAR (l-lll)+(ll-lll) levels in samples from from healthy individuals and/or individuals with untreated prostate cancer: a first reference level being the median value of uPAR (l-lll)+(ll-lll) in samples from healthy individuals, a second reference level being the 95 th percentile of uPAR (l-lll)+(ll-lll) in samples from healthy individuals, a third reference level being the 97.5 th percentile of uPAR (l-lll)+(ll- III) in samples from healthy individuals in individuals a fourth reference level being the median value of one or more uPAR forms in individuals with previously untreated prostate cancer, a fifth reference level being the 95 th percentile of uPAR (l-lll)+(ll-ll) comprises a set
  • Another embodiment relates to a method as described herein, wherein the median value of uPAR(l-lll)+(ll-lll) is a plasma level of above 1 12 pmol/L, the 95 th percentile of uPAR (l-lll)+(ll-lll) is a plasma level of 210 pmol/l.
  • Another embodiment relates to a method as described herein wherein the median value of uPAR (l-lll)+(ll-lll) in healthy individuals is a plasma level of 80 pmol/L, the 5 th percentile of uPAR (l-lll)+(ll-lll) is a plasma level of 70 pmol/L, the 95 percentile of uPAR (l-lll)+(ll-lll) is a plasma level of 108 pmol/L
  • Another embodiment relates to a method as described herein, wherein the determined level of one or more uPAR forms in the sample above one or more of the reference levels provides the classification of the prostate cancer and thus evidence of therapy to be initiated, continued, terminated or replaced.
  • Another embodiment relates to a method as described herein, wherein the
  • classification of the prostate cancer is provided by comparing the determined level of one or more uPAR forms from the sample with the one or more reference levels of one or more uPAR forms, wherein the higher the level of one or more uPAR forms the more severe the prostate cancer is classified as and whether a therapy of parenteral estrogen or of total andogen blockade should initiated, continued, or replaced.
  • Another embodiment relates to a method as described herein, wherein the
  • classification of the prostate cancer is provided by comparing the determined level of one or more uPAR forms from the sample with the one or more reference levels of one or more uPAR forms, wherein the lower the level of one or more uPAR forms the less severe the prostate cancer is classified as and whether a therapy of parenteral estrogen or of total andogen blockade should initiated, continued, or replaced.
  • Another embodiment relates to a method for classifying the severity based on the aggressiveness of said prostate cancer and/or determining a therapy and/or monitoring a therapeutic treatment of a prostate cancer in a subject, said method comprising:
  • a level of one or more uPAR forms in the sample above the reference level indicates the presence of a prostate cancer and/or the severity of said cancer is deduced from said comparison and/or deducing the progress and/or state of said cancer by said comparison, and/or based thereon determining a therapy to be initiated, continued, terminated or replaced.
  • Another embodiment relates to a method as described herein for classifying the severity of a prostate cancer based on the aggressiveness of said prostate cancer in a subject, said method comprising
  • a level of one or more uPAR forms in the sample being increased to at least a factor 1 .1 compared to the reference level of one or more uPAR forms indicates that the prostate cancer has evolved to a more severe stage of the prostate cancer
  • Another embodiment relates to a method as described herein wherein the method is a method for determining a therapy for a prostate cancer in a subject, said method comprising:
  • Another embodiment relates to a method as described herein wherein the method is a method for monitoring therapeutic treatment of a prostate cancer in a subject, said subject being treated for the specific disease, said method comprising
  • determining the level of one or more uPAR forms in a sample obtained from the subject ii) comparing the level of one or more uPAR forms with one or more reference levels of one or more uPAR forms; said reference levels being one or more previously determined levels of one or more uPAR forms from the same subject wherein the level of one or more uPAR forms with respect to the reference levels indicates the progress and/or state of said prostate cancer, and therefore the degree of efficacy of the ongoing therapeutic treatment;
  • a level of one or more uPAR forms in the sample being increased to at least a factor 1 .1 compared to the reference level of one or more uPAR forms indicates that the prostate cancer has evolved to a more severe stage of the prostate cancer, and thus requires a therapy of parenteral estrogen to be initiated;
  • Another embodiment relates to a method as described herein wherein the one or more uPAR forms is selected from the group consisting of uPAR (l-lll), uPAR(l-lll)+ uPAR(ll- III) and uPAR (l).
  • Another embodiment relates to a method as described herein wherein the one or more uPAR forms is uPAR (l-lll).
  • Another embodiment relates to a method as described herein wherein the one or more uPAR forms is uPAR (l-lll)+uPAR(ll-lll).
  • Another embodiment relates to a method as described herein wherein the one or more uPAR forms is uPAR (I).
  • Another embodiment relates to a method as described herein wherein the prostate cancer has been diagnosed prior to, during or after the measurement of the previously determined levels of one or more uPAR forms.
  • Another embodiment relates to a method as described herein wherein the prostate cancer is a previously diagnosed prostate cancer. Another embodiment relates to a method as described herein wherein the one or more reference levels of one or more uPAR forms has been determined after diagnosing the prostate cancer. Another embodiment relates to a method as described herein wherein a level of one or more uPAR forms in the sample being increased to at least a factor of 1 .1 or more compared to the one or more uPAR forms reference level indicates the presence of a prostate cancer and/or that the prostate cancer has evolved to a more severe stage of the cancer based on the aggressiveness of said prostate cancer, more preferably increased to at least a factor of 1 .2, such as e.g. a factor of 1 .3, or a factor 1 .4;
  • compared to the reference level of one or more uPAR forms indicates the presence of a prostate cancer and/or the severity of said cancer has evolved to a more severe stage of said cancer and optionally requires a therapy of high efficacy to be initiated and/or requires a therapy with higher efficacy than the ongoing therapy to be initiated.
  • Another embodiment relates to a method as described herein wherein a level of one or more uPAR forms in the sample being increased by at least 25% compared to the one or more uPAR forms reference level indicates that the prostate cancer has evolved to a more severe stage of the prostate cancer and optionally requires a therapy of parenteral estrogen to be initiated and/or requires a therapy with higher efficacy than the ongoing therapy to be initiated.
  • Another embodiment relates to a method as described herein, wherein the treatment is surgery, and/or treatment with chemotherapeutic and/or biological agents.
  • Another embodiment relates to a method as described herein wherein a level of one or more uPAR forms in the sample being increased by at least 25% compared to the one or more uPAR forms reference level indicates that a prostate cancer have evolved to a more severe stage of the cancer.
  • Another embodiment relates to a method as described herein wherein the determined level of one or more uPAR forms in the sample above one or more of the reference levels provides the classification of the prostate cancer.
  • Another embodiment relates to a method as described herein wherein the classification of the prostate cancer is provided by comparing the determined one or more uPAR forms level from the sample with the one or more reference levels of one or more uPAR forms, wherein the higher the level of one or more uPAR forms the more severe, based on the aggressiveness of said prostate cancer, the prostate cancer is classified as.
  • Another embodiment relates to a method as described herein, wherein the
  • immunoassay utilizes a detectable label selected from the group consisting of radioisotopes, enzymes, fluorescent molecules, chemiluminescent molecules, bioluminescent molecules and colloidal metals to measure one or more uPAR forms.
  • Another embodiment relates to a method as described herein, wherein the subject is a mammal, preferably a human.
  • Another embodiment relates to a use as described herein, comprising using of one or more uPAR forms a biomarker for classifying the severity of a prostate cancer.
  • Another embodiment relates to device as described herein, wherein the device comprises a single reference level, representing a cut-off value.
  • Another embodiment relates to device as described herein wherein the device comprises means for comparing the measured level of one or more uPAR forms with at a set of age adjusted reference levels of one or more uPAR forms.
  • Another embodiment relates to a method of diagnosing prostate cancer, wherein the prostate cancer has been diagnosed prior to, during or after the measurement of the previously determined one or more uPAR forms levels. Another embodiment relates to a method wherein the prostate cancer is a previously diagnosed disease or disorder.
  • kits of parts as described herein wherein the kit further comprises means for assaying additional biomarkers selected from the group consisting of prostate-specific antigen (PSA), the aminoterminal propeptide of type III procollagen (P-III-NP) and YKL-40.
  • PSA prostate-specific antigen
  • P-III-NP aminoterminal propeptide of type III procollagen
  • YKL-40 YKL-40
  • Another embodiment relates to a kit of parts as described herein comprising at least one device according to claim nn.
  • Figure 1 A-F Kaplan-Meier estimates of survival probabilities for the entire study population (A,D,G), the subpopulation of patients treated by TAB ( ⁇ , ⁇ , ⁇ ) and the subpopulation treated by PEP (C,F,I) for the indicated uPAR forms.
  • Patients are stratified in tertiles according to their serum levels of the uPAR forms (1 st tertile: yellow, 2 nd tertile: blue, 3 rd tertile). The numbers of patients at risk at 0, 24, 48 and 72 months are shown below the axis and the number of deaths to the left. P-values are calculated by the log rank test for trend.
  • FIG. 2 Kaplan-Meier plots for overall survival of the subgroup of prostate cancer patients with pre-treatment uPAR(l-lll)+uPAR(ll-lll) levels above the median (1 1 1 .9 fmol/ml).
  • the two curves represent patients treated with either intramuscular injections of polyestradiol phosphate (PEP) or total androgen blockade (TAB), respectively.
  • PEP polyestradiol phosphate
  • TAB total androgen blockade
  • the p-value shown is for the log rank test.
  • the number of patients at risk at 0, 24 and 48 months in each stratum are shown below the axis of the Kaplan-Meier plots with the number of events (deaths) shown to the left.
  • FIG. 3A and 3B Dipstick embodiments seen from above. Dipstick support material (1 .) with assay field (2.) for use with the biological sample and one control or standard field (3. in Figure 3A) or multiple control or standard fields (4a. to 4.e. in Figure 3B). Standards of a single (for 3.) or various (one concentration for each field in increasing or decreasing order, e.g.) concentrations of one or more uPAR forms may be applied to the control or standard fields to enable reading a positive / negative result with the stick portrayed in fig. 3A or assessing an approximate concentration of one or more uPAR forms in the biological sample compared to which of the control fields in Fig. 3B. References
  • Chodak GW A critical review of maximal androgen blockade for advanced
  • Hedlund PO, Henriksson P Parenteral estrogen versus total androgen ablation in the treatment of advanced prostate carcinoma: effects on overall survival and cardiovascular mortality.
  • SPCG Prostatic Cancer Group
  • urokinase receptor is required for efficient vitronectin binding: receptor cleavage prevents ligand interaction.. FEBS Lett 420:79-85, 1997
  • Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain. J Biol Chem 267:18224-18229, 1992 19. Behrendt N, Ronne E, Dano K. Domain interplay in the urokinase receptor.
  • Plebani M Herszenyi L, Carraro P, et al.: Urokinase-type plasminogen activator receptor in gastric cancer: tissue expression and prognostic role. Clin Exp Metastasis 15:418-425, 1997
  • LNCaP hormone-resistant cell line in prostate cancer research.
  • TMPRSS2 ERG gene fusion associated with lethal pof protease systems and cell types. Oncogene. 2007 Jul 5;26(31 ):4596-9. Epub 2007 Jan 22.
  • TAB total androgen blockade
  • PEP polyestradiol phosphate
  • the parenteral estrogen was administered as intramuscular injections of 240 mg PEP every second week for the first 8 weeks (five doses) followed by maintenance dose of 240 mg every month (4) Seventy-five of the patients in this study had been treated with TAB and 56 with PEP. There was no difference in the overall survival of the TAB and PEP- treated patients (see Figure 2A), in accordance with the original SPCG5 study of altogether 915 patients (4, 31 ) Staging at the time of inclusion was based on histological and/or cytological findings, digital rectal examination, and the assessment of bone metastasis (M1 ) by plain X-ray skeletal surveys and radio-isotopic bone imaging. Further details concerning the study are described elsewhere.
  • TNM classification the patients were staged as T1 -4, Nx, M+, and according to the WHO PS, 69 had a WHO PS 0, 46 WHO PS 1 , and 15 WHO PS 2.
  • the EOD was calculated from pre-treatment bone scans according to Soloway (9): score 1 in 37 patients, score 2 in 82 patients and score 3 in 10 patients (2 missing).
  • Tumors were graded according to the WHO grading system: 22 had grade 1 , 51 had grade 2 and 52 grade 3 tumors (6 missing).
  • the median pre- treatment PSA level was 266 ⁇ g/L (range 10-7730). The patients were followed until death or up to January 31 , 2000. The median follow-up was 4.9 (3.7-9.2) years. Time to death was measured from the date of treatment initiation.
  • TR-FIA time-resolved fluoroimmunoassays specific for the detection of intact uPAR(l-lll)
  • TR-FIA 1 uPAR(l-lll)+uPAR(ll-lll)
  • TR-FIA 2 uPAR(l)
  • Rank statistics were used to calculate correlation coefficients and to test hypotheses on location. Tests of independence were done using the chi-square test.
  • the levels of uPAR(l-lll) and uPAR(l-lll)+uPAR(ll-lll) were log-transformed ( 2 log e ) and treated as continuous variables for the uni- and multivariate analyses of survival and the uPAR(l) was also log e transformed with uPAR(l) levels below the quantification limit set to this value for these analyses.
  • the Kaplan-Meier method was used to estimate survival probabilities, and the log-rank test was used to test for equality of strata or trend grouping the biomarkers by their respective tertiles. For uPAR(l) the lower cut point was the quantification limit.
  • the Cox proportional hazards model was applied for univariate analysis as well as for multivariate analysis of OS. Tests for interaction
  • Serum levels of the different uPAR forms and their association to other variables Serum levels of the different uPAR forms and their association to other variables.
  • median (range) serum level of uPAR(l- lll)+uPAR(ll-lll) was 1 12 (62-266) pmol/L, of uPAR(l-lll) 45 (19-108) pmol/L, and of uPAR(l) 27 (20-182) pmol/L (in 48 of the cases uPAR(l) was below the limit of quantification of 20 pmol/L and allocated this value).
  • the rank correlations between uPAR(l-lll)+uPAR(ll-lll) and uPAR(l-lll), between uPAR(l-lll)+uPAR(ll-lll) and uPAR(l), and between uPAR(l-lll) and uPAR(l) were 0.74, 0.41 and 0.43, respectively.
  • the serum levels of the uPAR forms stratified by clinical parameters are presented in Table 1 . Significant association was found between uPAR(l-lll)+uPAR(ll-lll) and WHO tumor grade, and between uPAR(l-lll)+uPAR(ll-lll) and WHO PS .
  • Table 3 uPAR serum values in 131 patients with metastatic prostate cancer.
  • Table 5 factor increase of the levels of one or more uPAR forms in men with metastatic prostate cancer:
  • Table 6 Coefficients of variation for the intra and inter individual variation estimated from 23 normal individuals sampled 6 times in 3 periods 6 months apart. The inter class correlation is also shown (measures the inter-individual variation compared to the total variation). The estimates were obtained using a random effects model with levels of uPAR on the log scale and back transformed.
  • Table 7 Descriptive statistics for the 6 month value minus the baseline level

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Abstract

La présente invention concerne des procédés de classification de la gravité d'un cancer de la prostate, de choix d'un traitement contre le cancer de la prostate chez un sujet et/ou de suivi de la progression de la maladie avant, pendant et après l'administration d'un traitement, dans le cadre desquels si une concentration prédéterminée en une ou plusieurs formes du récepteur de l'activateur de la plasminogène urokinase (u PAR) se situe au-dessus d'une concentration de référence, il y a nécessité à administrer un traitement. La présente invention concerne, en outre, un nécessaire et un dispositif pouvant être utilisés dans le cadre des procédés selon l'invention et comprenant un moyen de mesure de la concentration d'un échantillon en une ou plusieurs formes du récepteur u PAR et un moyen permettant de comparer la concentration mesurée en ladite ou lesdites formes du récepteur u PAR avec au moins une concentration de référence en une ou plusieurs formes du récepteur u PAR.
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