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WO2010015659A1 - Cancer markers and methods for their detection - Google Patents

Cancer markers and methods for their detection Download PDF

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Publication number
WO2010015659A1
WO2010015659A1 PCT/EP2009/060162 EP2009060162W WO2010015659A1 WO 2010015659 A1 WO2010015659 A1 WO 2010015659A1 EP 2009060162 W EP2009060162 W EP 2009060162W WO 2010015659 A1 WO2010015659 A1 WO 2010015659A1
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Prior art keywords
polypeptide
seq
prostate cancer
protein
amino acid
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Ceased
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PCT/EP2009/060162
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French (fr)
Inventor
Miren Zumárraga Uribesalgo
Saioa Dominguez Hormaetxe
Antonio Martinez Martinez
Laureano Simon Buela
Daniel Nagore Casas
Itsaso Hormaeche Berciano
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Proteomika SL
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Proteomika SL
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Publication of WO2010015659A1 publication Critical patent/WO2010015659A1/en
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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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • the present invention relates to methods and products for detecting or assessing cancer, particularly in vitro methods for detecting the presence of markers of prostate cancer in a urine sample from a subject, and to products, such as kits, for use in such methods.
  • Prostate cancer is the second most common cancer and also the second leading cause of death in men.
  • GLOBOCAN International Agency for the Investigation of Cancer
  • GLOBOCAN in the year 2002 680,000 new cases of prostate cancer were diagnosed in the world, and over 200,000 men died due to this disease. It is estimated that about the 30% of all men will develop a prostate carcinoma during their lifetime, and its prevalence in men by the age of 80 is of 80%.
  • PSA Prostate Specific Antigen
  • This glycoprotein produced by the cells of the prostate is organ specific but not cancer specific, as it is also related to other prostatic benign alterations such as prostatitis and prostatic hyperplasia. This leads to a lack of specificity of this method that results in a 75% of false positives.
  • PSA is used for prostate cancer screening all over the world, and it is estimated that about 45 million PSA tests are carried out every year. Men with a positive result in their PSA test require a prostatic biopsy to confirm the diagnosis of cancer, but 75% of these biopsies result negative.
  • prostatic biopsies can be practised on a patient's lifetime while the PSA test results remain positive. Unnecessary biopsies are clearly undesirable.
  • tumour suppressor genes which act to block uncontrolled cell growth
  • tumour cells expression levels of the so-called "oncogenes” which act to induce malignant growth
  • the present inventors have now found that certain proteins can be detected in urine and act as biomarkers for prostate cancer.
  • the present invention which employs urinary biomarkers for detection and monitoring of prostate cancer is considered more attractive as compared with, for example, use of serum biomarkers.
  • Attempts to identify specific markers for prostate cancer in serum samples have been found to be largely unproductive due to the broad dynamic range of protein concentrations and certain highly abundant proteins which account for >98% of the total serum protein content.
  • the present invention which makes use of urine samples, is less prone to problems related to protein dynamic range. Changes in protein patterns in urine between groups are thought to be highly representative of the prostate cancer pathology.
  • Urine sample collection is noninvasive, which is a clear benefit in a clinical setting for both the patient and the clinician.
  • the provision of urinary protein markers for prostate cancer has the potential to result in improvement for patient management in the field of prostate cancer.
  • the present inventors have now found that the protein concentration, particularly relative protein concentration, of each of the proteins ABHEB, ALDOB, AMBP, APOAl, BHMTl, CATD, CNDP2, EPCR, FOLHl, GALM, GELS, GO45, GTPB2, IGHA2, KLK3, PGBM (particularly its fragment, Endorepellin), PPAP, RETBP, SAP3, SCTM l, SODC, TPPl, TTHY, VMOl and ZA2G is altered in urine samples collected from patients with prostate cancer when compared with urine samples collected from individuals that do not have prostate cancer.
  • the invention provides a method for detecting or monitoring prostate cancer or susceptibility to prostate cancer in a test subject, comprising : determining an amount (relative or absolute), such as relative or absolute protein concentration, of one or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) marker proteins in a urine sample obtained from the test subject; and comparing said amount (relative or absolute) with a standard value, wherein the one or more marker proteins are selected from:
  • Endorepellin polypeptide both Endorepellin polypeptide and GELS polypeptide, GELS polypeptide, ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOAl polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTM l polypeptide, SODC polypeptide, TPPl polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide; and/or a fragment of any one of said polypeptides.
  • the method may be non-invasive because a urine sample is readily collected and can be conveniently analysed. Moreover, changes in amount, particularly relative amount and more particularly relative protein concentration, are expected to be highly representative of the prostate cancer pathology.
  • the method may comprise measuring one marker protein or a panel of two or more, such as 3, 4, 5, 6, 7, 8, 9, 10 or more, marker proteins as defined herein.
  • the use of a panel of marker proteins as defined herein may provide further positive predictive value due, for example, to statistical analysis of multiple marker protein changes in amount, particularly relative amount, such as relative protein concentration. Use of a panel of marker proteins may improve the sensitivity and/or specificity of the assay.
  • the method of the one or more marker proteins may be selected from the group consisting of: Endorepellin polypeptide, GELS polypeptide and both Endorepellin polypeptide and GELS polypeptide.
  • Endorepellin polypeptide and GELS polypeptide may be advantageously combined as marker proteins in accordance with this and other aspects of the invention. It is believed that use of both Endorepellin polypeptide and GELS polypeptide improves sensitivity and/or specificity as compared with use of Endorepellin polypeptide or GELS polypeptide in isolation.
  • the urinary marker proteins defined herein may offer greater specificity for detection of prostate cancer in comparison with the detection of serum PSA.
  • Many factors cause an elevated serum PSA value (the threshold typically used in current testing is 4 ng/ml of serum). All patients with a serum PSA higher than this threshold are normally biopsied in current testing, despite the fact that up to 75% of those with elevated serum PSA in fact do not have prostate cancer.
  • the methods of the present invention aim to reduce this high false positive rate by providing higher specificity, whilst also providing high sensitivity.
  • the protein markers described above show altered relative protein concentration in urine samples from subjects having prostate cancer compared with healthy controls. A number of the protein markers exhibit elevated relative protein concentration in urine of prostate cancer subjects, while a number exhibit lower relative protein concentration in urine of prostate cancer subjects.
  • the one or more marker proteins are selected from: ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GELS polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide; and/or a fragment thereof, wherein the relative amount is the amount of the marker protein relative to a reference protein also present in the urine sample, and wherein a decreased relative amount of said one or more marker proteins in the urine sample obtained from the test subject compared with a standard value corresponding to the relative amount of the same marker protein in a urine sample obtained from a control subject not having prostate cancer indicates that the test subject has or is susceptible to prostate cancer.
  • a lower amount such as a lower relative amount or a lower absolute amount, particularly a lower relative protein concentration or lower absolute protein concentration, may be 10%, 25%, 50% or 75% lower than the standard value.
  • the average decrease in relative protein concentration for each marker protein is found in prostate cancer patient urine is shown in Table 1 as a "fold-change".
  • the precise threshold for the level of decrease and the precise numerical value used as the standard value for the marker protein of interest may be selected by the skilled person as appropriate for the subject or population under consideration.
  • the skilled person may derive a suitable standard value and threshold of decrease in amount (relative or absolute) for the given marker protein by means of clinical tests or by reference to charts, tables or databases of pre-determined standard values and/or threshold levels of decrease.
  • the standard value for the marker protein is a pre-determined value that has been determined by measuring a representative number of control subjects from a population of subjects not having prostate cancer.
  • the one or more marker proteins are selected from : ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide; and/or a fragment thereof, wherein the relative amount is the amount of the marker protein relative to a reference protein also present in the urine sample, and wherein an increased relative amount of said one or more marker proteins in the urine sample obtained from the test subject compared with a standard value corresponding to the relative amount of the same marker protein in a urine sample obtained from a control subject not having prostate cancer indicates that the test subject has or is susceptible to prostate cancer.
  • a higher amount such as a higher relative amount or a higher absolute amount, particularly a higher relative protein concentration or higher absolute protein concentration, may be 10%, 25%, 50%, 100%, 200% or 500% higher than said reference value.
  • the average increase in relative protein concentration for each marker protein is shown in Table 1 as a "fold-change".
  • the precise threshold for the level of increase and the precise numerical value used as the standard value for the marker protein of interest may be selected by the skilled person as appropriate for the subject or population under consideration. For example, the skilled person may derive a suitable standard value and threshold of increase in relative amount for the given marker protein by means of clinical tests or by reference to charts, tables or databases of predetermined standard values and/or threshold levels of increase.
  • the standard value for the marker protein is a pre-determined value that has been determined by measuring a representative number of control subjects from a population of subjects not having prostate cancer.
  • "Absolute amount" of protein as used herein is intended to include measures of protein amount that are independent of other proteins.
  • absolute amount of protein may be the protein concentration expressed in terms of a quantity of said protein (e.g. micrograms or nanograms or picograms) per unit of urine sample (e.g. millilitres of volume). Determining an absolute amount of a marker protein, as used in accordance with the methods of the invention, specifically includes indirect as well as direct determination.
  • the actual concentration e.g.
  • determining an absolute amount of a marker protein may comprise measuring fluorescence, optical density or similar measure in an immunoassay. The determination may or may not involve reference to a "standard curve" comprising known amounts (e.g. a series of known concentrations) of the protein.
  • Determination of absolute amount of a marker protein may be preferred in certain circumstances. It is believed that determination of absolute amount of a marker protein as defined herein may be informative for prostate cancer detection or monitoring. It has been found that use of a urine protein (such as creatinine) as a reference (for example for total protein concentration) is not necessarily required to achieve an assay having acceptable sensitivity and/or specificity. Determination of absolute amount of a given protein does not require measurement of a reference protein or total protein in the sample. Thus, determination of absolute amount may be in some cases quicker involving fewer steps than determination of a relative amount of a marker protein. The comparative simplicity of determining absolute rather than relative protein amount is particularly advantageous in the setting of clinical testing and/or large-scale sample analysis. In certain preferred cases the method of the invention may comprise indirect determination of the absolute amount of a marker protein as defined herein by means of a single reading of, e.g. fluorescence or optical density, following an immunoassay as further detailed herein.
  • protein concentration in the urine of healthy control subjects and subjects with cancer is highly variable both between individuals and also for a given individual depending on diet, time of day and disease status among other factors.
  • proteinuria is associated with, among other conditions, disease of the kidney.
  • the present inventors have found that protein concentration in the urine may differ by over two orders of magnitude between subjects. Many of the subjects whose urine was used in the analysis described herein had haematuria (both cancer patients and other controls with non-malignant urological conditions), and this can elevate substantially the total protein content of urine.
  • haematuria both cancer patients and other controls with non-malignant urological conditions
  • the present inventors have found that consistent comparisons may be made by determining relative protein amount, such as relative protein concentration.
  • the relative protein concentration gives a measure of the concentration of the given marker protein relative to a reference protein.
  • Methods in accordance with the invention may comprise determining relative amount of a marker protein, particularly when the sample is from a subject having or suspected to have significantly variable urinary protein concentration (for example a subject having or suspected of having a disease or condition affecting kidney function, proteinuria and/or haematuria).
  • relative amount of protein as used herein is intended to include measures of protein amount that are related to (e.g. divided by) a measure of the amount of one or more other proteins.
  • relative amount of protein may be the protein concentration expressed in terms of a quantity of said protein (e.g. micrograms or nanograms or picograms) per unit of one or more other proteins in a urine sample (e.g. mass of a given marker protein per total mass of protein or mass of a given marker protein per mass of a reference protein). Determining a relative amount of a marker protein, as used in accordance with the methods of the invention, specifically includes indirect as well as direct determination. For example, the actual concentration of the marker protein and of the reference protein (e.g.
  • determining a relative amount of a marker protein may comprise measuring fluorescence, optical density or similar measure in an immunoassay. The determination may or may not involve reference to a "standard curve" comprising known amounts (e.g. a series of known concentrations) of the marker protein and/or a reference protein.
  • the relative protein concentration is relative to a reference protein, the concentration of which is substantially unchanged between subjects having prostate cancer, and controls subjects not having prostate cancer.
  • the relative protein concentration of a given marker protein as defined herein may be relative to the protein concentration of another of the marker proteins as defined herein.
  • a first group of the marker proteins defined herein exhibit decreased concentration in urine of prostate cancer patients (ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GELS polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide; and/or a fragment thereof), while a second group of the marker proteins defined herein exhibit increased concentration in urine of prostate cancer patients (ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide; and/or a fragment thereof).
  • the marker proteins of the first group and second group exhibit "inverse expression changes" in association with prostate cancer.
  • the present inventors have realised that the inverse expression changes of the two groups of marker proteins can be utilised to improve the specificity and/or sensitivity of the methods of the invention.
  • the relative amount of a marker protein from the first group may be relative to the amount of a reference protein chosen from the second group. This will enhance the degree of decrease in relative amount that is associated with prostate cancer.
  • the relative amount of a marker protein from the second group may be relative to the amount of a reference protein chosen from the first group. This will enhance the degree of increase in relative amount that is associated with prostate cancer.
  • the method of the first aspect of the invention may comprise: determining the relative amount of one or more marker proteins selected from: ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GELS polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide; and/or a fragment thereof, in a urine sample obtained from the test subject, wherein the relative amount is the amount of the marker protein relative to a reference protein selected from: ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide; or a fragment thereof; and
  • the method of the first aspect of the invention may comprise: determining the relative amount of one or more marker proteins selected from: ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide; and/or a fragment thereof, in a urine sample obtained from the test subject, wherein the relative amount is the amount of the marker protein relative to a reference protein selected from: ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GELS polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTM l polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide; or a fragment thereof; and comparing the relative amount
  • preferred marker proteins may be selected based on likelihood values independently of whether a particular marker is up-regulated or down-regulated in urine of prostate cancer subjects. That is to say, improvements in assay performance (e.g. improved sensitivity and/or selectivity) may be achieved by combining two or more marker proteins whether the two or more marker proteins are all up-regulated, all down- regulated or a mixture of up-regulated and down-regulated in prostate cancer.
  • the one or more marker proteins may comprise at least two marker proteins as defined herein.
  • the at least two marker proteins may comprise two or more marker proteins that are both down-regulated in prostate cancer and/or two or more marker proteins that are both up- regulated in prostate cancer.
  • the one or more marker proteins and/or the reference protein may be selected so as to enhance the sensitivity and/or specificity of the method of this aspect of the invention.
  • a marker protein may be chosen which exhibits, based on the fold-change shown in Table 1, a substantial decrease or a substantial increase in relative amount in urine from prostate cancer patients.
  • a reference protein may be chosen which exhibits, based on the fold-change shown in Table 1, a substantial increase or a substantial decrease in relative amount in urine from prostate cancer patients. It is particular preferred that both the one or more marker proteins and the reference protein are selected based on maximal or near-maximal inverse expression changes.
  • ALDOB polypeptide which exhibits a 12-fold increase
  • RETBP polypeptide which exhibits a 3-fold decrease
  • the marker protein and/or the reference protein is selected from those proteins shown in Table 1 that exhibit a fold-change (increase or decrease) of greater than 1.5-fold, 2-fold or 2.5- fold, or a fragment of any one of those proteins.
  • the one or more marker proteins of this and other aspects of the invention are preferably the human full-length polypeptides having the sequences identified by GI number and UniProt accession number in Table 1.
  • the "Genlnfo Identifier" or GI number is an integer number assigned to each nucleotide and protein sequence available through NCBI's GenBank database.
  • the GI number uniquely identifies a particular sequence; if the sequence changes in any way, a new GI number is assigned. Therefore, the GI numbers in Table 1 identify particular, fixed sequence versions. Each of the amino acid sequences identified by GI number in Table 1 is specifically incorporated herein by reference in its entirety.
  • ABHEB polypeptide may be the human ABHEB polypeptide having the amino acid sequence disclosed in UniProt accession No: Q96IU4 (including any one of isoforms Q96IU4-1 (SEQ ID NO: 1) and/or Q96IU4-2 (SEQ ID NO: 2));
  • ALDOB polypeptide may be the human ALDOB polypeptide having the amino acid sequence disclosed in UniProt accession No: P05062 (SEQ ID NO: 3);
  • AMBP polypeptide may be the human AMBP polypeptide having the amino acid sequence disclosed in UniProt accession No: P02760 (SEQ ID NO: 4);
  • APOAl polypeptide may be the human APOAl polypeptide having the amino acid sequence disclosed in UniProt accession No: P02647 (SEQ ID NO: 5);
  • BHMT polypeptide may be the human BHMT polypeptide having the amino acid sequence disclosed in UniProt accession No: Q93088 (SEQ ID NO: 6);
  • CATD polypeptide may
  • KLK3 polypeptide may be the human KLK3 polypeptide having the amino acid sequence disclosed in UniProt accession No: P07288 (SEQ ID NO: 25);
  • PGBM polypeptide may be the human PGBM polypeptide having the amino acid sequence disclosed in UniProt accession No: P98160 (SEQ ID NO: 26);
  • Endorepellin polypeptide may be the 25kDa fragment of the C-terminal end of the human PGBM polypeptide having the amino acid sequence disclosed in UniProt accession No: P98160 (SEQ ID NO: 26), preferably the Endorepellin polypeptide comprises or consists essentially of the LG3 domain of said PGBM polypeptide, in particular amino acid amino acid residues 4196- 4391 of the sequence disclosed in GI 24212664 and/or UniProt accession No: P98160, more preferably the Endorepellin polypeptide comprises or consists essentially of the C- terminal V domain of said PGBM polypeptide, in
  • the one or more marker proteins of this and other aspects of the invention may be a derivative, variant, orthologue, homologue or fragment of one or more of the human full-length polypeptides having the sequences identified by GI and/or UniProt accession number in Table 1.
  • a marker protein of the invention may be a derivative, variant, orthologue, homologue or fragment of one or more of the human full-length polypeptides having the sequences identified by GI and UniProt accession number in Table 1, which comprises an amino acid sequence having at least 70%, 80%, 90%, 95% or 99% sequence identity to the sequence, preferably to the full-length sequence, of a polypeptide identified by GI and UniProt accession number in Table 1. It is well-known that multiple allelic variants may exist for a given gene product.
  • one or more variants of a given protein may exist due to transcriptional or post-translational changes.
  • the one or more marker proteins of this and other aspects of the invention may be a naturally occurring allelic variant of a marker protein identified in Table 1.
  • the one or more marker proteins of this and other aspects of the invention may be a derivative, such as a chemically-altered derivative, of a marker protein identified in Table 1.
  • a method of the invention may include chemically altering the one or more marker proteins present in a sample in order to facilitate detection and/or measurement of the one or more marker proteins.
  • one or more marker proteins may be labelled prior to measurement in a method of the invention.
  • the one or more marker proteins of this and other aspects of the invention may be a fragment of: (i) a polypeptide having an amino acid sequence identified by GI and/or UniProt accession number in Table 1; or
  • the fragment may be a naturally occurring fragment that results from proteolysis of its parent protein.
  • the one or more marker proteins may include a fragment selected from : Endorepellin polypeptide (preferably Endorepellin polypeptide comprises the LG3 domain of PGBM, more preferably comprising or consisting essentially of a 191 residue fragment of the 4391 residue Perlecan protein, more preferably the Endorepellin polypeptide comprises or consists essentially of the contiguous sequence of amino acid residues 4196-4391 of the sequence disclosed in GI 24212664 and/or UniProt accession No: P98160, yet more preferably the Endorepellin polypeptide comprises or consists essentially of the C-terminal V domain of said PGBM polypeptide, in particular the contiguous sequence of amino acid residues 3687-4391 (SEQ ID NO: 37) of the sequence disclosed in GI 24212664 and/or UniProt accession No: P98160), Ganglioside GM2 activator short form (residues 34-193 of SAP3; SEQ ID NO: 39), Ganglioside GM2
  • the method of this and other aspects of the invention may comprise one or more steps of treating the urine sample to remove or reduce the level of contaminants, particularly solid contaminants such as free tissue and cells.
  • a urine sample may be subjected to centrifugation so as to remove tissue, cells and/or other solid contaminants prior to measuring the relative amount of the one or more marker proteins as defined herein.
  • the method may employ direct urine samples, which have been subjected to little or no preparative treatment, thereby saving on time and laboratory resources.
  • the method of this and other aspects of the invention may comprise extracting protein from a urine sample to obtain a protein extract.
  • the relative amount of the one or more marker proteins, in particular the relative protein concentration of said one or more marker proteins, in a protein extract obtained from a urine sample provides a measure of the relative amount of the marker protein in the urine sample because any concentration or dilution of the protein in the sample that occurs during the extraction is expected to affect the concentration of the one or more marker proteins to substantially the same extent as the total protein concentration and/or the protein concentration of a reference protein.
  • the relative amount of the one or more marker proteins is in effect "normalised". Measurements based on a protein extract from the urine sample therefore provide an accurate measure of the relative amount of the one or more marker proteins in the urine sample.
  • Protein extraction from the urine sample may be employed so that the absolute protein concentration of the extract can be adjusted so as to be optimal or close to optimal for the method used to measure relative protein concentration. Therefore, in some cases the method of the invention involves measuring the relative amount of said one or more marker proteins in a urine sample obtained from the test subject by extracting protein from said urine sample to obtain a protein extract, measuring the relative protein concentration of said one or more marker proteins in the protein extract, thereby deriving a measure of the relative protein concentration of said one or more marker proteins in the urine sample.
  • determining the amount (relative amount or absolute amount) of said one or more marker proteins in the urine sample may comprise: contacting said urine sample with at least one specific binding member that selectively binds to one of said marker proteins; and detecting and/or quantifying formation of a complex formed by said specific binding member and said marker protein.
  • the specific binding member may be an antibody molecule or a binding fragment thereof.
  • the antibody molecule may be selected from : a monoclonal antibody, a polyclonal antibody, an antibody binding fragment and a combibody. Further details of specific binding members, including antibody molecules, for use in accordance with the present invention are provided herein.
  • a wide variety of techniques are available for detecting formation of a complex formed by said specific binding member and said marker protein.
  • Preferred techniques include: Western blot; enzyme-linked immunosorbent assay (ELISA); radioimmunoassay (RIA); competitive enzyme immunoassay; double antibody sandwich ELISA (DAS-ELISA); liquid immunoarray technology (e.g. Luminex xMAP technology and Becton-Dickinson FACS technology, among others); immunocytochemistry; immunohistochemistry; antibody microarray detection; precipitation of colloidal gold; affinity chromatography; ligand binding assay; and lectin binding assay.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • DAS-ELISA double antibody sandwich ELISA
  • liquid immunoarray technology e.g. Luminex xMAP technology and Becton-Dickinson FACS technology, among others
  • immunocytochemistry immunohistochemistry
  • antibody microarray detection precipitation of colloidal gold
  • a preferred method in accordance with this aspect of the invention comprises:
  • a first specific binding member which selectively binds to a marker protein selected from Endorepellin polypeptide, both Endorepellin polypeptide and GELS polypeptide, GELS polypeptide, ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide, or a fragment thereof;
  • a marker protein selected from Endorepellin polypeptide, both Endorepellin polypeptide and GELS polypeptide, GELS polypeptide, ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3
  • a second specific binding member which selectively binds to a reference protein selected from ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide, or a fragment thereof;
  • a preferred method in accordance with this aspect of the invention comprises:
  • a urine sample obtained from the subject with a first specific binding member, which selectively binds to a marker protein selected from ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide, or a fragment thereof;
  • a marker protein selected from ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide, or a fragment thereof;
  • a second specific binding member which selectively binds to a reference protein selected from Endorepellin polypeptide, both Endorepellin polypeptide and GELS polypeptide, GELS polypeptide, ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide, or a fragment thereof;
  • a reference protein selected from Endorepellin polypeptide, both Endorepellin polypeptide and GELS polypeptide, GELS polypeptide, ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3
  • the method for detecting or monitoring cancer or susceptibility to a cancer in a test subject comprises: diagnosing cancer in the test subject; determining the stage, severity or progression of cancer in the test subject; assessing the absence of cancer in the test subject after surgical treatment has been performed on the test subject; establishing the prognosis of a cancer in the test subject; or monitoring the effect of therapeutic treatment of cancer in the test subject.
  • the test subject may have been not previously diagnosed with prostate cancer.
  • the test subject may have been previously diagnosed with prostate cancer and/or may have been previously treated for prostate cancer.
  • the methods of the invention are suitable for detecting the presence of prostate cancer, for the first time in a subject and are suitable for on-going monitoring of the status of a subject having prostate cancer.
  • the on-going monitoring may be used to inform treatment strategy and/or determine the prognosis of the subject.
  • On-going monitoring may involve assessing the amount (relative amount or absolute amount), such as relative protein concentration or absolute protein concentration, of one or more of said marker proteins in a plurality of urine samples obtained from the test subject at different times, for example when at least one of the urine samples is obtained prior to a therapeutic or surgical intervention performed on the test subject and at least one of said urine samples is obtained after a said intervention.
  • the present invention provides a prostate cancer diagnostic kit comprising :
  • the prostate cancer diagnostic kit in accordance with this aspect of the invention may comprise: a specific binding member that selectively binds to Endorepellin polypeptide and/or a specific binding member that selectively binds to GELS polypeptide.
  • the prostate cancer diagnostic kit in accordance with this aspect of the invention may comprise:
  • specific binding members each of which selectively binds to a marker protein selected from ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOAl polypeptide, BHMTl polypeptide, CNDP2 polypeptide, EPCR polypeptide, GALM polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 polypeptide, Endorepellin polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, TPPl polypeptide, TTHY polypeptide and VMOl polypeptide, or a fragment thereof; and one or more reagents for detecting said one or more specific binding members.
  • a marker protein selected from ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOAl polypeptide, BHMTl polypeptide, CNDP2 polypeptide, EPCR polypeptide, GALM polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 poly
  • the prostate cancer diagnostic kit of this aspect of the invention comprises: a specific binding member which selectively binds to a marker protein selected from ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GELS polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide, or a fragment thereof; and a specific binding member which selectively binds to a marker protein selected from ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide, or a fragment thereof.
  • a marker protein selected from ABHEB polypeptide, AMBP
  • the prostate cancer diagnostic kit of this aspect of the invention may comprise a plurality of said specific binding members which are provided in the form of an array on a substrate.
  • the specific binding member of the prostate cancer diagnostic kit of this aspect of the invention may comprise an antibody molecule or a binding fragment thereof.
  • the marker proteins are as defined in the first aspect of the invention.
  • the specific binding members comprise antibody molecules or antibody binding fragments thereof, as defined further herein.
  • the prostate cancer diagnostic kit of this aspect of the invention may comprise at least one reagent or article for processing, storing and/or transporting a urine sample.
  • said kit may comprise a reagent (e.g. trichloroacetic acid) for use in extracting protein from a urine sample.
  • the prostate cancer diagnostic kit of this aspect of the invention may comprise instructions, e.g. in the form of an insert, internal or external label, or data carrier, for carrying out a method in accordance with the first aspect of the invention.
  • the instructions may refer to use of said kit in the analysis of one or more urine samples.
  • the prostate cancer diagnostic kit of this aspect of the invention may comprise one or marker protein standards (e.g. for preparing a standard curve of known protein concentrations).
  • the present invention provides a specific binding member for use in a method of diagnosing prostate cancer (preferably in vitro), wherein said specific binding member selectively binds a marker protein as defined in accordance with the first aspect of the invention.
  • the specific binding member of this aspect of the invention selectively binds Endorepellin polypeptide.
  • the specific binding member of this aspect of the invention selectively binds GELS polypeptide.
  • the specific binding member of this aspect of the invention selectively binds a marker protein selected from : ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOAl polypeptide, BHMTl polypeptide, CNDP2 polypeptide, EPCR polypeptide, GALM polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 polypeptide, Endorepellin polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, TPPl polypeptide, TTHY polypeptide and VMOl polypeptide; or a fragment thereof.
  • the marker proteins are as defined in the first aspect of the invention.
  • the specific binding members comprise antibody molecules or antibody binding fragments thereof, as defined further herein.
  • the present invention provides use of a specific binding member which selectively binds a marker protein as defined in accordance with the first aspect of the invention in a method of diagnosing prostate cancer (preferably in vitro) or in the manufacture of a diagnostic agent for diagnosis of prostate cancer.
  • the specific binding member of this aspect of the invention selectively binds
  • the specific binding member of this aspect of the invention selectively binds GELS polypeptide. In certain preferred cases, the specific binding member of this aspect of the invention selectively binds a marker protein selected from: ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOAl polypeptide, BHMTl polypeptide, CNDP2 polypeptide, EPCR polypeptide, GALM polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 polypeptide, Endorepellin polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, TPPl polypeptide, TTHY polypeptide and VMOl polypeptide; or a fragment thereof.
  • the marker proteins are as defined in the first aspect of the invention.
  • the specific binding members comprise antibody molecules or antibody binding fragments thereof, as defined further herein.
  • the present invention provides a method of screening a test compound for the ability to interfere with or block the development or progression of prostate cancer, comprising:
  • BHMTl polypeptide BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 polypeptide, KLK3 polypeptide, PGBM polypeptide, Endorepellin polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide,
  • the marker proteins are as defined in the first aspect of the invention.
  • the marker proteins are orthologues or homologues of the human marker proteins defined in the first aspect of the invention, which orthologues or homologues comprise the polypeptide sequence native to the species of said test and control animal.
  • the test compound is preferably a known or suspected anti-cancer agent.
  • the test compound may be compound and/or an antibody molecule that binds to a marker protein selected from: ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOAl polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLH l polypeptide, GALM polypeptide, GELS polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 polypeptide, KLK3 polypeptide, PGBM polypeptide, Endorepellin polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TPPl polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide; or a fragment thereof.
  • a marker protein selected from: ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOAl polypeptide,
  • the method of this aspect of the invention may provide a simplified way of screening potential anti-cancer agents in vivo because the effect of a potential anti-cancer agent on the development or progression of prostate cancer in the animal model can be readily assessed by collecting one or more urine samples and carrying out a simple in vitro assay.
  • the method of this aspect of the invention is a method wherein the test compound is found to cause a difference between said relative or absolute amount (i) (e.g. protein concentration) and said relative or absolute amount (ii) (e.g. protein concentration).
  • the method may further comprise formulating the test compound into a composition comprising at least one further component.
  • the test compound is formulated into a pharmaceutical composition.
  • Figure 1 shows a bi-dimensional electrophoresis (2D) gel obtained from a urine sample and showing the two different studied areas A and B;
  • Figure 2 shows spot 1669 in the B area of the bi-dimensional electrophoresis (2D) gel obtained from urine samples of three healthy individuals (panel A), and of three prostate cancer suffering patients (panel B);
  • Figure 3 shows the intensity of spot 1669 in control and prostate cancer samples, with the number of samples for each group indicated;
  • Figure 4 shows results of statistical analysis of endorepellin and GELS immunoassays.
  • the ROC curves for endorepellin (squares), GELS (diamonds) and the combination of endorepellin and GELS (triangles) immunoassays are shown;
  • Figure 5 shows results of further statistical analysis of endorepellin and GELS immunoassays.
  • A. The box-plots show the distribution of the relative amount of endorepellin for control (left) and prostate cancer (right) groups.
  • B. The box-plots show the distribution of the relative amount of GELS for control (left) and prostate cancer
  • cancer refers to the disease that is typically characterised by abnormal or unregulated cell growth, capable of invading adjacent tissues and spreading to distant organs.
  • carcinoma refers to the tissue resulting from abnormal or unregulated cell growth.
  • prostate cancer refers to any malign proliferative disorder in prostate cells.
  • tissue refers to any abnormal mass of tissue generated by a neoplastic process, whether this is benign (non cancerous) or malignant (cancerous).
  • gene refers to a region of a molecular chain of deoxyribonucleotides that encodes a protein and may represent a portion of a coding sequence or a complete coding sequence.
  • protein indicates at least one molecular chain of amino acids linked through either covalent or non-covalent bonds.
  • the term includes all forms of post-translational protein modifications, for example glycosylation, phosphorylation or acetylation.
  • peptide and “polypeptide” refer to molecular chains of amino acids that represent a protein fragment.
  • protein and “peptide” are used interchangeably.
  • the term "specificity” refers to the measurement of false positives, where a specificity of 100% means there are no false positives (positive diagnosis of prostate cancer when the patient individual does not in fact have suffer prostate cancer).
  • sensitivity refers to the measurement of false negatives, where a sensitivity of 100% means there are no false negatives (negative diagnosis of prostate cancer when the patient in fact does have prostate cancer).
  • solid phase refers to a non-aqueous matrix to which the antibody can bind.
  • materials for the solid phase include but are not limited to glass, polysaccharides (for example agarose), polyacrylamide, polystyrene, polyvinylic alcohol and silicons.
  • solid phase forms are the well of a plate or a purification column.
  • subject refers to all species of animals classified as mammals and includes, but is not restricted to, domestic and farm animals, primates and humans.
  • the subject is preferably male.
  • the subject is a male human of any age or race.
  • the subject is a male human identified as being at risk of developing prostate cancer on the basis of, for example, age, lifestyle and/or genetic predisposition.
  • the one or more marker proteins as defined in the methods and products of the invention are preferably the human full-length polypeptides having the sequences identified by GI and UniProt accession number in Table 1.
  • the one or more marker proteins as contemplated herein in relation to any aspect of the present invention may be a derivative, variant, orthologue, homologue or fragment of one or more of the human full-length polypeptides having the sequences identified by GI and UniProt accession number in Table 1.
  • the derivative, variant, orthologue, homologue or fragment may comprise an amino acid sequence having at least 70%, 80%, 90%, 95% or 99% sequence identity to the sequence of, preferably the full-length sequence of, a polypeptide identified by GI and UniProt accession number in Table 1.
  • the one or more marker proteins may be a fragment of:
  • the fragment may be a naturally occurring fragment that results from proteolysis of its parent protein.
  • the one or more marker proteins may include a fragment selected from: Endorepellin (preferably, a 191 residue fragment of the 4391 residue Perlecan protein, more preferably the Endorepellin polypeptide comprises or consists essentially of the contiguous sequence of amino acid residues 4196-4391 of the sequence disclosed in GI 24212664 and/or UniProt accession No: P98160, more preferably the Endorepellin polypeptide comprises or consists essentially of the C- terminal V domain of said PGBM polypeptide, in particular the contiguous sequence of amino acid residues 3687-4391 of the sequence disclosed in GI 24212664 and/or UniProt accession No: P98160), Ganglioside GM2 activator short form (residues 34-193 of SAP3), Ganglioside GM2 activator long form (residues 32-193 of SAP3), cathepsin D heavy chain (residues 169-412 of CATD), and cathepsin
  • percentage sequence identity may be calculated using methods known in the art.
  • An example is the BLAST algorithm [Altschul et al., J. MoI. Biol. Vol. 215, pp. 403-410, (1990)].
  • a particularly useful BLAST program is the WU-BLAST-2 program [Altschul et al., Methods in Enzymology, Vol. 266, pp. 460-480 (1996)], which may be used with default parameters.
  • Abhydrolase domain-containing protein 14B is a cytosolic and nuclear protein coded by the gene ABHD14B, that may interact with TAFl. By alternative splicing two isoforms of this protein can be produced (isoforms 1 and 2).
  • Fructose bisphosphate aldolase B is a cytosolic protein encoded by the ALDOB gene that catalyzes the conversion of D-fructose 1,6-bisphosphate to glycerone phosphate and D-glyceraldehyde 3-phosphate during glycolysis. Changes in expression of this protein have been observed in lung and liver cancer tissues (Li et al., 2006).
  • Alpha-1-microglobulin is encoded by the AMBP gene, and is produced in liver and secreted to plasma. It consists of two different proteins that are separated by proteolysis: alpha-1-microglobulin, that appears in different fluids like plasma, urine and cerebrospinal fluid, and inter-alpha-trypsin inhibitor, that occurs in plasma and urine and inhibits trypsin, plasmin and lysosomal granulocytic elastase.
  • Apolipoprotein A-I belongs to the apolipoprotein A1/A4/E family and participates in the reverse transport of cholesterol from tissues to the liver for excretion by promoting cholesterol efflux from tissues and by acting as a cofactor for the lecithin cholesterol acyltransferase (LCAT).
  • Betaine-homocysteine S-methyltransferase is a cytosolic protein encoded by the BHMT gene, which is involved in the amine and polyamine degradation pathway by converting betaine and homocysteine to dimethylglycine and methionine. This protein is specific of liver and kidney, and is believed not to have been previously related to any cancer type.
  • Cathepsin D is an acid protease which belongs to the peptidase Al family and activates intracellular protein breakdown. It is believed to be involved in the pathogenesis of several diseases such as prostate cancer (Hara et al., 2002), breast, pancreas, lung and bladder cancer and possibly Alzheimer's disease.
  • Cytosolic non-specific dipeptidase belongs to the peptidase M20 family, and binds zinc ions as cofactor. Underexpression of this protein has been observed in liver tumour cells (Zhang et al., 2006).
  • Endothelial protein C receptor is a membrane protein that binds activated protein C, enhances protein C activation by the thrombin-thrombodulin complex and regulates blood coagulation by participating in the protein C pathway.
  • the expression of EPCR is very high in arteries and veins of heart and lung, medium in capillaries of lung and skin and absent in the endothelium of small vessels of liver and kidney. The upregulation of this protein has been described in ovarian cancer (Wang et al., 2005).
  • Prostate specific membrane antigen (PSMA also known as FOLHl) is an enzyme with both folate hydrolase and N-acetylated-alpha-linked-acidic dipeptidase (NAALADase) activity.
  • PSMA-I to 6 Six different isoforms (PSMA-I to 6) produced by alternative splicing have been postulated. It is a single pass type II membrane protein, but the isoform PSMA ' is cytosolic.
  • FOLHl is expressed in a number of tissues, including small intestine, brain, kidney, liver, spleen, colon, trachea, spinal cord and the capillary endothelium of a variety of tumors, but its highest expression is found in prostate.
  • the cytosolic isoform appears in normal tissue, while the membrane-bound isoform, PSMA-I, appears in prostate primary tumours.
  • FOLHl is involved in the prostate tumour progression, and its presence in blood and prostatic tissue is used as diagnostic and prognostic marker of prostate cancer.
  • Aldose 1-epimerase is a cytosolic enzyme involved in the hexose metabolism that converts alpha-aldoses to beta-anomers. There are believed to be no previous reports of any link between GALM and any cancer.
  • Gelsolin is an actin-regulating protein that is regulated by calcium ions. GELS binds to the plus ends of actin monomers or filaments, preventing monomer exchange. It can promote the assembly of monomers into filaments (nucleation) as well as sever filaments already formed. Two different isoforms are produced by alternative initiation, isoform 1 that is secreted to plasma, and the cytosolic isoform 2. Overexpression of this protein has been observed in hormone-resistant prostate cancer tissues (Culig et al., 2005). Differences in the levels of this protein have been observed in pancreatic cancer serum and bladder cancer urine (WO 2007/042256).
  • Golgin-45 is an ubiquitous protein required for normal Golgi structure and for protein transport from the endoplasmic reticulum (ER) through the Golgi apparatus to the cell surface.
  • Isoform 1 is located in Golgi apparatus lumen, and isoform 2, produced by alternative splicing, is cytoplasmatic. Its upregulation has been observed in promyelocytic leukemia.
  • GTPB2 GTP binding protein-1
  • Ig alpha-2 chain C region (IGHA2) is the major immunoglobulin class in body secretions. It may serve both to defend against local infection and to prevent access of foreign antigens to the general immunologic system.
  • Kallikrein-3 is a secreted protein from the peptidase Sl family that hydrolyzes seminogelin-1 leading to the liquefaction of the seminal coagulum.
  • Levels in serum are elevated related to prostate cancer and other prostatic alterations. Its measure in blood is used for prostate cancer diagnosis.
  • the present inventors have surprisingly found that the relative protein concentration of KLK3 in urine is lower in subjects with prostate cancer than subjects without prostate cancer. This illustrates the finding that it is not possible to predict how concentration of a marker protein, even a protein known to have altered serum concentration associated with prostate cancer, will be altered, if at all, in urine.
  • Perlecan is a secreted protein that is an integral component of basement membranes. It is responsible for the fixed negative electrostatic charge and is involved in the charge-selective ultrafiltration properties. It serves as an attachment substrate for cells. It has been related to angiogenesis and to several types of cancer such as ovarian, endometrial, cervical and pancreatic cancers. Its upregulation has been observed in prostate cancer cell lines (Datta et al., 2006).
  • Perlecan or "PGBM polypeptide” preferably refers to the contiguous sequence (SEQ ID NO: 38) of amino acid residues 22-4391 of the sequence disclosed in GI 24212664 and/or UniProt accession No. P98160 (SEQ ID NO: 26) (approx. 470 kDa).
  • Endorepellin is a 25KDa fragment of the C-terminal end of perlecan, released from the rest of the protein by the action of metalloproteases, and that has an inhibitory effect in the angiogenesis.
  • Endorepellin or “Endorepellin polypeptide” preferably comprises the LG3 domain of PGBM, more preferably comprising or consisting essentially of a 191 residue fragment of the 4391 residue Perlecan protein, yet more preferably the Endorepellin polypeptide comprises or consists essentially of the contiguous sequence of amino acid residues 4196-4391 of the sequence disclosed in GI 24212664 and/or UniProt accession No: P98160, still more preferably the Endorepellin polypeptide comprises or consists essentially of the C-terminal V domain of said PGBM polypeptide, in particular amino acid residues 3687-4391 of the sequence disclosed in GI 24212664 and/or UniProt accession No: P98160.
  • endorepellin has been described (Mongiat et al., 2003; the entire contents of which are expressly disclosed herein by reference).
  • sequence of the LG3 fragment of PGBM and endorepellin has been described (Gonzalez et al., 2005; the entire contents of which are expressly disclosed herein by reference).
  • Prostatic acid phosphatase belongs to the family of the histidine acid phosphatase family and it is encoded by the ACPP gene. This protein has been described as prostate cancer stage marker in prostatic tissue (Merrick et al., 2005) and as a target for immunotherapy against prostate cancer (Fong et al., 2001).
  • Plasma retinol-binding protein belongs to the lipocalin family and is the specific carrier for retinol (vitamin A alcohol) in the blood. It delivers retinol from the liver stores to the peripheral tissues. In plasma, the RBP-retinol complex interacts with transthyretin which prevents its loss by filtration through the kidney glomeruli.
  • vitamin A blocks secretion of the binding protein posttranslationally and results in defective delivery and supply to the epidermal cells. It has been related to bladder cancer (Basu et al., 1982) and cervical cancer (Audisio el al., 1985).
  • Ganglioside GM2 activator is a lysosomal protein that binds gangliosides and stimulates ganglioside GM2 degradation. It stimulates only the breakdown of ganglioside GM2 and glycolipid GA2 by beta-hexosaminidase A. It extracts single GM2 molecules from membranes and presents them in soluble form to beta-hexosaminidase A for cleavage of N-acetyl-D-galactosamine and conversion to GM3. It is believed that there have been no previous reports linking SAP3 to cancer.
  • SCTM l Secreted and transmembrane protein 1
  • SODC Superoxide dismutase Copper-Zinc
  • Tripeptidyl peptidase 1 is a lysosomal protein belonging to the peptidase S53 family. It is a serine protease with tripeptidyl peptidase activity, that releases N- terminal peptides from polypeptides and also has endopeptidase activity. TPPl may act as a non-specific lysosomal peptidase which generates tripeptides from the breakdown products produced by lysosomal proteinases. It has been described that its distribution is altered in tumoral tissues and other pathological tissues (Kida et al., 2001).
  • Transthyretin is a secreted thyroid hormone-binding protein that probably transports thyroxine from the bloodstream to the brain. Underexpression of this protein has been observed in pancreatic (Ehmann et al., 2007) and ovarian cancer (Moore et al., 2006).
  • Vitelline membrane outer layer protein 1 homolog belongs to the VMOl family and is postulated to be a secreted protein. It is believed that VMOl has not previously been linked to cancer or any other pathology.
  • Zinc-alpha-2-glycoprotein is a secreted protein present in liver, epithelium of various glands, blood plasma, urine, sweat, seminal plasma and saliva.
  • ZA2G stimulates lipid degradation in adipocytes and causes the extensive fat losses associated with some advanced cancers. It has been related to pancreas and liver cancers. In prostate cancer, its levels in plasma have been related to tumour stage, metastasis and recurrence (Hale et al., 2001).
  • the marker proteins are listed in Table 1, with the exception of Endorepellin, which as noted above is a 25 kDa fragment of the C-terminal end of perlecan (PGBM).
  • Table 1 also shows the change in relative protein concentration of each marker protein in urine samples from subjects with prostate cancer as compared with controls not having prostate cancer. The change is shown for each marker protein as either an elevation or a lowering together with the average fold-change in relative protein concentration.
  • the range of observed protein concentration values for each marker protein as a proportion of total protein ( ⁇ g protein/100 ⁇ g total protein) is also shown in Table 1 (range shown is that for all subjects).
  • the term “antibody” or “antibody molecule” includes any immunoglobulin whether natural or partly or wholly synthetically produced.
  • the term “antibody” or “antibody molecule” includes monoclonal antibodies and polyclonal antibodies (including polyclonal antisera). Antibodies may be intact or fragments derived from full antibodies (see below). Antibodies may be human antibodies, humanised antibodies or antibodies of non-human origin. "Monoclonal antibodies” are homogeneous, highly specific antibody populations directed against a single antigenic site or “determinant” of the target molecule. "Polyclonal antibodies” include heterogeneous antibody populations that are directed against different antigenic determinants of the target molecule.
  • the term “antiserum” or “antisera” refers to blood serum containing antibodies obtained from immunized animals.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CHl domains; (ii) the Fd fragment consisting of the VH and CHl domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab') 2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; (viii) bispecific single chain Fv dimers (WO 93/11161) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94/ 13804; 58). Fv, scFv or diabody molecules may be stabilised by the incorporation of
  • selective binds may be used herein to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s).
  • the term is also applicable where e.g. an antigen-binding site is specific for a particular epitope that is carried by a number of antigens, in which case the specific binding member carrying the antigen-binding site will be able to bind to the various antigens carrying the epitope.
  • the methods and kits of the present invention may employ immunological detection of the one or more marker proteins as defined herein.
  • a wide range of immunological assays are available to detect and quantify formation of specific antigen-antibody complexes; numerous competitive or non-competitive protein-binding assays have been described previously and a large number of these are commercially available.
  • the marker proteins of the invention can be quantified with antibodies such as, for example: monoclonal antibodies, polyclonal antibodies, either intact or recombinant fragments of these, combibodies and Fab or scFv fragments of antibodies, specific for the selected proteins of the invention; these antibodies are human, humanised or of animal origin.
  • the antibodies used in these assays can be labelled or unlabelled; the antibodies can be used in a wide range of assays.
  • Marker molecules that can be used to label antibodies include radionucleotides, enzymes, fluorophores, chemoluminescent reagents, enzymatic substrates or cofactors, enzymatic inhibitors, particles, colorants and derivatives. The higher the antibody binding specificity, the lower the antigen concentration that can be detected.
  • the techniques used for the detection and/or quantification of the complexes formed by antibodies and proteins are selected from the group comprising : Western blot, ELISA (Enzyme-Linked Immunosorbent assay), RIA (Radioimmunoassay), Competitive EIA (Competitive Enzyme Immunoassay), DAS-ELISA (Double Antibody Sandwich-ELISA), liquid immunoarray technology (e.g.
  • Luminex xMAP technology or Becton-Dickinson FACS technology immunocytochemical or immunohistochemical techniques, techniques based on the use of biochips or protein microarrays that include specific antibodies, assays based on the precipitation of colloidal gold in formats such as dipsticks; or by affinity chromatography techniques, ligand binding assays and lectin binding assays.
  • Certain preferred embodiments of the methods and kits of the invention employ protein microarrays or double antibody sandwich ELISA (DAS-ELISA).
  • DAS-ELISA double antibody sandwich ELISA
  • any antibody, or combination of antibodies can be used, provided such antibodies are specific against one or more epitopes of the marker proteins of the invention.
  • an antibody specific for a marker protein as defined herein is attached to the surface of a solid phase support, placed in contact with the sample to be analysed and incubated for a specific time and in appropriate conditions to form an antigen-antibody complex.
  • an indicator reagent for example a monoclonal or polyclonal antibody, or a fragment thereof, bound to a signal generating molecule, is incubated with the antigen-antibody complexes in appropriate conditions of time and temperature.
  • the presence of the marker proteins of the invention in the sample is detected and, if present, quantified and the signal generated is measured.
  • the amount of the one or more marker proteins of the invention present in the sample is proportional to the signal.
  • a method for obtaining and analyzing the total protein content of human urine samples and monitoring the presence of differentially expressed proteins within the sample involves the manipulation and preparation of the sample, the use of 2D electrophoresis to separate proteins within the sample, the selection of differentially expressed proteins by means of image analysis and statistics and the use of the selected proteins to generate protein-specific antibodies to be used as prostate cancer markers. Assay methods are also disclosed which are useful for monitoring the presence of the tumour markers with the antibodies in urine samples.
  • Comparative proteome analysis was performed between samples obtained from healthy individuals (controls) and patients subsequently diagnosed of prostate cancer in order to identify proteins differentially expressed in prostate cancer compared to the expression in control individuals.
  • the urine samples analysed were collected prior to the actual diagnosis of prostate cancer in the subjects.
  • the proteins that showed differential expression were identified by peptide mass fingerprinting using mass spectrometry and database search.
  • the invention may be better understood from the following non-limiting examples wherein differentially-expressed proteins are prepared from urine samples, polyclonal and monoclonal antibodies are made and tested for reactivity and sensitivity, and immunoassays are performed in the same body fluid.
  • the method may include preparation of a standard ("dose response") curve for a given marker protein.
  • preparation of a standard curve may be unnecessary.
  • Urine samples (181 in total) were collected from individuals with a PSA value measured in blood higher than 4 ng/ml that were visiting a urology unit in order to have a prostatic biopsy.
  • the urology units were those of hospitals belonging to the Basque Public Health Network ("Osakidetza") or the Instituto Oncol ⁇ gico de Gipuzkoa. These samples were classified as follows: a) No Carcinoma (97 samples) including healthy patients and patient with other prostatic alterations, as prostatitis and prostatic hyperplasia. b) Prostate cancer patients (87 samples): patients diagnosed with the disease at different stages of development.
  • the volume necessary to precipitate 400 ⁇ g of protein was calculated taking into account that the efficacy of protein precipitation with 10% v/v of trichloroacetic acid (TCA) is 75%.
  • TCA trichloroacetic acid
  • the rest of the urine sample was frozen again at -8O 0 C and stored for further 2D electrophoresis (if needed).
  • TCA and urine were mixed for 1 hour on ice, and then centrifuged at 16000 g for 20 min at 4 0 C to obtain the pellet of precipitated proteins. This pellet was washed with acetone stored at -2O 0 C and dried by solvent evaporation.
  • the first dimension was IEF (isoelectric focusing) where proteins separate by their charge (pi); the second dimension was SDS-PAGE where proteins separate by their molecular weight.
  • the dried pellet of proteins was resuspended with 450 ⁇ l of rehydratation buffer (Urea 7M, Thiourea 2M, CHAPS 2%, IPG buffer 2%, bromophenol blue 0.002%) for 1 hour at room temp.
  • IPG buffer (Amersham, ref# 17-600-88) was used so that the IEF ranged from pH 3-10.
  • the EttanTM IPGphorTM Isoelectric Focusing System from Amersham was used following the manufacturer's directions. The IEF was performed in immobilized pH gradients, named IPG strips, purchased from
  • solubilised proteins focused in the first dimension in the strips after 10 hours of active rehydratation of the gel at 30 V. Then the voltage was increased to 10000 V, the intensity never being higher than 50 ⁇ A per gel. The IEF was finished when the voltage reached 90000 V/hour.
  • Progenesis PG220 software version 2006, from Nonlinear Dynamics (UK) was used to analyze image files in a 300 dpi (dot per inch) format and 8 bits/channel. To increase its resolution the analysis was performed in two discrete areas of the gels. From each area, named as A and B (see Figure 1) gels were selected so that the samples belonging to the different groups would be represented and the statistical analysis was performed using the values obtained from the selected gels.
  • the Progenesis PG220 software transformed the information of the flat image into a 3D image, where the intensity of each spot correlated with its volume. With the software, tables of intensities for each spot were obtained in each gel. These raw data were the basis for the subsequent statistical analysis.
  • TSV Total Spot Volume
  • Proteins for immunization were cloned and expressed in E. c ⁇ // using a cloning vector.
  • proteins for immunization may be obtained from commercial sources.
  • the released proteins are directly used to immunize rabbits or mice as described below.
  • the purified proteins will be used to immunize rabbits for polyclonal or mice for monoclonal antibodies.
  • Antibodies will be raised using standard methodologies; immunizing animals with the protein of interest diluted in Freund's complete adjuvant (Gibco, Grand Island, N. Y.) first, and then every month during three months with the protein in incomplete adjuvant. Rabbit or mice sera (prior to fusion, in this case) will be used as polyclonal antisera to show if they are reactive to the protein preparations by standard western blot technique.
  • antibodies such as recombinant monoclonal antibodies, having binding specificity for a marker protein as defined herein may be obtained from commercial sources.
  • Protein samples (100 ⁇ g of total protein) were mixed with SDS-PAGE gel loading buffer supplemented with 5% ⁇ -mercaptoethanol and incubated at 100 0 C for 5 min, before being loaded on a polyacrylamide gel (the percentage of polyacrylamide may be varied depending on the molecular weight of the protein of interest).
  • electrophoresis proteins were transferred to nitrocellulose membranes. Duplicate gels were run and blotted. The membrane was probed with antibodies raised against the selected marker proteins as defined herein.
  • membranes were hybridised with a secondary antibody conjugated with peroxidase (Sigma RTM) and the chemoluminescent signal was detected using the ECL system (Millipore RTM) with high performance chemiluminescence film (Hyperfilm ECL, Millipore).
  • protein samples obtained from different urines were mixed with SDS-PAGE gel loading buffer supplemented with 5% ⁇ -mercaptoethanol and incubated at 100 0 C for 5 min, before being loaded on polyacrylamide gel. Following electrophoresis proteins were transferred to nitrocellulose membranes. Duplicate gels were run and blotted. The membrane was probed with antibodies raised against a specific marker protein as defined herein.
  • membranes were hybridised with a secondary antibody conjugated with peroxidase (Sigma RTM) and the chemoluminescent signal was detected using the ECL system (Millipore RTM) with high performance chemiluminescence film (Hyperfilm ECL, Millipore).
  • Figure 2 shows spot 1669 in the B area of bidimensional electrophoresis (2D) gel obtained from urine samples of three healthy individuals (Figure 2A), and from three prostate cancer patients (Figure 2B). It can be seen that the intensity of spot 1669 is clearly decreased in the samples of the patients with cancer when compared to the healthy urine samples. These differences showed to be significant after statistical analysis with Progenesis PG220.
  • Figure 3 shows the intensity of spot 1669 in control and cancer samples. The number of samples for each group is indicated. Table 2 below shows the p, fold change value and area under ROC curve of the statistical analysis for spot 1669 in samples of cancer in comparison samples of a non-cancer patients (CV). The fold change of spot 1669 was normalized by the total spot volume (TSV) for each individual gel.
  • TSV total spot volume
  • Sandwich type immunoassays have been developed in order to detect two of the prostate cancer markers selected based on the comparative proteomic analysis described above.
  • the two prostate cancer markers are: endorepellin (fragment derived from the carboxyl terminus of perlecan PGBM) and gelsolin (high molecular mass isoforms corresponding to the full length unprocessed protein).
  • the immunoassay for gelsolin specifically detects the full length protein (high molecular mass isoforms around 8OkDa, GELS).
  • Gelsolin is processed by a caspase and present in the urine as two groups of isoforms: those with high molecular mass (around 8OkDa) corresponding to the full length protein (GELS), and those with lower molecular mass (around 4OkDa) corresponding to the processed C-terminal domain of gelsolin.
  • the specific isoforms described here correspond to GELS.
  • the expression profile of the total amount of gelsolin in the urine (GELS + the rest of low molecular mass isoforms) behaves differently from GELS as a biomarker for the diagnosis of prostate cancer. Therefore, it has been found that the full length protein GELS, rather than the total amount of gelsolin, has the greater diagnostic capability.
  • the fragment selected in 2D electrophoresis (2DE) was the LG3 domain of this protein (a 25 kDa fragment of perlecan) and therefore, the antibodies used in the immunoassay were specific for this domain.
  • the immunoassays developed were validated in 72 urine samples. These samples were already included in the comparative proteomic analysis performed for the identification of prostate cancer markers. All the samples were collected from individuals with PSA concentration in serum higher than 4 ng/ml and subjected to prostatic biopsy in order to confirm the diagnosis. These 72 samples were classified as follows:
  • the immunoassays quantify the amount of GELS and endorepellin in each of the 72 subjects.
  • the results obtained were statistically analyzed to determine the role of both markers (individually and in combination) in the classification of each sample in the control or cancer group.
  • the results of the combination of GELS and endorepellin were analyzed using a binomial logistic regression. From figures 4 and 5, we can conclude that the ROC curves as well as the distribution of the values for control and cancer patients show an improvement when the endorepellin and GELS are combined (compared to the individual assays). This suggests that endorepellin and GELS may be advantageously combined as markers for prostate cancer.
  • Diagnostic data were computed for the markers endorepellin, GELS and for the combination of endorepellin and GELS.
  • the assays were performed in 22 controls and 50 prostate cancer urine samples.
  • AUC represents the values for the area under the ROC curves; PPV and NPV correspond to the positive predictive values and negative predictive values, respectively.
  • TP True positives
  • TN true negatives
  • FP false positives
  • FN false negatives
  • kits 4 and 5 and Tables 3 and 4 the diagnostic potential of a kit that utilises the combination of endorepellin and GELS detection, such as a kit comprising specific immunoassays for the detection of endorepellin and GELS, significantly improves over the individual immunoassays. These results support the advantageous combination of endorepellin and GELS as markers for use in a diagnostic test for prostate cancer.

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Abstract

Methods and products for detecting or assessing prostate cancer, particularly in vitro methods for detecting the presence of markers of prostate cancer in a urine sample from a subject, and related products, such as kits, for use in such methods are disclosed. The markers may be selected from: Endorepellin polypeptide, both Endorepellin polypeptide and GELS polypeptide, GELS polypeptide, ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOA1 polypeptide, BHMT1 polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLH1 polypeptide, GALM polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTM1 polypeptide, SODC polypeptide, TPP1 polypeptide, TTHY polypeptide, VMO1 polypeptide and ZA2G polypeptide; and/or a fragment of any one of said polypeptides.

Description

Cancer Markers and Methods For Their Detection
Field of the invention
The present invention relates to methods and products for detecting or assessing cancer, particularly in vitro methods for detecting the presence of markers of prostate cancer in a urine sample from a subject, and to products, such as kits, for use in such methods.
Background to the invention
Despite all the advances that have been achieved during the last 20 years, cancer is still one of the leading causes of mortality worldwide. Prostate cancer is the second most common cancer and also the second leading cause of death in men. Based on data from the International Agency for the Investigation of Cancer, GLOBOCAN, in the year 2002 680,000 new cases of prostate cancer were diagnosed in the world, and over 200,000 men died due to this disease. It is estimated that about the 30% of all men will develop a prostate carcinoma during their lifetime, and its prevalence in men by the age of 80 is of 80%.
The only non-invasive method available today for the diagnosis of prostate cancer is the measure of blood PSA (Prostate Specific Antigen). This glycoprotein produced by the cells of the prostate, is organ specific but not cancer specific, as it is also related to other prostatic benign alterations such as prostatitis and prostatic hyperplasia. This leads to a lack of specificity of this method that results in a 75% of false positives. PSA is used for prostate cancer screening all over the world, and it is estimated that about 45 million PSA tests are carried out every year. Men with a positive result in their PSA test require a prostatic biopsy to confirm the diagnosis of cancer, but 75% of these biopsies result negative. Several prostatic biopsies can be practised on a patient's lifetime while the PSA test results remain positive. Unnecessary biopsies are clearly undesirable.
Alteration of gene expression levels is tightly associated with uncontrolled cell growth and de-differentiation, common features of all cancers. The expression levels of the so- called "tumour suppressor genes", which act to block uncontrolled cell growth, are repressed in tumour cells; and expression levels of the so-called "oncogenes", which act to induce malignant growth, are elevated in tumour cells. Many of these genes have been associated with prostate cancer development, including Bcl-2, Cadherin-1, Catenin b, cyclinDl, EphA2, hyaluronidase, p53 and VEGF (Drobnjaket al., 2000; Zeng et al., 2003; Lokeshwar et al., 2001; Mackey et al., 1998; Miyake et al., 2004; Zellweger et al., 2003; and Miyake et al., 2005).
The alteration of the levels of some proteins has been observed in blood or urine, and have been related to presence, stage or progression of prostate cancer; these include: Cathepsin B, Caveolin-1, GSTPl, Hyaluronidase-1, KLK-2, PSCA, PSMA and UROC28 (Tricoli et al., 2004; An et al., 2000; Kurek et al., 2004; Tahir et al., 2003; Lokeshwar et al., 2001; Sinha et al., 2001; and Tasken et al., 2005).
There is clearly a need for further biomarkers of prostate cancer, in addition to presently-used measurement of the serum concentration of PSA.
Disclosure of the Invention
The present inventors have now found that certain proteins can be detected in urine and act as biomarkers for prostate cancer. The present invention, which employs urinary biomarkers for detection and monitoring of prostate cancer is considered more attractive as compared with, for example, use of serum biomarkers. Attempts to identify specific markers for prostate cancer in serum samples have been found to be largely unproductive due to the broad dynamic range of protein concentrations and certain highly abundant proteins which account for >98% of the total serum protein content. By contrast, the present invention, which makes use of urine samples, is less prone to problems related to protein dynamic range. Changes in protein patterns in urine between groups are thought to be highly representative of the prostate cancer pathology.
It is important to note that the profile of alteration of biomarkers, where established, in serum does not necessarily correlate with that in urine. Urine sample collection is noninvasive, which is a clear benefit in a clinical setting for both the patient and the clinician. The provision of urinary protein markers for prostate cancer has the potential to result in improvement for patient management in the field of prostate cancer.
The present inventors have now found that the protein concentration, particularly relative protein concentration, of each of the proteins ABHEB, ALDOB, AMBP, APOAl, BHMTl, CATD, CNDP2, EPCR, FOLHl, GALM, GELS, GO45, GTPB2, IGHA2, KLK3, PGBM (particularly its fragment, Endorepellin), PPAP, RETBP, SAP3, SCTM l, SODC, TPPl, TTHY, VMOl and ZA2G is altered in urine samples collected from patients with prostate cancer when compared with urine samples collected from individuals that do not have prostate cancer.
Accordingly, in a first aspect the invention provides a method for detecting or monitoring prostate cancer or susceptibility to prostate cancer in a test subject, comprising : determining an amount (relative or absolute), such as relative or absolute protein concentration, of one or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) marker proteins in a urine sample obtained from the test subject; and comparing said amount (relative or absolute) with a standard value, wherein the one or more marker proteins are selected from:
Endorepellin polypeptide, both Endorepellin polypeptide and GELS polypeptide, GELS polypeptide, ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOAl polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTM l polypeptide, SODC polypeptide, TPPl polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide; and/or a fragment of any one of said polypeptides.
The method may be non-invasive because a urine sample is readily collected and can be conveniently analysed. Moreover, changes in amount, particularly relative amount and more particularly relative protein concentration, are expected to be highly representative of the prostate cancer pathology. The method may comprise measuring one marker protein or a panel of two or more, such as 3, 4, 5, 6, 7, 8, 9, 10 or more, marker proteins as defined herein. In some cases, the use of a panel of marker proteins as defined herein may provide further positive predictive value due, for example, to statistical analysis of multiple marker protein changes in amount, particularly relative amount, such as relative protein concentration. Use of a panel of marker proteins may improve the sensitivity and/or specificity of the assay.
In certain preferred cases, the method of the one or more marker proteins may be selected from the group consisting of: Endorepellin polypeptide, GELS polypeptide and both Endorepellin polypeptide and GELS polypeptide. As described herein, particularly with reference to Example 2, Endorepellin polypeptide and GELS polypeptide may be advantageously combined as marker proteins in accordance with this and other aspects of the invention. It is believed that use of both Endorepellin polypeptide and GELS polypeptide improves sensitivity and/or specificity as compared with use of Endorepellin polypeptide or GELS polypeptide in isolation.
The present inventors have found that the urinary marker proteins defined herein may offer greater specificity for detection of prostate cancer in comparison with the detection of serum PSA. Many factors cause an elevated serum PSA value (the threshold typically used in current testing is 4 ng/ml of serum). All patients with a serum PSA higher than this threshold are normally biopsied in current testing, despite the fact that up to 75% of those with elevated serum PSA in fact do not have prostate cancer. The methods of the present invention aim to reduce this high false positive rate by providing higher specificity, whilst also providing high sensitivity.
As detailed further herein (see Table 1), the protein markers described above show altered relative protein concentration in urine samples from subjects having prostate cancer compared with healthy controls. A number of the protein markers exhibit elevated relative protein concentration in urine of prostate cancer subjects, while a number exhibit lower relative protein concentration in urine of prostate cancer subjects.
Accordingly, in the certain methods of the present invention the one or more marker proteins are selected from: ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GELS polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide; and/or a fragment thereof, wherein the relative amount is the amount of the marker protein relative to a reference protein also present in the urine sample, and wherein a decreased relative amount of said one or more marker proteins in the urine sample obtained from the test subject compared with a standard value corresponding to the relative amount of the same marker protein in a urine sample obtained from a control subject not having prostate cancer indicates that the test subject has or is susceptible to prostate cancer.
As used herein, a lower amount, such as a lower relative amount or a lower absolute amount, particularly a lower relative protein concentration or lower absolute protein concentration, may be 10%, 25%, 50% or 75% lower than the standard value. The average decrease in relative protein concentration for each marker protein is found in prostate cancer patient urine is shown in Table 1 as a "fold-change". The precise threshold for the level of decrease and the precise numerical value used as the standard value for the marker protein of interest may be selected by the skilled person as appropriate for the subject or population under consideration. For example, the skilled person may derive a suitable standard value and threshold of decrease in amount (relative or absolute) for the given marker protein by means of clinical tests or by reference to charts, tables or databases of pre-determined standard values and/or threshold levels of decrease. Preferably, the standard value for the marker protein is a pre-determined value that has been determined by measuring a representative number of control subjects from a population of subjects not having prostate cancer.
Furthermore, in the certain methods of the present invention the one or more marker proteins are selected from : ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide; and/or a fragment thereof, wherein the relative amount is the amount of the marker protein relative to a reference protein also present in the urine sample, and wherein an increased relative amount of said one or more marker proteins in the urine sample obtained from the test subject compared with a standard value corresponding to the relative amount of the same marker protein in a urine sample obtained from a control subject not having prostate cancer indicates that the test subject has or is susceptible to prostate cancer.
As used herein, a higher amount, such as a higher relative amount or a higher absolute amount, particularly a higher relative protein concentration or higher absolute protein concentration, may be 10%, 25%, 50%, 100%, 200% or 500% higher than said reference value. The average increase in relative protein concentration for each marker protein is shown in Table 1 as a "fold-change". The precise threshold for the level of increase and the precise numerical value used as the standard value for the marker protein of interest may be selected by the skilled person as appropriate for the subject or population under consideration. For example, the skilled person may derive a suitable standard value and threshold of increase in relative amount for the given marker protein by means of clinical tests or by reference to charts, tables or databases of predetermined standard values and/or threshold levels of increase. Preferably, the standard value for the marker protein is a pre-determined value that has been determined by measuring a representative number of control subjects from a population of subjects not having prostate cancer. "Absolute amount" of protein as used herein is intended to include measures of protein amount that are independent of other proteins. For example, absolute amount of protein may be the protein concentration expressed in terms of a quantity of said protein (e.g. micrograms or nanograms or picograms) per unit of urine sample (e.g. millilitres of volume). Determining an absolute amount of a marker protein, as used in accordance with the methods of the invention, specifically includes indirect as well as direct determination. For example, the actual concentration (e.g. expressed in mass of protein per unit volume of sample) may be determined or an indirect measure that correlates with the absolute amount or absolute protein concentration. For example, determining an absolute amount of a marker protein may comprise measuring fluorescence, optical density or similar measure in an immunoassay. The determination may or may not involve reference to a "standard curve" comprising known amounts (e.g. a series of known concentrations) of the protein.
Determination of absolute amount of a marker protein may be preferred in certain circumstances. It is believed that determination of absolute amount of a marker protein as defined herein may be informative for prostate cancer detection or monitoring. It has been found that use of a urine protein (such as creatinine) as a reference (for example for total protein concentration) is not necessarily required to achieve an assay having acceptable sensitivity and/or specificity. Determination of absolute amount of a given protein does not require measurement of a reference protein or total protein in the sample. Thus, determination of absolute amount may be in some cases quicker involving fewer steps than determination of a relative amount of a marker protein. The comparative simplicity of determining absolute rather than relative protein amount is particularly advantageous in the setting of clinical testing and/or large-scale sample analysis. In certain preferred cases the method of the invention may comprise indirect determination of the absolute amount of a marker protein as defined herein by means of a single reading of, e.g. fluorescence or optical density, following an immunoassay as further detailed herein.
However, absolute protein concentration in the urine of healthy control subjects and subjects with cancer is highly variable both between individuals and also for a given individual depending on diet, time of day and disease status among other factors. For example, proteinuria is associated with, among other conditions, disease of the kidney. The present inventors have found that protein concentration in the urine may differ by over two orders of magnitude between subjects. Many of the subjects whose urine was used in the analysis described herein had haematuria (both cancer patients and other controls with non-malignant urological conditions), and this can elevate substantially the total protein content of urine. However, the present inventors have found that consistent comparisons may be made by determining relative protein amount, such as relative protein concentration. The relative protein concentration gives a measure of the concentration of the given marker protein relative to a reference protein. Thus, determination of relative, rather than absolute, amount of a marker protein as defined herein may be preferred in cases where it is desired to correct for or "normalise" the baseline protein amount. Methods in accordance with the invention may comprise determining relative amount of a marker protein, particularly when the sample is from a subject having or suspected to have significantly variable urinary protein concentration (for example a subject having or suspected of having a disease or condition affecting kidney function, proteinuria and/or haematuria).
"Relative amount" of protein as used herein is intended to include measures of protein amount that are related to (e.g. divided by) a measure of the amount of one or more other proteins. For example, relative amount of protein may be the protein concentration expressed in terms of a quantity of said protein (e.g. micrograms or nanograms or picograms) per unit of one or more other proteins in a urine sample (e.g. mass of a given marker protein per total mass of protein or mass of a given marker protein per mass of a reference protein). Determining a relative amount of a marker protein, as used in accordance with the methods of the invention, specifically includes indirect as well as direct determination. For example, the actual concentration of the marker protein and of the reference protein (e.g. expressed in mass of protein per unit volume of sample) may be determined or an indirect measure that correlates with the amount of the marker protein and an indirect measure that correlates with the amount of the reference protein may be determined and used to derive the relative protein amount indirectly. In some cases, determining a relative amount of a marker protein may comprise measuring fluorescence, optical density or similar measure in an immunoassay. The determination may or may not involve reference to a "standard curve" comprising known amounts (e.g. a series of known concentrations) of the marker protein and/or a reference protein.
In some cases the relative protein concentration is relative to a reference protein, the concentration of which is substantially unchanged between subjects having prostate cancer, and controls subjects not having prostate cancer. Preferably, the relative protein concentration of a given marker protein as defined herein may be relative to the protein concentration of another of the marker proteins as defined herein. As noted above, a first group of the marker proteins defined herein exhibit decreased concentration in urine of prostate cancer patients (ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GELS polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide; and/or a fragment thereof), while a second group of the marker proteins defined herein exhibit increased concentration in urine of prostate cancer patients (ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide; and/or a fragment thereof). Thus, the marker proteins of the first group and second group exhibit "inverse expression changes" in association with prostate cancer. The present inventors have realised that the inverse expression changes of the two groups of marker proteins can be utilised to improve the specificity and/or sensitivity of the methods of the invention. In particular, the relative amount of a marker protein from the first group may be relative to the amount of a reference protein chosen from the second group. This will enhance the degree of decrease in relative amount that is associated with prostate cancer. Likewise, the relative amount of a marker protein from the second group may be relative to the amount of a reference protein chosen from the first group. This will enhance the degree of increase in relative amount that is associated with prostate cancer.
Accordingly, in some cases, the method of the first aspect of the invention may comprise: determining the relative amount of one or more marker proteins selected from: ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GELS polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide; and/or a fragment thereof, in a urine sample obtained from the test subject, wherein the relative amount is the amount of the marker protein relative to a reference protein selected from: ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide; or a fragment thereof; and comparing said relative amount with a standard value corresponding to the relative amount of the same marker protein relative to the same reference protein in a urine sample obtained from a control subject not having prostate cancer, and wherein a decreased relative amount of said one or more marker proteins in the urine sample obtained from the test subject compared with the standard value indicates that the test subject has or is susceptible to prostate cancer.
In some cases, the method of the first aspect of the invention may comprise: determining the relative amount of one or more marker proteins selected from: ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide; and/or a fragment thereof, in a urine sample obtained from the test subject, wherein the relative amount is the amount of the marker protein relative to a reference protein selected from: ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GELS polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTM l polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide; or a fragment thereof; and comparing said relative amount with a standard value corresponding to the relative amount of the same marker protein relative to the same reference protein in a urine sample obtained from a control subject not having prostate cancer, and wherein an increased relative amount of said one or more marker proteins in the urine sample obtained from the test subject compared with the standard value indicates that the test subject has or is susceptible to prostate cancer.
By means of binomial logistic regression analysis methods employed herein, it has been found that preferred marker proteins may be selected based on likelihood values independently of whether a particular marker is up-regulated or down-regulated in urine of prostate cancer subjects. That is to say, improvements in assay performance (e.g. improved sensitivity and/or selectivity) may be achieved by combining two or more marker proteins whether the two or more marker proteins are all up-regulated, all down- regulated or a mixture of up-regulated and down-regulated in prostate cancer.
Accordingly, in some cases of this and other aspects of the invention, the one or more marker proteins may comprise at least two marker proteins as defined herein. The at least two marker proteins may comprise two or more marker proteins that are both down-regulated in prostate cancer and/or two or more marker proteins that are both up- regulated in prostate cancer.
In some cases, the one or more marker proteins and/or the reference protein may be selected so as to enhance the sensitivity and/or specificity of the method of this aspect of the invention. In particular, a marker protein may be chosen which exhibits, based on the fold-change shown in Table 1, a substantial decrease or a substantial increase in relative amount in urine from prostate cancer patients. Likewise, a reference protein may be chosen which exhibits, based on the fold-change shown in Table 1, a substantial increase or a substantial decrease in relative amount in urine from prostate cancer patients. It is particular preferred that both the one or more marker proteins and the reference protein are selected based on maximal or near-maximal inverse expression changes. For example, ALDOB polypeptide, which exhibits a 12-fold increase, and RETBP polypeptide, which exhibits a 3-fold decrease, in urine from prostate cancer patients may be advantageously selected as marker protein and reference protein, respectively, or vice versa. The skilled person is readily able to select further such combinations based on the fold-change values shown in Table 1. Preferably, the marker protein and/or the reference protein is selected from those proteins shown in Table 1 that exhibit a fold-change (increase or decrease) of greater than 1.5-fold, 2-fold or 2.5- fold, or a fragment of any one of those proteins.
The one or more marker proteins of this and other aspects of the invention are preferably the human full-length polypeptides having the sequences identified by GI number and UniProt accession number in Table 1.
The "Genlnfo Identifier" or GI number is an integer number assigned to each nucleotide and protein sequence available through NCBI's GenBank database. The GI number uniquely identifies a particular sequence; if the sequence changes in any way, a new GI number is assigned. Therefore, the GI numbers in Table 1 identify particular, fixed sequence versions. Each of the amino acid sequences identified by GI number in Table 1 is specifically incorporated herein by reference in its entirety.
The UniProt accession numbers used herein, particularly in Table 1, refer to UniProtKB release 14.0 available 22 July 2008 (incorporating Swiss-Prot release 56.0). Each of the amino acid sequences identified by UniProt accession number, and where applicable the isoform suffix -1, -2, etc., in Table 1 is specifically incorporated herein by reference in its entirety. Thus, ABHEB polypeptide may be the human ABHEB polypeptide having the amino acid sequence disclosed in UniProt accession No: Q96IU4 (including any one of isoforms Q96IU4-1 (SEQ ID NO: 1) and/or Q96IU4-2 (SEQ ID NO: 2)); ALDOB polypeptide may be the human ALDOB polypeptide having the amino acid sequence disclosed in UniProt accession No: P05062 (SEQ ID NO: 3); AMBP polypeptide may be the human AMBP polypeptide having the amino acid sequence disclosed in UniProt accession No: P02760 (SEQ ID NO: 4); APOAl polypeptide may be the human APOAl polypeptide having the amino acid sequence disclosed in UniProt accession No: P02647 (SEQ ID NO: 5); BHMT polypeptide may be the human BHMT polypeptide having the amino acid sequence disclosed in UniProt accession No: Q93088 (SEQ ID NO: 6); CATD polypeptide may be the human CATD polypeptide having the amino acid sequence disclosed in UniProt accession No: P07339 (SEQ ID NO: 7); CNDP2 polypeptide may be the human CNDP2 polypeptide having the amino acid sequence disclosed in UniProt accession No: Q96KP4 (SEQ ID NO: 8); EPCR polypeptide may be the human EPCR polypeptide having the amino acid sequence disclosed in UniProt accession No: Q9UNN8 (SEQ ID NO: 9); FOLHl polypeptide may be the human FOLH l polypeptide having the amino acid sequence disclosed in UniProt accession No: Q04609 (including any one of isoforms PSMA-I (Q04609-1; SEQ ID NO: 10), PSMA-2 (Q04609-2; SEQ ID NO: 11), PSMA-3 (Q04609-3; SEQ ID NO: 12), PSMA-4 (Q04609-4; SEQ ID NO: 13), PSMA-5 (Q04609-5; SEQ ID NO: 14) and PSMA-6 (Q04609-6; SEQ ID NO: 15)); GALM polypeptide may be the human GALM polypeptide having the amino acid sequence disclosed in UniProt accession No: Q96C23 (SEQ ID NO: 16); GELS polypeptide may be the human GELS polypeptide having the amino acid sequence disclosed in UniProt accession No: P06396 (SEQ ID NO: 17); GO45 polypeptide may be the human GO45 polypeptide having the amino acid sequence disclosed in UniProt accession No: Q9H2G9 (including any one of isoforms Q9H2G9-1 (SEQ ID NO: 18) and Q9H2G9-2 (SEQ ID NO: 19)); GTPB2 polypeptide may be the human GTPB2 polypeptide having the amino acid sequence disclosed in UniProt accession No: Q9BX10 (SEQ ID NOs: 20 - 23); IGHA2 polypeptide may be the human IGHA2 polypeptide having the amino acid sequence disclosed in UniProt accession No:
P01877 (SEQ ID NO: 24); KLK3 polypeptide may be the human KLK3 polypeptide having the amino acid sequence disclosed in UniProt accession No: P07288 (SEQ ID NO: 25); PGBM polypeptide may be the human PGBM polypeptide having the amino acid sequence disclosed in UniProt accession No: P98160 (SEQ ID NO: 26); Endorepellin polypeptide may be the 25kDa fragment of the C-terminal end of the human PGBM polypeptide having the amino acid sequence disclosed in UniProt accession No: P98160 (SEQ ID NO: 26), preferably the Endorepellin polypeptide comprises or consists essentially of the LG3 domain of said PGBM polypeptide, in particular amino acid amino acid residues 4196- 4391 of the sequence disclosed in GI 24212664 and/or UniProt accession No: P98160, more preferably the Endorepellin polypeptide comprises or consists essentially of the C- terminal V domain of said PGBM polypeptide, in particular amino acid residues 3687- 4391 (SEQ ID NO: 37) of the sequence disclosed in GI 24212664 and/or UniProt accession No: P98160; PPAP polypeptide may be the human PPAP polypeptide having the amino acid sequence disclosed in UniProt accession No: P15309 (SEQ ID NO: 27); RETBP polypeptide may be the human RETBP polypeptide having the amino acid sequence disclosed in UniProt accession No: P02753 (SEQ ID NO: 28); SAP3 polypeptide may be the human SAP3 polypeptide having the amino acid sequence disclosed in
UniProt accession No: P17900 (SEQ ID NO: 29); SCTMl polypeptide may be the human SCTM l polypeptide having the amino acid sequence disclosed in UniProt accession No: Q8WVN6 (SEQ ID NO: 30); SODC polypeptide may be the human SODC polypeptide having the amino acid sequence disclosed in UniProt accession No: P00441 (SEQ ID NO: 31); TPPl polypeptide may be the human TPPl polypeptide having the amino acid sequence disclosed in UniProt accession No: 014773 (including any one of isoforms 014773-1 (SEQ ID NO: 32) and 014773-2 (SEQ ID NO: 33)); TTHY polypeptide may be the human TTHY polypeptide having the amino acid sequence disclosed in UniProt accession No: P02766 (SEQ ID NO: 34); VMOl polypeptide may be the human VMOl polypeptide having the amino acid sequence disclosed in UniProt accession No: Q7Z5L0 (SEQ ID NO: 35); and Za2G polypeptide may be the human ZA2G polypeptide having the amino acid sequence disclosed in UniProt accession No: P25311 (SEQ ID NO: 36).
The one or more marker proteins of this and other aspects of the invention may be a derivative, variant, orthologue, homologue or fragment of one or more of the human full-length polypeptides having the sequences identified by GI and/or UniProt accession number in Table 1. A marker protein of the invention may be a derivative, variant, orthologue, homologue or fragment of one or more of the human full-length polypeptides having the sequences identified by GI and UniProt accession number in Table 1, which comprises an amino acid sequence having at least 70%, 80%, 90%, 95% or 99% sequence identity to the sequence, preferably to the full-length sequence, of a polypeptide identified by GI and UniProt accession number in Table 1. It is well-known that multiple allelic variants may exist for a given gene product. Furthermore, one or more variants of a given protein may exist due to transcriptional or post-translational changes. The one or more marker proteins of this and other aspects of the invention may be a naturally occurring allelic variant of a marker protein identified in Table 1. The one or more marker proteins of this and other aspects of the invention may be a derivative, such as a chemically-altered derivative, of a marker protein identified in Table 1. In some cases a method of the invention may include chemically altering the one or more marker proteins present in a sample in order to facilitate detection and/or measurement of the one or more marker proteins. For example, it is specifically contemplated that one or more marker proteins may be labelled prior to measurement in a method of the invention.
The one or more marker proteins of this and other aspects of the invention may be a fragment of: (i) a polypeptide having an amino acid sequence identified by GI and/or UniProt accession number in Table 1; or
(ii) a derivative, variant, homologue of (i) having an amino acid sequence with at least 70%, 80%, 90%, 95% or 99% sequence identity to (i), wherein said fragment comprises at least 20, 50, 100, 150, 200, 250 or 500 amino acids. The fragment may be a naturally occurring fragment that results from proteolysis of its parent protein. For example, the one or more marker proteins may include a fragment selected from : Endorepellin polypeptide (preferably Endorepellin polypeptide comprises the LG3 domain of PGBM, more preferably comprising or consisting essentially of a 191 residue fragment of the 4391 residue Perlecan protein, more preferably the Endorepellin polypeptide comprises or consists essentially of the contiguous sequence of amino acid residues 4196-4391 of the sequence disclosed in GI 24212664 and/or UniProt accession No: P98160, yet more preferably the Endorepellin polypeptide comprises or consists essentially of the C-terminal V domain of said PGBM polypeptide, in particular the contiguous sequence of amino acid residues 3687-4391 (SEQ ID NO: 37) of the sequence disclosed in GI 24212664 and/or UniProt accession No: P98160), Ganglioside GM2 activator short form (residues 34-193 of SAP3; SEQ ID NO: 39), Ganglioside GM2 activator long form (residues 32-193 of SAP3; SEQ ID NO: 40), cathepsin D heavy chain (residues 169-412 of CATD; SEQ ID NO: 41), and cathepsin D light chain (residues 65- 161 of CATD; SEQ ID NO: 42), any one of the 40-45KDa fragments of GELS that correspond to the N-terminal cleaved portion from GELS by thermolysin in presence of calcium ions, any one of five fragments of RETBP that result from different cleavage (residues 19-201 (SEQ ID NO: 43), residues 19-200 (SEQ ID NO: 44), residues 19-199 (SEQ ID NO: 45), residues 19-197 (SEQ ID NO: 46) and/or residues 19-194 (SEQ ID NO: 47) of RETBP) and a 14-16 KDa N-terminal fragment of SCTMl.
The method of this and other aspects of the invention may comprise one or more steps of treating the urine sample to remove or reduce the level of contaminants, particularly solid contaminants such as free tissue and cells. In some cases a urine sample may be subjected to centrifugation so as to remove tissue, cells and/or other solid contaminants prior to measuring the relative amount of the one or more marker proteins as defined herein. Thus, in preferred embodiments, it may not be necessary to perform a protein extraction. The method may employ direct urine samples, which have been subjected to little or no preparative treatment, thereby saving on time and laboratory resources.
In certain cases the method of this and other aspects of the invention may comprise extracting protein from a urine sample to obtain a protein extract. The relative amount of the one or more marker proteins, in particular the relative protein concentration of said one or more marker proteins, in a protein extract obtained from a urine sample provides a measure of the relative amount of the marker protein in the urine sample because any concentration or dilution of the protein in the sample that occurs during the extraction is expected to affect the concentration of the one or more marker proteins to substantially the same extent as the total protein concentration and/or the protein concentration of a reference protein. Thus, the relative amount of the one or more marker proteins is in effect "normalised". Measurements based on a protein extract from the urine sample therefore provide an accurate measure of the relative amount of the one or more marker proteins in the urine sample. Protein extraction from the urine sample may be employed so that the absolute protein concentration of the extract can be adjusted so as to be optimal or close to optimal for the method used to measure relative protein concentration. Therefore, in some cases the method of the invention involves measuring the relative amount of said one or more marker proteins in a urine sample obtained from the test subject by extracting protein from said urine sample to obtain a protein extract, measuring the relative protein concentration of said one or more marker proteins in the protein extract, thereby deriving a measure of the relative protein concentration of said one or more marker proteins in the urine sample.
In the method of this and other aspects of the invention, determining the amount (relative amount or absolute amount) of said one or more marker proteins in the urine sample may comprise: contacting said urine sample with at least one specific binding member that selectively binds to one of said marker proteins; and detecting and/or quantifying formation of a complex formed by said specific binding member and said marker protein. The specific binding member may be an antibody molecule or a binding fragment thereof. For example, the antibody molecule may be selected from : a monoclonal antibody, a polyclonal antibody, an antibody binding fragment and a combibody. Further details of specific binding members, including antibody molecules, for use in accordance with the present invention are provided herein.
A wide variety of techniques are available for detecting formation of a complex formed by said specific binding member and said marker protein. Preferred techniques include: Western blot; enzyme-linked immunosorbent assay (ELISA); radioimmunoassay (RIA); competitive enzyme immunoassay; double antibody sandwich ELISA (DAS-ELISA); liquid immunoarray technology (e.g. Luminex xMAP technology and Becton-Dickinson FACS technology, among others); immunocytochemistry; immunohistochemistry; antibody microarray detection; precipitation of colloidal gold; affinity chromatography; ligand binding assay; and lectin binding assay.
A preferred method in accordance with this aspect of the invention comprises:
(i) contacting a urine sample obtained from the subject with a first specific binding member, which selectively binds to a marker protein selected from Endorepellin polypeptide, both Endorepellin polypeptide and GELS polypeptide, GELS polypeptide, ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide, or a fragment thereof;
(ii) contacting said urine sample with a second specific binding member, which selectively binds to a reference protein selected from ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide, or a fragment thereof;
(iii) detecting and/or quantifying the formation of a first complex formed by the first specific binding member and the marker protein to which it binds and of a second complex formed by the second specific binding member and the reference protein to which it binds, thereby deriving a measurement of the relative amount of said marker protein relative to said reference protein;
(iv) comparing said relative amount of said marker protein with a standard value that corresponds to the relative amount of the same marker protein relative to the same reference protein in a urine sample obtained from a subject not having prostate cancer, wherein a relative amount of said marker protein which is decreased by 10%, 20%, 50% or greater compared with the standard value indicates that the subject has prostate cancer.
A preferred method in accordance with this aspect of the invention comprises:
(i) contacting a urine sample obtained from the subject with a first specific binding member, which selectively binds to a marker protein selected from ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide, or a fragment thereof;
(ii) contacting said urine sample with a second specific binding member, which selectively binds to a reference protein selected from Endorepellin polypeptide, both Endorepellin polypeptide and GELS polypeptide, GELS polypeptide, ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide, or a fragment thereof;
(iii) detecting and/or quantifying the formation of a first complex formed by the first specific binding member and the marker protein to which it binds and of a second complex formed by the second specific binding member and the reference protein to which it binds, thereby deriving a measurement of the relative amount of said marker protein relative to said reference protein;
(iv) comparing said relative amount of said marker protein with a standard value that corresponds to the relative amount of the same marker protein relative to the same reference protein in a urine sample obtained from a subject not having prostate cancer, wherein a relative amount of said marker protein which is increased by 10%, 20%, 50% or more compared with the standard value indicates that the subject has prostate cancer.
In certain cases, the method for detecting or monitoring cancer or susceptibility to a cancer in a test subject, in accordance with this aspect of the invention, comprises: diagnosing cancer in the test subject; determining the stage, severity or progression of cancer in the test subject; assessing the absence of cancer in the test subject after surgical treatment has been performed on the test subject; establishing the prognosis of a cancer in the test subject; or monitoring the effect of therapeutic treatment of cancer in the test subject. In this and other aspects of the invention, the test subject may have been not previously diagnosed with prostate cancer. Alternatively, the test subject may have been previously diagnosed with prostate cancer and/or may have been previously treated for prostate cancer. The methods of the invention are suitable for detecting the presence of prostate cancer, for the first time in a subject and are suitable for on-going monitoring of the status of a subject having prostate cancer. In the latter case, the on-going monitoring may be used to inform treatment strategy and/or determine the prognosis of the subject. On-going monitoring may involve assessing the amount (relative amount or absolute amount), such as relative protein concentration or absolute protein concentration, of one or more of said marker proteins in a plurality of urine samples obtained from the test subject at different times, for example when at least one of the urine samples is obtained prior to a therapeutic or surgical intervention performed on the test subject and at least one of said urine samples is obtained after a said intervention.
In a further aspect the present invention provides a prostate cancer diagnostic kit comprising :
1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more specific binding members each of which selectively binds to a marker protein as defined above with reference to the first aspect of the invention; and one or more reagents for detecting said one or more specific binding members.
In some cases, the prostate cancer diagnostic kit in accordance with this aspect of the invention may comprise: a specific binding member that selectively binds to Endorepellin polypeptide and/or a specific binding member that selectively binds to GELS polypeptide.
In some cases, the prostate cancer diagnostic kit in accordance with this aspect of the invention may comprise:
1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more specific binding members each of which selectively binds to a marker protein selected from ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOAl polypeptide, BHMTl polypeptide, CNDP2 polypeptide, EPCR polypeptide, GALM polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 polypeptide, Endorepellin polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, TPPl polypeptide, TTHY polypeptide and VMOl polypeptide, or a fragment thereof; and one or more reagents for detecting said one or more specific binding members. In some cases the prostate cancer diagnostic kit of this aspect of the invention comprises: a specific binding member which selectively binds to a marker protein selected from ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GELS polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide, or a fragment thereof; and a specific binding member which selectively binds to a marker protein selected from ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide, or a fragment thereof.
The prostate cancer diagnostic kit of this aspect of the invention may comprise a plurality of said specific binding members which are provided in the form of an array on a substrate.
The specific binding member of the prostate cancer diagnostic kit of this aspect of the invention may comprise an antibody molecule or a binding fragment thereof. The marker proteins are as defined in the first aspect of the invention. Preferably, the specific binding members comprise antibody molecules or antibody binding fragments thereof, as defined further herein.
The prostate cancer diagnostic kit of this aspect of the invention may comprise at least one reagent or article for processing, storing and/or transporting a urine sample. For example, said kit may comprise a reagent (e.g. trichloroacetic acid) for use in extracting protein from a urine sample. Alternatively or additionally, the prostate cancer diagnostic kit of this aspect of the invention may comprise instructions, e.g. in the form of an insert, internal or external label, or data carrier, for carrying out a method in accordance with the first aspect of the invention. In particular, the instructions may refer to use of said kit in the analysis of one or more urine samples. Alternatively or additionally, the prostate cancer diagnostic kit of this aspect of the invention may comprise one or marker protein standards (e.g. for preparing a standard curve of known protein concentrations).
In a further aspect the present invention provides a specific binding member for use in a method of diagnosing prostate cancer (preferably in vitro), wherein said specific binding member selectively binds a marker protein as defined in accordance with the first aspect of the invention. In certain preferred cases, the specific binding member of this aspect of the invention selectively binds Endorepellin polypeptide. In certain preferred cases, the specific binding member of this aspect of the invention selectively binds GELS polypeptide.
In certain preferred cases, the specific binding member of this aspect of the invention selectively binds a marker protein selected from : ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOAl polypeptide, BHMTl polypeptide, CNDP2 polypeptide, EPCR polypeptide, GALM polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 polypeptide, Endorepellin polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, TPPl polypeptide, TTHY polypeptide and VMOl polypeptide; or a fragment thereof. The marker proteins are as defined in the first aspect of the invention. Preferably, the specific binding members comprise antibody molecules or antibody binding fragments thereof, as defined further herein.
In a further aspect the present invention provides use of a specific binding member which selectively binds a marker protein as defined in accordance with the first aspect of the invention in a method of diagnosing prostate cancer (preferably in vitro) or in the manufacture of a diagnostic agent for diagnosis of prostate cancer. In certain preferred cases, the specific binding member of this aspect of the invention selectively binds
Endorepellin polypeptide. In certain preferred cases, the specific binding member of this aspect of the invention selectively binds GELS polypeptide. In certain preferred cases, the specific binding member of this aspect of the invention selectively binds a marker protein selected from: ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOAl polypeptide, BHMTl polypeptide, CNDP2 polypeptide, EPCR polypeptide, GALM polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 polypeptide, Endorepellin polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, TPPl polypeptide, TTHY polypeptide and VMOl polypeptide; or a fragment thereof. The marker proteins are as defined in the first aspect of the invention. Preferably, the specific binding members comprise antibody molecules or antibody binding fragments thereof, as defined further herein.
In a further aspect the present invention provides a method of screening a test compound for the ability to interfere with or block the development or progression of prostate cancer, comprising :
(a) treating a test animal which has, or is susceptible to, prostate cancer with said test compound; (b) assessing
(i) an amount (relative amount or absolute amount) of one or more marker proteins selected from Endorepellin polypeptide, both Endorepellin polypeptide and GELS polypeptide, GELS polypeptide, ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOAl polypeptide,
BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 polypeptide, KLK3 polypeptide, PGBM polypeptide, Endorepellin polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide,
TPPl polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide, or a fragment thereof, in a urine sample obtained from the test animal, and
(ii) an amount (relative or absolute) of said one or more marker proteins in a urine sample obtained from a control animal which has, or is susceptible to, prostate cancer, which control animal has not been treated with said test compound; and
(c) comparing said relative or absolute amount (i) with said relative or absolute amount (ii), whereby a difference between said relative or absolute amount (i) and said relative or absolute amount (ii) indicates that the test compound has the ability to interfere with or block the development or progression of prostate cancer.
The marker proteins are as defined in the first aspect of the invention. Preferably, the marker proteins are orthologues or homologues of the human marker proteins defined in the first aspect of the invention, which orthologues or homologues comprise the polypeptide sequence native to the species of said test and control animal. The test compound is preferably a known or suspected anti-cancer agent. The test compound may be compound and/or an antibody molecule that binds to a marker protein selected from: ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOAl polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLH l polypeptide, GALM polypeptide, GELS polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 polypeptide, KLK3 polypeptide, PGBM polypeptide, Endorepellin polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TPPl polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide; or a fragment thereof. The method of this aspect of the invention may provide a simplified way of screening potential anti-cancer agents in vivo because the effect of a potential anti-cancer agent on the development or progression of prostate cancer in the animal model can be readily assessed by collecting one or more urine samples and carrying out a simple in vitro assay. Preferably, the method of this aspect of the invention is a method wherein the test compound is found to cause a difference between said relative or absolute amount (i) (e.g. protein concentration) and said relative or absolute amount (ii) (e.g. protein concentration). When a test compound is found to cause a difference between said relative or absolute amount (i) and said relative or absolute amount (ii) in the method of this aspect of the invention, the method may further comprise formulating the test compound into a composition comprising at least one further component. Preferably, the test compound is formulated into a pharmaceutical composition.
These and further aspects and embodiments of the invention are described in further detail below and with reference to the accompanying examples and figures.
Description of the figures
Figure 1 shows a bi-dimensional electrophoresis (2D) gel obtained from a urine sample and showing the two different studied areas A and B;
Figure 2 shows spot 1669 in the B area of the bi-dimensional electrophoresis (2D) gel obtained from urine samples of three healthy individuals (panel A), and of three prostate cancer suffering patients (panel B);
Figure 3 shows the intensity of spot 1669 in control and prostate cancer samples, with the number of samples for each group indicated;
Figure 4 shows results of statistical analysis of endorepellin and GELS immunoassays. The ROC curves for endorepellin (squares), GELS (diamonds) and the combination of endorepellin and GELS (triangles) immunoassays are shown;
Figure 5 shows results of further statistical analysis of endorepellin and GELS immunoassays. A. The box-plots show the distribution of the relative amount of endorepellin for control (left) and prostate cancer (right) groups. B. The box-plots show the distribution of the relative amount of GELS for control (left) and prostate cancer
(right) groups. C. The box-plot shows the probability values obtained from the logistic binomial regression of the combination of both immunoassays (endorepellin and GELS) for controls (left) and prostate cancer (right).
Detailed description of the invention
The following definitions are employed herein.
The term "cancer" refers to the disease that is typically characterised by abnormal or unregulated cell growth, capable of invading adjacent tissues and spreading to distant organs.
The term "carcinoma" refers to the tissue resulting from abnormal or unregulated cell growth.
The term "prostate cancer" refers to any malign proliferative disorder in prostate cells.
The term "tumour" refers to any abnormal mass of tissue generated by a neoplastic process, whether this is benign (non cancerous) or malignant (cancerous).
The term "gene" refers to a region of a molecular chain of deoxyribonucleotides that encodes a protein and may represent a portion of a coding sequence or a complete coding sequence.
The term "protein" indicates at least one molecular chain of amino acids linked through either covalent or non-covalent bonds. The term includes all forms of post-translational protein modifications, for example glycosylation, phosphorylation or acetylation.
The terms "peptide" and "polypeptide" refer to molecular chains of amino acids that represent a protein fragment. The terms "protein" and "peptide" are used interchangeably.
The phrase "increased levels" means that the levels measured in patients with prostate cancer are higher than the levels measured in a control population of individuals with no history of prostate cancer.
In relation to assay methods, the term "specificity", refers to the measurement of false positives, where a specificity of 100% means there are no false positives (positive diagnosis of prostate cancer when the patient individual does not in fact have suffer prostate cancer).
The term "sensitivity", as used herein, refers to the measurement of false negatives, where a sensitivity of 100% means there are no false negatives (negative diagnosis of prostate cancer when the patient in fact does have prostate cancer).
The term "solid phase", as it is used in the present invention refers to a non-aqueous matrix to which the antibody can bind. Examples of materials for the solid phase include but are not limited to glass, polysaccharides (for example agarose), polyacrylamide, polystyrene, polyvinylic alcohol and silicons. Examples of solid phase forms are the well of a plate or a purification column.
The term "subject" refers to all species of animals classified as mammals and includes, but is not restricted to, domestic and farm animals, primates and humans. The subject is preferably male. Yet more preferably, the subject is a male human of any age or race. In some embodiments of the present invention, the subject is a male human identified as being at risk of developing prostate cancer on the basis of, for example, age, lifestyle and/or genetic predisposition.
The one or more marker proteins as defined in the methods and products of the invention are preferably the human full-length polypeptides having the sequences identified by GI and UniProt accession number in Table 1. However, the one or more marker proteins as contemplated herein in relation to any aspect of the present invention may be a derivative, variant, orthologue, homologue or fragment of one or more of the human full-length polypeptides having the sequences identified by GI and UniProt accession number in Table 1. The derivative, variant, orthologue, homologue or fragment may comprise an amino acid sequence having at least 70%, 80%, 90%, 95% or 99% sequence identity to the sequence of, preferably the full-length sequence of, a polypeptide identified by GI and UniProt accession number in Table 1. The one or more marker proteins may be a fragment of:
(i) a polypeptide having an amino acid sequence identified by GI and/or UniProt accession number in Table 1; or
(ii) a derivative, variant, homologue of (i) having an amino acid sequence with at least 70%, 80%, 90%, 95% or 99% sequence identity to (i), wherein said fragment comprises at least 20, 50, 100, 150, 200, 250 or 500 amino acids. The fragment may be a naturally occurring fragment that results from proteolysis of its parent protein. For example, the one or more marker proteins may include a fragment selected from: Endorepellin (preferably, a 191 residue fragment of the 4391 residue Perlecan protein, more preferably the Endorepellin polypeptide comprises or consists essentially of the contiguous sequence of amino acid residues 4196-4391 of the sequence disclosed in GI 24212664 and/or UniProt accession No: P98160, more preferably the Endorepellin polypeptide comprises or consists essentially of the C- terminal V domain of said PGBM polypeptide, in particular the contiguous sequence of amino acid residues 3687-4391 of the sequence disclosed in GI 24212664 and/or UniProt accession No: P98160), Ganglioside GM2 activator short form (residues 34-193 of SAP3), Ganglioside GM2 activator long form (residues 32-193 of SAP3), cathepsin D heavy chain (residues 169-412 of CATD), and cathepsin D light chain (residues 65-161 of CATD), any one of the 40-45KDa fragments of GELS that correspond to the N-terminal cleaved portion from GELS by thermolysin in presence of calcium ions, any one of five fragments of RETBP that result from different cleavage (residues 19-201, residues 19- 200, residues 19-199, residues 19-197 and/or residues 19-194 of RETBP) and a 14-16 KDa N-terminal fragment of SCTMl.
As used herein, "percentage sequence identity" may be calculated using methods known in the art. An example is the BLAST algorithm [Altschul et al., J. MoI. Biol. Vol. 215, pp. 403-410, (1990)]. A particularly useful BLAST program is the WU-BLAST-2 program [Altschul et al., Methods in Enzymology, Vol. 266, pp. 460-480 (1996)], which may be used with default parameters.
Abhydrolase domain-containing protein 14B (ABHEB) is a cytosolic and nuclear protein coded by the gene ABHD14B, that may interact with TAFl. By alternative splicing two isoforms of this protein can be produced (isoforms 1 and 2).
Fructose bisphosphate aldolase B (ALDOB) is a cytosolic protein encoded by the ALDOB gene that catalyzes the conversion of D-fructose 1,6-bisphosphate to glycerone phosphate and D-glyceraldehyde 3-phosphate during glycolysis. Changes in expression of this protein have been observed in lung and liver cancer tissues (Li et al., 2006).
Alpha-1-microglobulin (AMBP) is encoded by the AMBP gene, and is produced in liver and secreted to plasma. It consists of two different proteins that are separated by proteolysis: alpha-1-microglobulin, that appears in different fluids like plasma, urine and cerebrospinal fluid, and inter-alpha-trypsin inhibitor, that occurs in plasma and urine and inhibits trypsin, plasmin and lysosomal granulocytic elastase. Apolipoprotein A-I (APOAl) belongs to the apolipoprotein A1/A4/E family and participates in the reverse transport of cholesterol from tissues to the liver for excretion by promoting cholesterol efflux from tissues and by acting as a cofactor for the lecithin cholesterol acyltransferase (LCAT).
Betaine-homocysteine S-methyltransferase (BHMTl) is a cytosolic protein encoded by the BHMT gene, which is involved in the amine and polyamine degradation pathway by converting betaine and homocysteine to dimethylglycine and methionine. This protein is specific of liver and kidney, and is believed not to have been previously related to any cancer type.
Cathepsin D (CATD) is an acid protease which belongs to the peptidase Al family and activates intracellular protein breakdown. It is believed to be involved in the pathogenesis of several diseases such as prostate cancer (Hara et al., 2002), breast, pancreas, lung and bladder cancer and possibly Alzheimer's disease.
Cytosolic non-specific dipeptidase (CNDP2) belongs to the peptidase M20 family, and binds zinc ions as cofactor. Underexpression of this protein has been observed in liver tumour cells (Zhang et al., 2006).
Endothelial protein C receptor (EPCR) is a membrane protein that binds activated protein C, enhances protein C activation by the thrombin-thrombodulin complex and regulates blood coagulation by participating in the protein C pathway. The expression of EPCR is very high in arteries and veins of heart and lung, medium in capillaries of lung and skin and absent in the endothelium of small vessels of liver and kidney. The upregulation of this protein has been described in ovarian cancer (Wang et al., 2005).
Prostate specific membrane antigen (PSMA also known as FOLHl) is an enzyme with both folate hydrolase and N-acetylated-alpha-linked-acidic dipeptidase (NAALADase) activity. Six different isoforms (PSMA-I to 6) produced by alternative splicing have been postulated. It is a single pass type II membrane protein, but the isoform PSMA ' is cytosolic. FOLHl is expressed in a number of tissues, including small intestine, brain, kidney, liver, spleen, colon, trachea, spinal cord and the capillary endothelium of a variety of tumors, but its highest expression is found in prostate. In prostate, the cytosolic isoform appears in normal tissue, while the membrane-bound isoform, PSMA-I, appears in prostate primary tumours. FOLHl is involved in the prostate tumour progression, and its presence in blood and prostatic tissue is used as diagnostic and prognostic marker of prostate cancer.
Aldose 1-epimerase (GALM) is a cytosolic enzyme involved in the hexose metabolism that converts alpha-aldoses to beta-anomers. There are believed to be no previous reports of any link between GALM and any cancer.
Gelsolin (GELS) is an actin-regulating protein that is regulated by calcium ions. GELS binds to the plus ends of actin monomers or filaments, preventing monomer exchange. It can promote the assembly of monomers into filaments (nucleation) as well as sever filaments already formed. Two different isoforms are produced by alternative initiation, isoform 1 that is secreted to plasma, and the cytosolic isoform 2. Overexpression of this protein has been observed in hormone-resistant prostate cancer tissues (Culig et al., 2005). Differences in the levels of this protein have been observed in pancreatic cancer serum and bladder cancer urine (WO 2007/042256).
Golgin-45 (GO45) is an ubiquitous protein required for normal Golgi structure and for protein transport from the endoplasmic reticulum (ER) through the Golgi apparatus to the cell surface. Isoform 1 is located in Golgi apparatus lumen, and isoform 2, produced by alternative splicing, is cytoplasmatic. Its upregulation has been observed in promyelocytic leukemia.
GTP binding protein-1 (GTPB2) is a protein belonging to the GTPBl GTP-binding protein family, predominantly expressed in thymus, spleen, and testis, and at lower levels in brain, lung, kidney, and ovary. There are believed to be no previous reports linking this protein with cancer.
Ig alpha-2 chain C region (IGHA2) is the major immunoglobulin class in body secretions. It may serve both to defend against local infection and to prevent access of foreign antigens to the general immunologic system.
Kallikrein-3 (KLK3) is a secreted protein from the peptidase Sl family that hydrolyzes seminogelin-1 leading to the liquefaction of the seminal coagulum. Levels in serum are elevated related to prostate cancer and other prostatic alterations. Its measure in blood is used for prostate cancer diagnosis. The present inventors have surprisingly found that the relative protein concentration of KLK3 in urine is lower in subjects with prostate cancer than subjects without prostate cancer. This illustrates the finding that it is not possible to predict how concentration of a marker protein, even a protein known to have altered serum concentration associated with prostate cancer, will be altered, if at all, in urine.
Perlecan (PGBM) is a secreted protein that is an integral component of basement membranes. It is responsible for the fixed negative electrostatic charge and is involved in the charge-selective ultrafiltration properties. It serves as an attachment substrate for cells. It has been related to angiogenesis and to several types of cancer such as ovarian, endometrial, cervical and pancreatic cancers. Its upregulation has been observed in prostate cancer cell lines (Datta et al., 2006). As used herein, "Perlecan" or "PGBM polypeptide" preferably refers to the contiguous sequence (SEQ ID NO: 38) of amino acid residues 22-4391 of the sequence disclosed in GI 24212664 and/or UniProt accession No. P98160 (SEQ ID NO: 26) (approx. 470 kDa).
Endorepellin is a 25KDa fragment of the C-terminal end of perlecan, released from the rest of the protein by the action of metalloproteases, and that has an inhibitory effect in the angiogenesis. As used herein, "Endorepellin" or "Endorepellin polypeptide" preferably comprises the LG3 domain of PGBM, more preferably comprising or consisting essentially of a 191 residue fragment of the 4391 residue Perlecan protein, yet more preferably the Endorepellin polypeptide comprises or consists essentially of the contiguous sequence of amino acid residues 4196-4391 of the sequence disclosed in GI 24212664 and/or UniProt accession No: P98160, still more preferably the Endorepellin polypeptide comprises or consists essentially of the C-terminal V domain of said PGBM polypeptide, in particular amino acid residues 3687-4391 of the sequence disclosed in GI 24212664 and/or UniProt accession No: P98160.
The identification and characterisation of endorepellin has been described (Mongiat et al., 2003; the entire contents of which are expressly disclosed herein by reference). The sequence of the LG3 fragment of PGBM and endorepellin has been described (Gonzalez et al., 2005; the entire contents of which are expressly disclosed herein by reference).
Prostatic acid phosphatase (PPAP) belongs to the family of the histidine acid phosphatase family and it is encoded by the ACPP gene. This protein has been described as prostate cancer stage marker in prostatic tissue (Merrick et al., 2005) and as a target for immunotherapy against prostate cancer (Fong et al., 2001). Plasma retinol-binding protein (RETBP) belongs to the lipocalin family and is the specific carrier for retinol (vitamin A alcohol) in the blood. It delivers retinol from the liver stores to the peripheral tissues. In plasma, the RBP-retinol complex interacts with transthyretin which prevents its loss by filtration through the kidney glomeruli. A deficiency of vitamin A blocks secretion of the binding protein posttranslationally and results in defective delivery and supply to the epidermal cells. It has been related to bladder cancer (Basu et al., 1982) and cervical cancer (Audisio el al., 1985).
Ganglioside GM2 activator (SAP3) is a lysosomal protein that binds gangliosides and stimulates ganglioside GM2 degradation. It stimulates only the breakdown of ganglioside GM2 and glycolipid GA2 by beta-hexosaminidase A. It extracts single GM2 molecules from membranes and presents them in soluble form to beta-hexosaminidase A for cleavage of N-acetyl-D-galactosamine and conversion to GM3. It is believed that there have been no previous reports linking SAP3 to cancer.
Secreted and transmembrane protein 1 (SCTM l) secreted protein that is detected at the highest levels in peripheral blood leukocytes and breast cancer cell lines. Found in leukocytes of the myeloid lineage, with the strongest expression observed in granulocytes and no detectable expression in lymphocytes.
Superoxide dismutase Copper-Zinc (SODC) is a cytoplasmatic protein that destroys the toxic radicals that are normally produced within the cells. SODC binds 1 copper and 1 zinc ion per subunit. Alterations of its expression have been described in several types of cancer and some other diseases. Underexpression of this protein has been observed in prostatic tumorous tissues (Aydin et al., 2000) and also alterations in erythrocyte levels of SODC in prostate cancer patients (Bostwick et al., 2006).
Tripeptidyl peptidase 1 (TPPl) is a lysosomal protein belonging to the peptidase S53 family. It is a serine protease with tripeptidyl peptidase activity, that releases N- terminal peptides from polypeptides and also has endopeptidase activity. TPPl may act as a non-specific lysosomal peptidase which generates tripeptides from the breakdown products produced by lysosomal proteinases. It has been described that its distribution is altered in tumoral tissues and other pathological tissues (Kida et al., 2001).
Transthyretin (TTHY) is a secreted thyroid hormone-binding protein that probably transports thyroxine from the bloodstream to the brain. Underexpression of this protein has been observed in pancreatic (Ehmann et al., 2007) and ovarian cancer (Moore et al., 2006).
Vitelline membrane outer layer protein 1 homolog (VMOl) belongs to the VMOl family and is postulated to be a secreted protein. It is believed that VMOl has not previously been linked to cancer or any other pathology.
Zinc-alpha-2-glycoprotein (ZA2G) is a secreted protein present in liver, epithelium of various glands, blood plasma, urine, sweat, seminal plasma and saliva. ZA2G stimulates lipid degradation in adipocytes and causes the extensive fat losses associated with some advanced cancers. It has been related to pancreas and liver cancers. In prostate cancer, its levels in plasma have been related to tumour stage, metastasis and recurrence (Hale et al., 2001).
The marker proteins are listed in Table 1, with the exception of Endorepellin, which as noted above is a 25 kDa fragment of the C-terminal end of perlecan (PGBM). Table 1 also shows the change in relative protein concentration of each marker protein in urine samples from subjects with prostate cancer as compared with controls not having prostate cancer. The change is shown for each marker protein as either an elevation or a lowering together with the average fold-change in relative protein concentration. The range of observed protein concentration values for each marker protein as a proportion of total protein (μg protein/100 μg total protein) is also shown in Table 1 (range shown is that for all subjects).
Table 1
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
measurements refer to the Endorepellin fragment of PGBM
As used herein with reference to all aspects of the invention, the term "antibody" or "antibody molecule" includes any immunoglobulin whether natural or partly or wholly synthetically produced. The term "antibody" or "antibody molecule" includes monoclonal antibodies and polyclonal antibodies (including polyclonal antisera). Antibodies may be intact or fragments derived from full antibodies (see below). Antibodies may be human antibodies, humanised antibodies or antibodies of non-human origin. "Monoclonal antibodies" are homogeneous, highly specific antibody populations directed against a single antigenic site or "determinant" of the target molecule. "Polyclonal antibodies" include heterogeneous antibody populations that are directed against different antigenic determinants of the target molecule. The term "antiserum" or "antisera" refers to blood serum containing antibodies obtained from immunized animals.
It has been shown that fragments of a whole antibody can perform the function of binding antigens. Thus reference to antibody herein, and with reference to the methods, arrays and kits of the invention, covers a full antibody and also covers any polypeptide or protein comprising an antibody binding fragment. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CHl domains; (ii) the Fd fragment consisting of the VH and CHl domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; (viii) bispecific single chain Fv dimers (WO 93/11161) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94/ 13804; 58). Fv, scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and VL domains. Minibodies comprising a scFv joined to a CH3 domain may also be made.
In relation to a "specific binding member", such as an antibody molecule, the term
"selectively binds" may be used herein to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s). The term is also applicable where e.g. an antigen-binding site is specific for a particular epitope that is carried by a number of antigens, in which case the specific binding member carrying the antigen-binding site will be able to bind to the various antigens carrying the epitope.
The methods and kits of the present invention may employ immunological detection of the one or more marker proteins as defined herein. A wide range of immunological assays are available to detect and quantify formation of specific antigen-antibody complexes; numerous competitive or non-competitive protein-binding assays have been described previously and a large number of these are commercially available. Hence, the marker proteins of the invention can be quantified with antibodies such as, for example: monoclonal antibodies, polyclonal antibodies, either intact or recombinant fragments of these, combibodies and Fab or scFv fragments of antibodies, specific for the selected proteins of the invention; these antibodies are human, humanised or of animal origin. The antibodies used in these assays can be labelled or unlabelled; the antibodies can be used in a wide range of assays. Marker molecules that can be used to label antibodies include radionucleotides, enzymes, fluorophores, chemoluminescent reagents, enzymatic substrates or cofactors, enzymatic inhibitors, particles, colorants and derivatives. The higher the antibody binding specificity, the lower the antigen concentration that can be detected.
In certain embodiments of the invention the techniques used for the detection and/or quantification of the complexes formed by antibodies and proteins are selected from the group comprising : Western blot, ELISA (Enzyme-Linked Immunosorbent assay), RIA (Radioimmunoassay), Competitive EIA (Competitive Enzyme Immunoassay), DAS-ELISA (Double Antibody Sandwich-ELISA), liquid immunoarray technology (e.g. Luminex xMAP technology or Becton-Dickinson FACS technology), immunocytochemical or immunohistochemical techniques, techniques based on the use of biochips or protein microarrays that include specific antibodies, assays based on the precipitation of colloidal gold in formats such as dipsticks; or by affinity chromatography techniques, ligand binding assays and lectin binding assays.
Certain preferred embodiments of the methods and kits of the invention employ protein microarrays or double antibody sandwich ELISA (DAS-ELISA). In these immunoassays any antibody, or combination of antibodies can be used, provided such antibodies are specific against one or more epitopes of the marker proteins of the invention. As an example of one of the many possible formats of this assay, an antibody specific for a marker protein as defined herein is attached to the surface of a solid phase support, placed in contact with the sample to be analysed and incubated for a specific time and in appropriate conditions to form an antigen-antibody complex. After washing in appropriate conditions to eliminate non-specific complexes, an indicator reagent, for example a monoclonal or polyclonal antibody, or a fragment thereof, bound to a signal generating molecule, is incubated with the antigen-antibody complexes in appropriate conditions of time and temperature. The presence of the marker proteins of the invention in the sample is detected and, if present, quantified and the signal generated is measured. The amount of the one or more marker proteins of the invention present in the sample is proportional to the signal.
The following is presented by way of example and is not to be construed as a limitation to the scope of the claims.
Example 1 - Urinary prostate cancer markers
In the description which follows, a method for obtaining and analyzing the total protein content of human urine samples and monitoring the presence of differentially expressed proteins within the sample is disclosed. The method involves the manipulation and preparation of the sample, the use of 2D electrophoresis to separate proteins within the sample, the selection of differentially expressed proteins by means of image analysis and statistics and the use of the selected proteins to generate protein-specific antibodies to be used as prostate cancer markers. Assay methods are also disclosed which are useful for monitoring the presence of the tumour markers with the antibodies in urine samples.
Comparative proteome analysis was performed between samples obtained from healthy individuals (controls) and patients subsequently diagnosed of prostate cancer in order to identify proteins differentially expressed in prostate cancer compared to the expression in control individuals. The urine samples analysed were collected prior to the actual diagnosis of prostate cancer in the subjects. The proteins that showed differential expression were identified by peptide mass fingerprinting using mass spectrometry and database search.
The invention may be better understood from the following non-limiting examples wherein differentially-expressed proteins are prepared from urine samples, polyclonal and monoclonal antibodies are made and tested for reactivity and sensitivity, and immunoassays are performed in the same body fluid. The method may include preparation of a standard ("dose response") curve for a given marker protein. However, as the measurement of one or more marker proteins may be relative to other proteins measured by the same method, preparation of a standard curve may be unnecessary.
To identify differentially expressed proteins along progression of prostate cancer, the protein profiles of healthy urines with those of patients suffering prostate cancer were compared.
1. Manipulation of urine samples and separation of proteins.
Urine samples (181 in total) were collected from individuals with a PSA value measured in blood higher than 4 ng/ml that were visiting a urology unit in order to have a prostatic biopsy. The urology units were those of hospitals belonging to the Basque Public Health Network ("Osakidetza") or the Instituto Oncolόgico de Gipuzkoa. These samples were classified as follows: a) No Carcinoma (97 samples) including healthy patients and patient with other prostatic alterations, as prostatitis and prostatic hyperplasia. b) Prostate cancer patients (87 samples): patients diagnosed with the disease at different stages of development.
All samples were accompanied by a biopsy that is key for their classification as control or cancer patients. Urine samples were frozen at -8O0C and shipped to the lab in dry ice without breaking the cold chain. Samples were kept at -8O0C until they were processed. The samples were very heterogenic, ranging from light yellow urines to red ones with blood crumps, transparent or containing tissues in suspension. The total volume was also very variable, from 5 to 100 ml. The protein concentration of the samples ranged from 20 μg/ml to 2 mg/ml (150 μg/ml was the average concentration). To obtain the protein content, samples were thawed in cold water and centrifuged at 2000 g for 7 min at 40C. The supernatant was used to determine protein concentration. The volume necessary to precipitate 400 μg of protein was calculated taking into account that the efficacy of protein precipitation with 10% v/v of trichloroacetic acid (TCA) is 75%. The rest of the urine sample was frozen again at -8O0C and stored for further 2D electrophoresis (if needed). TCA and urine were mixed for 1 hour on ice, and then centrifuged at 16000 g for 20 min at 40C to obtain the pellet of precipitated proteins. This pellet was washed with acetone stored at -2O0C and dried by solvent evaporation.
To perform the two dimension (2D) electrophoresis experiments, the first dimension was IEF (isoelectric focusing) where proteins separate by their charge (pi); the second dimension was SDS-PAGE where proteins separate by their molecular weight. In order to carry out the first dimension, the dried pellet of proteins was resuspended with 450 μl of rehydratation buffer (Urea 7M, Thiourea 2M, CHAPS 2%, IPG buffer 2%, bromophenol blue 0.002%) for 1 hour at room temp. IPG buffer (Amersham, ref# 17-600-88) was used so that the IEF ranged from pH 3-10. For the IEF, the Ettan™ IPGphor™ Isoelectric Focusing System from Amersham was used following the manufacturer's directions. The IEF was performed in immobilized pH gradients, named IPG strips, purchased from
Amersham (ref# 17-6002-45). The solubilised proteins focused in the first dimension in the strips after 10 hours of active rehydratation of the gel at 30 V. Then the voltage was increased to 10000 V, the intensity never being higher than 50 μA per gel. The IEF was finished when the voltage reached 90000 V/hour.
For the second dimension, 26 x 20 cm 12.5% acrylamide where polymerized in the lab using the Ettan DALT twelve Gel Caster from Amersham. Gels were run in the Ettan DALT twelve Large Format Vertical System and following the manufacturer's instructions until the electrophoresis front left the gel. Gels were silver nitrate dyed using the dye kit from Amersham (17-1150-01) following the manufacturer's directions. Gels were then digitised for subsequent image analysis of the protein spots (see Figure IA) before being dried by vacuum and stored. For some urine samples more than one 2D-gel was run. 2. Analysis of the protein spots on the gels.
Every gel was scanned to obtain the map of spots for image analysis. Progenesis PG220 software, version 2006, from Nonlinear Dynamics (UK) was used to analyze image files in a 300 dpi (dot per inch) format and 8 bits/channel. To increase its resolution the analysis was performed in two discrete areas of the gels. From each area, named as A and B (see Figure 1) gels were selected so that the samples belonging to the different groups would be represented and the statistical analysis was performed using the values obtained from the selected gels. The Progenesis PG220 software transformed the information of the flat image into a 3D image, where the intensity of each spot correlated with its volume. With the software, tables of intensities for each spot were obtained in each gel. These raw data were the basis for the subsequent statistical analysis.
To perform the statistical analysis of each individual spot and compare its intensity in two different groups, a normal distribution had to be proven in these blocks of data. To compare the spots belonging to two groups, a Student's test or a Mann-Whitney test was applied depending on the number of samples in each group, and a p value was obtained : this was the theoretical error of assigning a spot to one subgroup or to another one. Only the spots with p values < 0.05 were selected. The fold change or average ratio of these spots was calculated when referred to a protein that would ideally be constant in every sample. As this was not the case, the fold change of every significant spot in each group of samples had been calculated in relation to the proteins with the closer to the unit fold change. Also the n value or number of samples was taken into account. Before statistical analysis the data were normalised by different values:
1. Total protein content of each gel, measured by the Progenesis PG220 software as Total Spot Volume (TSV); and
2. The volume of 6 spots in each gel (in 6 separate normalizations). These spots are not constant in urine, and it is believed that they have never before been used as reference proteins in urine.
Finally, 14 different normalizations were applied, and statistical analysis performed separately for each.
26 spots that show statistical significance and consistency in their fold change when compared with different groups of urines were identified. The identification of the spots was carried out by MALDI-ToF spectrometry. Urine samples of which the 2D electrophoresis had shown statistically significant spots were submitted again to 2D electrophoresis and the spots were excised from the gel. The proteins were digested and all peptides resulting from the digestion were analyzed by MALDI-ToF. This enabled the unambiguous identification of the proteins (see Table 1).
3. Immunization Protocols
Proteins for immunization were cloned and expressed in E. cσ// using a cloning vector. Alternatively, proteins for immunization may be obtained from commercial sources. For the generation of antibodies against recombinant proteins, the released proteins are directly used to immunize rabbits or mice as described below.
For the generation of antibodies against recombinant proteins, the purified proteins will be used to immunize rabbits for polyclonal or mice for monoclonal antibodies. Antibodies will be raised using standard methodologies; immunizing animals with the protein of interest diluted in Freund's complete adjuvant (Gibco, Grand Island, N. Y.) first, and then every month during three months with the protein in incomplete adjuvant. Rabbit or mice sera (prior to fusion, in this case) will be used as polyclonal antisera to show if they are reactive to the protein preparations by standard western blot technique.
Alternatively, antibodies, such as recombinant monoclonal antibodies, having binding specificity for a marker protein as defined herein may be obtained from commercial sources.
4. Western blotting experiments
Protein samples (100 μg of total protein) were mixed with SDS-PAGE gel loading buffer supplemented with 5% β-mercaptoethanol and incubated at 1000C for 5 min, before being loaded on a polyacrylamide gel (the percentage of polyacrylamide may be varied depending on the molecular weight of the protein of interest). Following electrophoresis proteins were transferred to nitrocellulose membranes. Duplicate gels were run and blotted. The membrane was probed with antibodies raised against the selected marker proteins as defined herein. Finally, membranes were hybridised with a secondary antibody conjugated with peroxidase (Sigma RTM) and the chemoluminescent signal was detected using the ECL system (Millipore RTM) with high performance chemiluminescence film (Hyperfilm ECL, Millipore).
To probe the reactivity and specificity of the generated antibodies against the selected proteins, protein samples obtained from different urines (100 μg of total protein) were mixed with SDS-PAGE gel loading buffer supplemented with 5% β-mercaptoethanol and incubated at 1000C for 5 min, before being loaded on polyacrylamide gel. Following electrophoresis proteins were transferred to nitrocellulose membranes. Duplicate gels were run and blotted. The membrane was probed with antibodies raised against a specific marker protein as defined herein. Finally, membranes were hybridised with a secondary antibody conjugated with peroxidase (Sigma RTM) and the chemoluminescent signal was detected using the ECL system (Millipore RTM) with high performance chemiluminescence film (Hyperfilm ECL, Millipore).
As an example, Figure 2 shows spot 1669 in the B area of bidimensional electrophoresis (2D) gel obtained from urine samples of three healthy individuals (Figure 2A), and from three prostate cancer patients (Figure 2B). It can be seen that the intensity of spot 1669 is clearly decreased in the samples of the patients with cancer when compared to the healthy urine samples. These differences showed to be significant after statistical analysis with Progenesis PG220. Figure 3 shows the intensity of spot 1669 in control and cancer samples. The number of samples for each group is indicated. Table 2 below shows the p, fold change value and area under ROC curve of the statistical analysis for spot 1669 in samples of cancer in comparison samples of a non-cancer patients (CV). The fold change of spot 1669 was normalized by the total spot volume (TSV) for each individual gel.
Table 2
Figure imgf000042_0001
Example 2 - Diagnostic immunoassays
1. Immunoassay development (endorepellin and full length gelsolin)
Sandwich type immunoassays have been developed in order to detect two of the prostate cancer markers selected based on the comparative proteomic analysis described above. The two prostate cancer markers are: endorepellin (fragment derived from the carboxyl terminus of perlecan PGBM) and gelsolin (high molecular mass isoforms corresponding to the full length unprocessed protein).
i. In this example the immunoassay for gelsolin specifically detects the full length protein (high molecular mass isoforms around 8OkDa, GELS). Gelsolin is processed by a caspase and present in the urine as two groups of isoforms: those with high molecular mass (around 8OkDa) corresponding to the full length protein (GELS), and those with lower molecular mass (around 4OkDa) corresponding to the processed C-terminal domain of gelsolin. The specific isoforms described here correspond to GELS. In addition, the expression profile of the total amount of gelsolin in the urine (GELS + the rest of low molecular mass isoforms) behaves differently from GELS as a biomarker for the diagnosis of prostate cancer. Therefore, it has been found that the full length protein GELS, rather than the total amount of gelsolin, has the greater diagnostic capability.
ii. Regarding endorepellin, the fragment selected in 2D electrophoresis (2DE) was the LG3 domain of this protein (a 25 kDa fragment of perlecan) and therefore, the antibodies used in the immunoassay were specific for this domain.
The immunoassays developed were validated in 72 urine samples. These samples were already included in the comparative proteomic analysis performed for the identification of prostate cancer markers. All the samples were collected from individuals with PSA concentration in serum higher than 4 ng/ml and subjected to prostatic biopsy in order to confirm the diagnosis. These 72 samples were classified as follows:
i. 50 urine samples from patients diagnosed with prostate cancer ii. 22 samples from males without prostate cancer.
In order to reduce the percentage of false negatives in first biopsies among the control population (around 15-30 %), only men with negative results in at least 2 prostatic biopsies were included.
The immunoassays quantify the amount of GELS and endorepellin in each of the 72 subjects. The results obtained were statistically analyzed to determine the role of both markers (individually and in combination) in the classification of each sample in the control or cancer group. The results of the combination of GELS and endorepellin were analyzed using a binomial logistic regression. From figures 4 and 5, we can conclude that the ROC curves as well as the distribution of the values for control and cancer patients show an improvement when the endorepellin and GELS are combined (compared to the individual assays). This suggests that endorepellin and GELS may be advantageously combined as markers for prostate cancer.
Diagnostic data were computed for the markers endorepellin, GELS and for the combination of endorepellin and GELS. The assays were performed in 22 controls and 50 prostate cancer urine samples.
Table 3 - Diagnostic values for endorepellin and GELS
Marker AUC Sensitivity Specificity PPV NPV
90 59 83 72
Endorepellin 0.81 52 82 87 43
98 23 74 83
GELS 0.68 59 66 79 43
94 64 85 82
Endorepellin+GELS 0.84 70 82 90 55
AUC represents the values for the area under the ROC curves; PPV and NPV correspond to the positive predictive values and negative predictive values, respectively.
Table 4 - Contingency tables
True positives (TP), true negatives (TN), false positives (FP) and false negatives (FN) values obtained from the combination of endorepellin and GELS biomarkers for the diagnosis of prostate cancer, compare to the results obtained from the prostatic biopsies. The values correspond to the maximum sensitivity (left) and maximum specificity (right).
Sens: 94%; Spe: 64%; Sens: 70%; Spe: 82%; PPV: 85%; NPV: 82% PPy: 90%; NPV: 55%
Biopsy Biopsy
Test Test
+ + _
47 8 35
+ TP FP TP FP
14 15 18
- -
FN TN Total FN TN Tota
50 22 72 50 22 7? As shown by, in particular, Figures 4 and 5 and Tables 3 and 4, the diagnostic potential of a kit that utilises the combination of endorepellin and GELS detection, such as a kit comprising specific immunoassays for the detection of endorepellin and GELS, significantly improves over the individual immunoassays. These results support the advantageous combination of endorepellin and GELS as markers for use in a diagnostic test for prostate cancer.
All references cited herein are incorporated by reference in their entirety.
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Claims

Claims
1. A method for detecting or monitoring prostate cancer or susceptibility to prostate cancer in a test subject, comprising : determining an amount of one or more marker proteins in a urine sample obtained from the test subject; and comparing said amount with a standard value, wherein the one or more marker proteins are selected from:
Endorepellin polypeptide, both Endorepellin polypeptide and GELS polypeptide, GELS polypeptide, ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOAl polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTM l polypeptide, SODC polypeptide, TPPl polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide; and/or a fragment of any one of said polypeptides.
2. A method according to claim 1, wherein said amount is a relative amount.
3. A method according to claim 1, wherein said amount is an absolute amount.
4. A method according to any one of claims 1 to 3, wherein said one or more marker proteins comprise Endorepellin polypeptide and/or GELS polypeptide.
5. A method according to any one of claims 1 to 3, wherein said one or more marker proteins comprise at least two marker proteins including Endorepellin polypeptide and
GELS polypeptide.
6. A method according to claim 2, wherein the one or more marker proteins are selected from: ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GELS polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide; and/or a fragment thereof, and wherein the relative amount is the amount of the marker protein relative to a reference protein also present in the urine sample, and wherein a decreased relative amount of said one or more marker proteins in the urine sample obtained from the test subject compared with a standard value corresponding to the relative amount of the same marker protein in a urine sample obtained from a control subject not having prostate cancer indicates that the test subject has or is susceptible to prostate cancer.
7. A method according to claim 6, wherein the reference protein is selected from :
ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide; or a fragment thereof.
8. A method according to claim 6 or claim 7, wherein a decrease in the relative amount of said one or more marker proteins of 10%, 20%, 50% or greater compared with the standard value indicates that the test subject has or is susceptible to prostate cancer.
9. A method according to claim 2, wherein the one or more marker proteins are selected from: ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide,
EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide; and/or a fragment thereof, and wherein the relative amount is the amount of the marker protein relative to a reference protein also present in the urine sample, and wherein an increased relative amount of said one or more marker proteins in the urine sample obtained from the test subject compared with a standard value corresponding to the relative amount of the same marker protein in a urine sample obtained from a control subject not having prostate cancer indicates that the test subject has or is susceptible to prostate cancer.
10. A method according to claim 9, wherein the reference protein is selected from : ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GELS polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide; or a fragment thereof.
11. A method according to claim 9 or claim 10, wherein an increase in the relative amount of said one or more marker proteins of 10%, 20%, 50% or greater compared with the standard value indicates that the test subject has or is susceptible to prostate cancer.
12. A method according to any preceding claim, wherein the standard value for the marker protein is a pre-determined value that has been determined by measuring a representative number of control subjects from a population of subjects not having prostate cancer.
13. A method according to any preceding claim, wherein said one or more marker proteins and/or said reference protein are selected from:
(i) Endorepellin polypeptide comprising or consisting essentially of the contiguous sequence of amino acid residues 3687-4391 (SEQ ID NO: 37) of the PGBM sequence disclosed in UniProt accession No: P98160 (SEQ ID NO: 26);
GELS polypeptide having the amino acid sequence disclosed in UniProt accession No: P06396 (SEQ ID NO: 17);
ABHEB polypeptide having the amino acid sequence disclosed in UniProt accession No: Q96IU4-1 (SEQ ID NO: 1) or Q96IU4-2 (SEQ ID NO: 2); ALDOB polypeptide having the amino acid sequence disclosed in UniProt accession No: P05062 (SEQ ID NO: 3);
AMBP polypeptide having the amino acid sequence disclosed in UniProt accession No: P02760 (SEQ ID NO: 4);
APOAl polypeptide having the amino acid sequence disclosed in UniProt accession No: P02647 (SEQ ID NO: 5);
BHMT polypeptide having the amino acid sequence disclosed in UniProt accession No: Q93088 (SEQ ID NO: 6);
CATD polypeptide having the amino acid sequence disclosed in UniProt accession No: P07339 (SEQ ID NO: 7); CNDP2 polypeptide having the amino acid sequence disclosed in UniProt accession No: Q96KP4 (SEQ ID NO: 8);
EPCR polypeptide having the amino acid sequence disclosed in UniProt accession No: Q9UNN8 (SEQ ID NO: 9);
FOLHl polypeptide having the amino acid sequence disclosed in UniProt accession No: Q04609-1 (SEQ ID NO: 10), Q04609-2 (SEQ ID NO: 11),
Q04609-3 (SEQ ID NO: 12), Q04609-4 (SEQ ID NO: 13), Q04609-5 (SEQ ID NO: 14) or Q04609-6 (SEQ ID NO: 15);
GALM polypeptide having the amino acid sequence disclosed in UniProt accession No: Q96C23 (SEQ ID NO: 16); GO45 polypeptide having the amino acid sequence disclosed in UniProt accession No: Q9H2G9-1 (SEQ ID NO: 18) or Q9H2G9-2 (SEQ ID NO: 19); GTPB2 polypeptide having the amino acid sequence disclosed in UniProt accession No: Q9BX10-1 (SEQ ID NO: 20), Q9BX10-2 (SEQ ID NO: 21), Q9BX10-3 (SEQ ID NO: 22) or Q9BX10-4 (SEQ ID NO: 23);
IGHA2 polypeptide having the amino acid sequence disclosed in UniProt accession No: P01877 (SEQ ID NO: 24);
KLK3 polypeptide having the amino acid sequence disclosed in UniProt accession No: P07288 (SEQ ID NO: 25);
PGBM polypeptide having the amino acid sequence disclosed in UniProt accession No: P98160 (SEQ ID NO: 26), preferably comprising or consisting essentially of the contiguous sequence of amino acid residues 22-4391 (SEQ ID
NO: 38) of the amino acid sequence disclosed in UniProt accession No: P98160 (SEQ ID NO: 26);
PPAP polypeptide having the amino acid sequence disclosed in UniProt accession No: P15309 (SEQ ID NO: 27); RETBP polypeptide having the amino acid sequence disclosed in UniProt accession No: P02753 (SEQ ID NO: 28);
SAP3 polypeptide having the amino acid sequence disclosed in UniProt accession No: P17900 (SEQ ID NO: 29);
SCTM l polypeptide having the amino acid sequence disclosed in UniProt accession No: Q8WVN6 (SEQ ID NO: 30);
SODC polypeptide having the amino acid sequence disclosed in UniProt accession No: P00441 (SEQ ID NO: 31);
TPPl polypeptide having the amino acid sequence disclosed in UniProt accession No: 014773-1 (SEQ ID NO: 32) or 014773-2 (SEQ ID NO: 33); TTHY polypeptide having the amino acid sequence disclosed in UniProt accession No: P02766 (SEQ ID NO: 34);
VMOl polypeptide having the amino acid sequence disclosed in UniProt accession No: Q7Z5L0 (SEQ ID NO: 35);
ZA2G polypeptide having the amino acid sequence disclosed in UniProt accession No: P25311 (SEQ ID NO: 36);
(ii) a variant, derivative or homologue of (i) having at least 70%, 80%, 90%, 95% or 99% sequence identity to an amino acid sequence of (i); and (iii) a fragment of (i) or (ii), wherein the fragment comprises at least 50, 100, 150 or 200 amino acids.
14. A method according to claim 13, wherein said fragment is selected from: endorepellin, consisting essentially of a 191 amino acid fragment of said PGBM polypeptide, preferably said fragment comprising the LG3 domain of said PGBM polypeptide, more preferably said fragment consisting essentially of the contiguous sequence of amino acid residues 3687-4391 (SEQ ID NO: 37) of the sequence disclosed in UniProt accession No: P98160 (SEQ ID NO: 26); ganglioside GM2 activator short form, consisting essentially of residues 34-193 of said SAP3 polypeptide (SEQ ID NO: 39); ganglioside GM2 activator long form, consisting essentially of residues 32-193 of said SAP3 polypeptide (SEQ ID NO: 40); cathepsin D heavy chain, consisting essentially of residues 169-412 of said CATD polypeptide (SEQ ID NO: 41); cathepsin D light chain, consisting essentially of residues 65-161 of said CATD polypeptide (SEQ ID NO: 42); any one of the 40-45kDa fragments of said GELS polypeptide that correspond to the N-terminal portion cleaved from said GELS polypeptide by thermolysin in presence of calcium ions; a fragment of said RETBP polypeptide consisting essentially of residues 19-201 (SEQ ID NO: 43), residues 19-200 (SEQ ID NO: 44), residues 19-199 (SEQ ID NO: 45), residues 19-197 (SEQ ID NO: 46) or residues 19-194 (SEQ ID NO: 47) of said RETBP polypeptide; and a 14-16 kDa N-terminal fragment of said SCTMl polypeptide.
15. A method according to any preceding claim, wherein determining the amount, relative amount or absolute amount of said one or more marker proteins in the urine sample comprises: contacting said urine sample with at least one specific binding member that selectively binds to one of said marker proteins; and detecting and/or quantifying formation of a complex formed by said specific binding member and said marker protein.
16. A method according to claim 15, wherein the specific binding member comprises an antibody molecule or a binding fragment thereof.
17. A method according to claim 15 or claim 16, wherein detecting formation of a complex formed by said specific binding member and said marker protein is carried out by use of a technique selected from : Western blot; enzyme-linked immunosorbent assay (ELISA); radioimmunoassay (RIA); competitive enzyme immunoassay; double antibody sandwich ELISA (DAS-ELISA); liquid immunoarray technology; immunocytochemistry; immunohistochemistry; antibody microarray detection; precipitation of colloidal gold; affinity chromatography; ligand binding assay; and lectin binding assay.
18. A method according to any preceding claim which comprises determining the amount, relative amount or absolute amount of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of said marker proteins in said urine sample.
19. A method for detecting or monitoring prostate cancer in a subject, comprising :
(i) contacting a urine sample obtained from the subject with a first specific binding member, which selectively binds to a marker protein selected from Endorepellin polypeptide, both Endorepellin polypeptide and GELS polypeptide, GELS polypeptide, ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide, or a fragment thereof;
(ii) contacting said urine sample with a second specific binding member, which selectively binds to a reference protein selected from ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide, or a fragment thereof;
(iii) detecting and/or quantifying the formation of a first complex formed by the first specific binding member and the marker protein to which it binds and of a second complex formed by the second specific binding member and the reference protein to which it binds, thereby deriving a measurement of the relative amount of said marker protein relative to said reference protein;
(iv) comparing said relative amount of said marker protein with a standard value that corresponds to the relative amount of the same marker protein relative to the same reference protein in a urine sample obtained from a subject not having prostate cancer, wherein a relative amount of said marker protein which is decreased by 10%, 20%, 50% or greater compared with the standard value indicates that the subject has prostate cancer.
20. A method for detecting or monitoring prostate cancer in a subject, comprising :
(i) contacting a urine sample obtained from the subject with a first specific binding member, which selectively binds to a marker protein selected from ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide, or a fragment thereof;
(ii) contacting said urine sample with a second specific binding member, which selectively binds to a reference protein selected from Endorepellin polypeptide, both Endorepellin polypeptide and GELS polypeptide, GELS polypeptide, ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide, or a fragment thereof; (iii) detecting and/or quantifying the formation of a first complex formed by the first specific binding member and the marker protein to which it binds and of a second complex formed by the second specific binding member and the reference protein to which it binds, thereby deriving a measurement of the relative amount of said marker protein relative to said reference protein; (iv) comparing said relative amount of said marker protein with a standard value that corresponds to the relative amount of the same marker protein relative to the same reference protein in a urine sample obtained from a subject not having prostate cancer, wherein a relative amount of said marker protein which is increased by 10%, 20%, 50% or more compared with the standard value indicates that the subject has prostate cancer.
21. A method according to any preceding claim, wherein the method is a method for diagnosing prostate cancer in the test subject; determining the stage, severity or progression of prostate cancer in the test subject; or monitoring the effect of therapeutic treatment of prostate cancer in the test subject.
22. A method according to any preceding claim, wherein said test subject has not previously been diagnosed with prostate cancer.
23. A method according to any one of claims 1 to 21, wherein said test subject has previously been diagnosed with prostate cancer and/or treated for prostate cancer.
24. A method according to any preceding claim, wherein the method comprises determining the amount, relative amount or absolute amount of one or more of said marker proteins in a plurality of urine samples obtained from the test subject at different times.
25. A method according to claim 24, wherein at least one of said urine samples has been obtained prior to a therapeutic or surgical intervention performed on the test subject and at least one of said urine samples has been obtained after a said intervention.
26. A prostate cancer diagnostic kit comprising :
1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more specific binding members each of which selectively binds to a marker protein as defined in any one of claims 1-14; and one or more reagents for detecting said one or more specific binding members.
27. A prostate cancer diagnostic kit according to claim 26, comprising a specific binding member that selectively binds to Endorepellin polypeptide.
28. A prostate cancer diagnostic kit according to claim 26 or claim 27, comprising a specific binding member that selectively binds to GELS polypeptide.
29. A prostate cancer diagnostic kit according to claim 26 comprising :
1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more specific binding members each of which selectively binds to a marker protein selected from ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOAl polypeptide, BHMTl polypeptide, CNDP2 polypeptide, EPCR polypeptide, GALM polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 polypeptide, Endorepellin polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, TPPl polypeptide, TTHY polypeptide and VMOl polypeptide, or a fragment thereof; and one or more reagents for detecting said one or more specific binding members.
30. A prostate cancer diagnostic kit according to claim 29, comprising : a specific binding member which selectively binds to a marker protein selected from ABHEB polypeptide, AMBP polypeptide, APOAl polypeptide, GELS polypeptide, GO45 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide, or a fragment thereof; and a specific binding member which selectively binds to a marker protein selected from ALDOB polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GTPB2 polypeptide, IGHA2 polypeptide and TPPl polypeptide, or a fragment thereof.
31. A prostate cancer diagnostic kit according to any one of claims 26 to 30, wherein a plurality of said specific binding members are provided in the form of an array on a substrate.
32. A prostate cancer diagnostic kit according to any one of claims 26 to 31, wherein the specific binding member comprises an antibody molecule or a binding fragment thereof.
33. A specific binding member for use in a method of diagnosing prostate cancer, wherein said specific binding member selectively binds a marker protein as defined in any one of claims 1 to 14.
34. A specific binding member for use in a method of diagnosing prostate cancer, wherein said specific binding member selectively binds a marker protein selected from : ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOAl polypeptide, BHMTl polypeptide, CNDP2 polypeptide, EPCR polypeptide, GALM polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 polypeptide, Endorepellin polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, TPPl polypeptide, TTHY polypeptide and VMOl polypeptide, or a fragment thereof.
35. A specific binding member according to claim 33 or claim 34, wherein the specific binding member comprises an antibody molecule or a binding fragment thereof.
36. Use of a specific binding member as defined in any one of claims 33 to 35 in a method of diagnosing prostate cancer.
37. Use of a specific binding member as defined in any one of claims 33 to 35 in the manufacture of a diagnostic agent for diagnosis of prostate cancer.
38. A method of screening a test compound for the ability to interfere with or block the development or progression of prostate cancer, comprising :
(a) treating a test animal which has, or is susceptible to, prostate cancer with said test compound;
(b) assessing (i) an amount of one or more marker proteins selected from
Endorepellin polypeptide, both Endorepellin polypeptide and GELS polypeptide, GELS polypeptide, ABHEB polypeptide, ALDOB polypeptide, AMBP polypeptide, APOAl polypeptide, BHMTl polypeptide, CATD polypeptide, CNDP2 polypeptide, EPCR polypeptide, FOLHl polypeptide, GALM polypeptide, GO45 polypeptide, GTPB2 polypeptide, IGHA2 polypeptide, KLK3 polypeptide, PGBM polypeptide, PPAP polypeptide, RETBP polypeptide, SAP3 polypeptide, SCTMl polypeptide, SODC polypeptide, TPPl polypeptide, TTHY polypeptide, VMOl polypeptide and ZA2G polypeptide, or a fragment thereof, in a urine sample obtained from the test animal, and
(ii) an amount of said one or more marker proteins in a urine sample obtained from a control animal which has, or is susceptible to, prostate cancer, which control animal has not been treated with said test compound; and
(c) comparing amount (i) with amount (ii), whereby a difference between amount (i) and amount (ii) indicates that the test compound has the ability to interfere with or block the development or progression of prostate cancer.
39. A method according to claim 38, wherein said amount is a relative amount or an absolute amount.
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