GB2634269A

GB2634269A - Biomarkers for prostate cancer

Info

Publication number: GB2634269A
Application number: GB2315205.1A
Authority: GB
Inventors: Alejandro Jose Mur Luis; John Rooke David
Original assignee: Aberystwyth University
Current assignee: Aberystwyth University
Priority date: 2023-10-04
Filing date: 2023-10-04
Publication date: 2025-04-09
Also published as: GB202315205D0; WO2025074104A2; WO2025074104A3

Abstract

A method for determining the presence of prostate cancer in a subject, the method comprising the steps of: (i) determining the level of one or more biomarkers in a sample from the subject; (ii) comparing the level of biomarker to the level of biomarker in a control sample to determine whether prostate cancer is present in the subject. The prostate cancer biomarkers are selected from: 3-hydroxyhexadecanoylcarnitine, alpha lipoic acid and N- heptanoylglycine. An immunological capture device and kit for detecting prostate cancer in a subject is also disclosed. Further disclosed is a method, immunological device and kit for determining the presence of benign prostatic hyperplasia (BPH) in a subject, the method comprising the steps of: (i) determining the level of one or more biomarkers in a sample from the subject; (ii) comparing the level of biomarker to the level of biomarker in a control sample to determine whether BPH is present in the subject. The BPH biomarkers are selected from N-Isobutyl-2,4,8,10,12- tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-D- myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.

Description

Biomarkers for Prostate Cancer

Field of the Invention

The present invention relates to biomarkers for detecting prostate cancer. In particular, the present invention relates to a method for determining the presence of prostate cancer in a subject. The present invention also relates to a device for detecting prostate cancer in a subject and a kit for detecting prostate cancer in a subject. The present invention also relates to a method, device and kit for detecting benign prostatic hyperplasia in a subject. The present invention further relates to differentiating between prostate cancer and benign prostatic hyperplasia.

Background to the Invention

Urological cancers consist of bladder (BC), prostate (PCa), renal and testicular cancers and are among the ten most common causes of cancer worldwide [1]. Prostate cancer is the second most common cause of cancer in men. In 2018 over 1 million new cases of PCa were diagnosed globally, with approximately 359,000 deaths caused by the disease [2]. Prostate, renal and bladder cancers can all present as painless blood in the urine (painless haematuria), which can have several causal factors, both benign and malignant. Prostate specific antigen (PSA) level testing, alongside the digital rectal exam (DRE), has been the mainstay of PCa screening for decades, with abnormalities triggering needle biopsy interventions. As the symptoms of early PCa are indistinguishable from those of benign prostatic hyperplasia (BPH) this could lead to unnecessary interventions. For example, elevations in PSA levels are commonly seen in both PCa and BPH and cannot be used to discriminate between them [3]. Further, BPH is relatively common in older men, with a prevalence of up to 50% in men over 50 years of age. DRE and PSA based diagnostic procedures are vulnerable to over-diagnosis [4, 5]. Taken together, this highlights the clinical need for a non-invasive test, which is capable of differentiating between cancerous and non-cancerous disease.

An easily obtained non-invasive sample would be much more comfortable and therefore less stressful for the patient. An inexpensive diagnostic which can be used in a non-clinical environment may increase diagnosis rates and early-diagnosis providing the opportunity for better clinical outcomes.

It is therefore an object of the present invention to obviate or mitigate one or more of the abovementioned problems.

Summary of the Invention

The present invention relates to a method for determining the presence of prostate cancer in a subject and is based, in part, on studies by the inventors in which they have shown that certain metabolites are present at significantly different levels in subjects with prostate cancer as compared to control subjects and subjects with benign prostatic hyperplasia (BPH) and are therefore suitable as biomarkers for prostate cancer.

In a first aspect of the present invention there is provided a method for determining the presence of prostate cancer in a subject, the method comprising the steps of: determining the level of one or more biomarkers in a sample from the subject; comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether prostate cancer is present in the subject; wherein the one or more biomarkers are selected from: 3-hydroxyhexadecanoylcarnitine, Lhistidinol, alpha lipoic acid and N-heptanoylglycine.

As discussed in more detail below, the present inventors have found that the level of the abovementioned biomarkers is significantly altered in subjects in which prostate cancer is present.

As shown below, the inventors have found that these biomarkers have individual diagnostic accuracies (area under the curve; AUC) of greater than or equal to 0.79. The inventors have therefore found that by comparing the level of the abovementioned biomarkers in a sample from a subject with the level in a control sample, the presence of prostate cancer in a subject can be determined with high sensitivity and specificity. These metabolite biomarkers are therefore useful for determining whether a subject has prostate cancer. By quickly and accurately identifying subjects affected by prostate cancer, before clinical symptoms become apparent, spread of the disease can be reduced and/or prevented and outcomes improved.

The levels of the metabolite biomarkers of the invention have been shown by the inventors to be consistently and significantly different between subjects with prostate cancer as compared to control subjects. These biomarkers can therefore be used to determine the presence of prostate cancer in a subject with extremely high sensitivity and specificity.

The method of the present invention comprises determining the level of one or more biomarkers in a sample from the subject. Preferably, the method of the present invention comprises determining the level of two, three or four, or more, biomarkers in a sample from the subject. By determining the level of multiple biomarkers in the sample and comparing multiple biomarkers with the levels in a control sample, the sensitivity and specificity of the determination (of whether a subject has prostate cancer) may be improved. The level of one or more biomarkers in the sample will be determined. Together these biomarker levels may be integrated and when considering their different levels together will form a "fingerprint" for the sample, thereby increasing the specificity of the method.

In embodiments of the invention, the method may comprise determining the level of two, three, or four of the following biomarkers, 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid and N-heptanoylglycine.

In embodiments in which the method involves determining the level of four biomarkers in a sample from the subject, the biomarkers may consist of 3-hydroxyhexadecanoylcarnitine, Lhistidinol, alpha lipoic acid and N-heptanoylglycine.

In preferred embodiments, the method of the present invention comprises the steps of: determining the level of four biomarkers in a sample from the subject; comparing the level of said four biomarkers with the level of said four biomarkers in a control sample to determine whether prostate cancer is present in the subject; wherein the biomarkers consist of 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid and N-heptanoylglycine.

The present inventors have found that using these biomarkers, in combination, offer a "signature" or "diagnostic fingerprint" which allows the highly accurate identification of prostate cancer. Individually, each biomarker has accuracies of greater than or equal to 0.79 (AUC value) and by utilising a combination of these biomarkers a highly accurate diagnosis can be made. A metabolomic signature of the four biomarkers gives accuracies of 87.5% (CI 0.7810.956).

In another aspect of the present invention there is provided a method for determining the presence of BPH in a subject, the method comprising the steps of: (iii) determining the level of one or more biomarkers in a sample from the subject; (iv) comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether BPH is present in the subject; wherein the one or more biomarkers are selected from: N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-Dmyo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.

As discussed in more detail below, the present inventors have found that the level of the abovementioned biomarkers is significantly altered in subjects in which BPH is present.

As shown below, the inventors have found that these biomarkers have individual diagnostic accuracies (area under the curve; AUC) of greater than or equal to 0.81. The inventors have therefore found that by comparing the level of the abovementioned biomarkers in a sample from a subject with the level in a control sample, the presence of BPH in a subject can be determined with high sensitivity and specificity. These metabolite biomarkers are therefore useful for determining whether a subject has BPH.

The levels of the metabolite biomarkers of the invention have been shown by the inventors to be consistently and significantly different between subjects with BPH as compared to control subjects. These biomarkers can therefore be used to determine the presence of BPH in a subject with extremely high sensitivity and specificity.

The method of the present invention comprises determining the level of one or more biomarkers in a sample from the subject. Preferably, the method of the present invention comprises determining the level of two, three or four, or more, biomarkers in a sample from the subject. The method may comprise determining the level of five, six, seven, eight, nine or more biomarkers in a sample from the subject. By determining the level of multiple biomarkers in the sample and comparing multiple biomarkers with the levels in a control sample, the sensitivity and specificity of the determination (of whether a subject has BPH) may be improved. The level of one or more biomarkers in the sample will be determined. Together these biomarker levels may be integrated and when considering their different levels together will form a "fingerprint" for the sample, thereby increasing the specificity of the method.

In embodiments of the invention, the method may comprise determining the level of two, three, four, five, six, seven, eight or nine of the following biomarkers:, N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-Dmyo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.

In embodiments in which the method involves determining the level of nine biomarkers in a sample from the subject, the biomarkers may consist of N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-Dmyo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.

In preferred embodiments, the method of the present invention comprises the steps of: (iii) determining the level of nine biomarkers in a sample from the subject; (iv) comparing the level of said nine biomarkers with the level of said nine biomarkers in a control sample to determine whether BPH is present in the subject; wherein the biomarkers consist of N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PENMe2(24:1 (15Z)/20:5(5Z,8Z,11Z,147,177)), 1-Phosphatidyl-D-myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.

The present inventors have found that using these biomarkers, in combination, offer a "signature" or "diagnostic fingerprint" which allows the highly accurate identification of BPH. Individually, each biomarker has accuracies of greater than or equal to 0.81 (AUC value) and 5 by utilising a combination of these biomarkers a highly accurate diagnosis can be made. A metabolomic signature of the nine biomarkers gives accuracies of 90.7% (CI 0.731-1).

In embodiments of the present invention, the method may be for distinguishing between subjects with benign prostatic hyperplasia (BPH) and subjects with prostate cancer.

Thus, additionally the present inventors have shown that a number of the metabolite biomarkers identified can distinguish between subjects with BPH and subjects with prostate cancer.

In such embodiments, the one or more biomarkers that indicate prostate cancer are selected from: 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid and N-heptanoylglycine. The one or more biomarkers that indicate BPH are selected from: N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-Dmyo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.

Therefore, the present invention provides a method for determining whether a subject has BPH or prostate cancer, the method comprising the steps of: determining the level of one or more biomarkers in a sample from the subject; comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether the subject has BPH or prostate cancer; wherein the one or more biomarkers that indicate prostate cancer are selected from: 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid and N-heptanoylglycine. The one or more biomarkers that indicate BPH are selected from: N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,117,147,177)), 1-Phosphatidyl-Dmyo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.

The present inventors have found that the biomarkers listed above are able to differentiate between subjects with prostate cancer, those with BPH and those without prostate cancer and/or BPH.

In embodiments of the invention, the method may comprise determining the level of up to 13 of the following biomarkers: 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid, N- heptanoylglycine., N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(1 5Z)/20:5(5Z,8Z,11Z,147,177)), 1-Phosphatidyl-D-myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.

In embodiments the method involves determining the level of 13 of the following biomarkers: 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid, N-heptanoylglycine., NIsobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-D-myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.

In embodiments, the method comprises the steps of: determining the level of 13 biomarkers in a sample from the subject; comparing the level of said 13 biomarkers with the level of said 13 biomarkers in a control sample to determine whether the subject has prostate cancer or BPH or neither; wherein the biomarkers consist of 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid, N-heptanoylglycine., N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,117,147,177)), 1-Phosphatidyl-D-myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid. And wherein the presence of any of the following biomarkers indicates the presence of prostate cancer: 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid, N-heptanoylglycine and wherein the presence of any of the following biomarkers indicates the presence of BPH: N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PENMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-D-myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.

The present inventors have found that using these biomarkers, in combination, offers a "signature" or "diagnostic fingerprint' which allows the highly accurate differentiation between subjects with prostate cancer and those with BPH. The presentation of the prostate cancer biomarker signature may indicate the presence of prostate cancer. The presentation of the BPH biomarker signature may indicate BPH. Utilising a combination of biomarkers enables a highly accurate diagnosis with accuracies of greater than 0.8 (specificity. 0.73, sensitivity 0.82) for prostate cancer diagnosis and for BPH diagnosis greater than 0.9 (specificity. 0.83, sensitivity 0.86).

Determining the level of biomarkers The method of the present invention involves determining the level of one or more biomarkers in a sample. The skilled person will appreciate that there are a number of ways in which these biomarker levels can be determined.

For example, the biomarker level may be determined using mass spectrometry, for example high resolution mass spectrometry (HR-MS), gas chromatography time-of-flight mass spectrometry (GC-MS), flow infusion electrospray high resolution mass spectrometry (FIEHRMS) or liquid chromatography-electrospray mass spectrometry (LC-MS).

GC-MS involves linking a gas chromatograph with a mass spectrometer. The gas chromatograph utilizes a capillary column where the chemical properties between the sampled chemicals in a mixture and their relative affinity for the stationary phase of the column will result in their separation along the column. This provides "retention time", information. The chemicals then enter the mass spectrometer which will show mass-to-charge ratios.

LC-MS links liquid chromatography (LC or High-Performance LC [HPLC]) with a mass spectrometer. The LC part physically separates chemicals between a liquid mixture of two immiscible phases, i.e., stationary and mobile. The chemicals then enter the mass spectrometer which will show mass-to-charge ratios.

In flow infusion, samples are injected directly into a solvent (usually methanol-water) line leading to a mass spectrometer.

Alternatively, the biomarker level could be determined using NMR, enzymatic assays (e.g., enzymatic reaction followed by colorimetric detection) or immunoassays (i.e., antibody binding based assays).

The most well-established immunoassay is the enzyme-linked immunosorbent assay (ELISA). The most used ELISA technique is the sandwich ELISA which measures the antigen using a capture (often associated within the well of a 96 well plate to allow high-throughput screening) and a detection antibody. Monoclonal or polyclonal antibodies can be used in sandwich or competitive ELISA systems. Other immunoassay systems include lateral flow or flow through systems. The use of such immunoassays for detecting the biomarker level would allow the method to be used as a Point of Care (POC) test, for example a POC device, allowing the presence of prostate cancer or BPH to be determined in locations away from a laboratory.

The step of determining the biomarker level (and comparing the biomarker level with the biomarker level in a control sample) may be performed using a POC test device, for example the device described below. For example, the step of determining the biomarker level (and comparing the biomarker level with the biomarker level in a control sample) may be performed using a flow through device or a lateral flow device. In embodiments, the step of determining the biomarker level (and comparing the biomarker level with the biomarker level in a control sample) may be performed using a lateral flow device, for example the device described below.

The term "biomarker level" as used herein is used to mean the amount of biomarker present in the samples compared to a baseline previously or concurrently established using appropriate controls (e.g., samples from age-matched healthy volunteers).

Comparing the level of biomarkers with a control sample Step (ii) of the method of the present invention involves comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether prostate cancer or BPH is present in the subject.

As will be appreciated by the skilled person, the origin of the control sample will depend upon the particular subject being tested. However, the control sample may be obtained from an age-matched subject and/or a subject of the same sex. Furthermore, since the comparison of biomarker levels may be used to determine whether prostate cancer or BPH is present in a subject, the control sample may be derived, for example, from a subject in which prostate cancer or BPH is not present but have another urological disease. That would be considered to be a symptom "control" in that they have symptoms which are similar to prostate cancer or BPH.

In the method of the present invention the biomarker level in a sample from a subject is compared with the biomarker level in a sample from a control. As will be appreciated by the skilled person, in the comparison step, the level of a biomarker in the sample from the subject is compared with a corresponding biomarker level in the control sample (e.g., in embodiments in which the metabolite 3-Hydroxyhexadecanoylcarnitine is utilised, the level of 3Hydroxyhexadecanoylcarnitine in the sample is compared with the level of 3Hydroxyhexadecanoylcarnitine in the control).

In embodiments of the invention, a biomarker level in the sample from the subject which is higher or lower than the biomarker level in the control sample indicates that prostate cancer or BPH is present in the subject.

In embodiments of the invention in which the one or more biomarkers are selected from: 3-hydroxyhexadecanoylcarnitine, alpha lipoic acid and N-heptanoylglycine a biomarker level in the sample from the subject which is higher than the biomarker level in the control sample indicates that prostate cancer is present in the subject.

In embodiments of the invention in which the one or more biomarkers is L-histidinol a biomarker level in the sample from the subject which is lower than the biomarker level in the control sample indicates that prostate cancer is present in the subject.

In embodiments of the invention, a biomarker level in the sample from the subject which is higher or lower than the biomarker level in the control sample indicates that BPH is present in the subject.

In embodiments of the invention in which the one or more biomarkers is N-Acetyl-DGlucosamine 6-Phosphate a biomarker level in the sample from the subject which is higher than the biomarker level in the control sample indicates that BPH is present in the subject.

In embodiments of the invention in which the one or more biomarkers is N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-Dmyo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, and cholic acid a biomarker level in the sample from the subject which is lower than the biomarker level in the control sample indicates that BPH is present in the subject.

As will be appreciated by the skilled person, when referring to a higher or lower biomarker level in a sample from the subject compared to a control sample, we mean a biomarker level which is significantly higher (at least two-fold higher) or significantly lower (at least 0.5-fold lower) than the biomarker level in a control sample.

In embodiments in which the biomarker level in the sample from the subject is higher than the biomarker level in the control sample, preferably the biomarker level in the sample from the subject is at least two-fold higher than in the control sample. More preferably, the biomarker level in the sample from the subject is at least three-fold, four-fold or five-fold higher than in the control sample. More preferably, the biomarker level in the sample from the subject is at least six-fold, seven-fold, eight-fold, nine-fold or ten-fold higher than in the control sample.

In embodiments in which the biomarker level in the sample from the subject is lower than the biomarker level in the control sample, preferably the biomarker level in the sample from the subject is at least two-fold lower than in the control sample. More preferably, the biomarker level in the sample from the subject is at least three-fold, four-fold or five-fold lower than in the control sample. More preferably, the biomarker level in the sample from the subject is at least six-fold, seven-fold, eight-fold, nine-fold or ten-fold lower than in the control sample.

Subject The present invention provides a method for determining the presence of prostate cancer in a subject. Prostate cancer is a disease which primarily affects humans but also a variety of animals such as dogs and cats for example. In embodiments of the present invention, the subject is a mammal, preferably a human.

The present invention also provides a method for determining the presence of BPH in a subject. BPH affects humans but also a variety of animals such as dogs and cats for example. In embodiments of the present invention, the subject is a mammal, preferably a human.

Other features The method of the present invention may further comprise providing a sample from a subject. In such embodiments the method of the present invention may comprise the steps of: providing a sample from a subject; (ii) determining the level of one or more biomarkers in the sample; and (iii) comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether prostate cancer is present in the subject; wherein the one or more biomarkers are selected from: 3-hydroxyhexadecanoylcarnitine, Lhistidinol, alpha lipoic acid and N-heptanoylglycine.

The method of the present invention may further comprise providing a sample from a subject. In such embodiments the method of the present invention may comprise the steps of: (i) providing a sample from a subject; (ii) determining the level of one or more biomarkers in the sample; and (iii) comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether BPH is present in the subject; wherein the one or more biomarkers are selected from: N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-Dmyo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.

Features of the abovementioned methods described herein apply equally to these methods.

The present invention comprises determining the level of one or more biomarkers in a sample from the subject. As will be appreciated by the skilled person, the sample may comprise a biological sample from the subject. The biological sample may have been obtained from a bodily fluid of the subject. The biological sample, may include, for example, urine, blood and blood components (e.g., serum), mucus, saliva, milk, vomit, faeces, sweat, semen, tears or pus. Preferably, the biological sample is a urine sample. This is particularly advantageous as urine samples are not invasive as with many tests for prostate cancer and are therefore well tolerated by subjects. What is more, such samples can be easily obtained. As will be appreciated by the skilled person, the method of the present invention is therefore an in vitro method for determining the presence prostate cancer in a subject, the method being carried out on a sample provided from a subject.

In a further aspect of the invention there is provided a method for determining the presence of prostate cancer in a subject, the method comprising the steps of (I) obtaining a sample from the subject; (ii) determining the level of one or more biomarkers in the sample; (hi) comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether prostate cancer is present in the subject; wherein the one or more biomarkers are selected from: 3-hydroxyhexadecanoylcarnitine, Lhistidinol, alpha lipoic acid and N-heptanoylglycine.

In a further aspect of the invention there is provided a method for determining the presence of BPH in a subject, the method comprising the steps of: (I) obtaining a sample from the subject; (h) determining the level of one or more biomarkers in the sample; (hi) comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether prostate cancer is present in the subject; wherein the one or more biomarkers are selected from: N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-Dmyo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.

The method of obtaining the sample from the subject will depend on the sample type and subject. In embodiments in which the sample is a urine sample, the subject may collect the sample in a urine collection container, for example.

There is also provided a method for determining the presence of prostate cancer in a subject, determining progression of prostate cancer or assessing response to therapy of a subject with prostate cancer. The method comprises the steps of: determining the level of one or more biomarkers in a sample from the subject; comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether the subject has prostate cancer, has prostate cancer which is progressing, or is responding to therapy for prostate cancer; wherein the one or more biomarkers are selected from: 3-hydroxyhexadecanoylcarnitine, Lhistidinol, alpha lipoic acid and N-heptanoylglycine.

There is also provided a method for determining the presence of BPH in a subject, determining progression of BPH or assessing response to therapy of a subject with BPH. The method comprises the steps of: determining the level of one or more biomarkers in a sample from the subject; comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether the subject has BPH, has BPH which is progressing, or is responding to therapy for BPH; wherein the one or more biomarkers are selected from: N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-Dmyo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.

There is also provided a method for determining the presence of prostate cancer in a subject, determining progression of prostate cancer or assessing response to therapy of a subject with prostate cancer. The method comprises the steps of: obtaining a sample from the subject; determining the level of one or more biomarkers in a sample from the subject; (iii) comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether the subject has prostate cancer, has prostate cancer which is progressing, or is responding to therapy for prostate cancer; wherein the one or more biomarkers are selected from: 3-hydroxyhexadecanoylcarnitine, Lhistidinol, alpha lipoic acid and N-heptanoylglycine.

There is also provided a method for determining the presence of BPH in a subject, determining progression of BPH or assessing response to therapy of a subject with BPH. The method comprises the steps of: obtaining a sample from the subject; (ii) determining the level of one or more biomarkers in a sample from the subject; (iii) comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether the subject has BPH, has BPH which is progressing, or is responding to therapy for BPH; wherein the one or more biomarkers are selected from: N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-Dmyo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.

In embodiments where the method is being used to determine whether prostate cancer or BPH is progressing, the method may comprise monitoring over time to determine whether prostate cancer or BPH has progressed. In such an embodiment, an initial biomarker level (as described above) may be compared with a biomarker level in a sample obtained later in time. The biomarker level(s) obtained may change over time, to be further removed (either higher or lower) from a control biomarker level. Alternatively, in examples where multiple biomarkers are utilised to determine prostate cancer, for example four biomarkers, in an initial analysis only two of the four biomarkers may be indicative of prostate cancer. In the later analysis an increased number of biomarkers may be indicative of prostate cancer, which may indicate that prostate cancer is progressing. In examples where multiple biomarkers are utilised to determine BPH, for example nine biomarkers, in an initial analysis only four of the nine biomarkers may be indicative of BPH. In the later analysis an increased number of biomarkers may be indicative of BPH, which may indicate that BPH is progressing.

In embodiments where the method is being used to determine whether a subject is responding to therapy, the method may comprise monitoring over time to determine whether a subject is responding to therapy. In such an embodiment, an initial biomarker analysis (which may be before therapy has commenced) may be compared with one or more biomarker analyses undertaken on samples obtained later in time (for example after therapy has commenced). The biomarker levels may change over time, to be further removed (either higher or lower) from a control biomarker level. Alternatively, in examples where multiple biomarkers are utilised, for example four biomarkers, in an initial analysis, four out of four biomarker levels may be indicative of prostate cancer. In a later analysis, fewer biomarkers may be indicative of prostate cancer, which may indicate that the subject is responding to therapy. Alternatively, in a later analysis, the number of biomarkers indicative of prostate cancer may remain the same or increase, which may indicate that a subject is not responding to therapy. In examples where multiple biomarkers are utilised to determine BPH, for example nine biomarkers, in an initial analysis, nine out of nine biomarker levels may be indicative of BPH. In a later analysis, fewer biomarkers may be indicative of BPH, which may indicate that the subject is responding to therapy. Alternatively, in a later analysis, the number of biomarkers indicative of BPH may remain the same or increase, which may indicate that a subject is not responding to therapy.

The method of the present invention may also be used to determine whether a drug is effective at treating prostate cancer or BPH, in a similar manner to that described above in relation to determining whether a subject is responding to therapy. The use of "effective" is used to indicate that a treatment reduces or alleviates signs or symptoms of prostate cancer or BPH, improves the clinical course of the disease, decreases the number or severity of exacerbations or reduces any other objective or subjective indicia of the disease. The method of the present invention can be used to determine whether drugs used to treat prostate cancer or BPH, in addition to other drugs developed to treat prostate cancer or BPH are effective.

Since a diagnosis of a disease is often not based on the results of a single test alone, the method of the present invention may be used to determine whether a subject is more likely than not to have prostate cancer or BPH based on comparison of one or more biomarker levels with a control biomarker level. Thus, for example, a subject with a putative diagnosis of prostate cancer or BPH may be diagnosed as being "more likely" or "less likely" to have prostate cancer or BPH in light of the information provided by the method of the present invention. The present invention may therefore be used to assist a clinician with the diagnosis of prostate cancer or BPH.

The method of the present invention may, in certain embodiments, comprise detecting other signs or symptoms of prostate cancer or BPH, conducting clinical tests of prostate cancer or BPH and/or measuring other prostate cancer markers or BPH biomarkers, for example other alternative biomarkers.

As will be appreciated by the skilled person, the above description is not limited to making an initial identification (or diagnosis) of prostate cancer or BPH in a subject but is also applicable to confirming a provisional diagnosis of prostate cancer or BPH or "ruling out" such a diagnosis.

The present invention may also be used to determine a suitable treatment for a subject, depending on whether testing indicates that they have prostate cancer or BPH. The method of the present invention may therefore comprise a step of determining a suitable treatment for the subject, if the subject has prostate cancer or a particular type of prostate cancer or BPH.

Devices The present invention also provides an immunological capture device for detecting prostate cancer in a subject, the device comprising a substrate carrying capture antibodies to one or more of the following biomarkers: 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid and N-heptanoylglycine.

In embodiments, the device comprises antibodies to one, two, three or four of the following biomarkers: 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid and Nheptanoylglycine.

In embodiments, the substrate carries capture antibodies to all listed biomarkers.

In other embodiments, the device comprises a substrate carrying capture antibodies to one or more of the following biomarkers: N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-D-myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid. Such a device can be useful in identifying subjects with BPH.

In embodiments the device comprises a substrate carrying capture antibodies to one or more of the following biomarkers: 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid and N-heptanoylglycine; and one or more of the following biomarkers: N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-Dmyo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid. Such a device would be useful in differentiating between subjects with prostate cancer and BPH.

The term "immunological capture device" as described herein is used to describe an immunoassay device which can be used to measure the presence of a biomarker in a sample through the use of an antibody. The antibody would be specific to the biomarker of interest, such that the antibody could "capture" the biomarker, through binding, if the biomarker is present. The antibodies may therefore be described as "capture antibodies".

In embodiments, the device comprises antibodies to one, two, three or four of the following biomarkers: 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid and N-heptanoglycine; and one, two, three, four, five, six, seven, eight or nine of the following NIsobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-D-myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.

In embodiments, the device comprises antibodies to all listed biomarkers.

The substrate of the present invention may be any suitable surface which can carry an antibody. For example, the substrate may be plastic, for example a plate (e.g., a multi-well plate). Alternatively, the substrate may be a porous substrate. The porous substrate may be any material which allows another medium to pass through it. Any suitable porous substrate could be used, for example a woven material, or a cellulosic material.

The substrate carries the capture antibodies. The antibodies may be carried within the substrate or on the surface of the substrate. The antibodies may form a chemical interaction with the surface of the substrate. The antibodies may be bound to the substrate.

In embodiments, the device is a lateral flow device. Alternatively, the device may be a flow through device or an ELISA device.

Advantageously the device is a rapid, simple, non-invasive diagnostic providing quick diagnosis of prostate cancer. It is easily accessible and can improve detection and control of prostate cancer. Dependent on the antibodies present in the device it can also detect BPH and differentiate between subjects with prostate cancer and those with BPH.

Lateral flow and flow through devices are particularly advantageous in that they can be used remotely to obtain rapid results in a simple manner.

A sample to be tested (i.e., a sample from the subject) may be applied to the immunological capture device, for example to the substrate of the immunological capture device.

A second antibody may also be applied the substrate of the immunological capture device. The second antibody may be specific to the biomarkers or to the capture antibodies (i.e., the antibodies carried on the substrate). The second antibody may have coloured particles attached. The coloured particles may be covalently linked to the second antibody. The coloured particles may be cellulose beads or plastic microparticles, for example. In alternative embodiments the second antibody may be linked to an enzyme, via bio-conjugation, for example. In such embodiments, a composition comprising a substrate which undergoes a colour change upon reaction with the enzyme, indicating the presence of the enzyme, may be added to the device during use. Suitable enzymes and compositions will be well known to the skilled person.

In embodiments in which the second antibody is specific to the biomarkers, binding of the second antibody to the biomarkers bound to the capture antibodies results in a colour change. The presence of the biomarkers can therefore be detected by a colour change.

In embodiments in which the second antibody is specific to the capture antibodies, binding of the second antibody to the capture antibody may take place when the biomarker is not present.

In such an embodiment, binding of the second antibody to the capture antibodies, results in a colour change. The absence of the biomarkers can therefore be detected by a colour change.

A reader, for example a lateral flow reader, may be used to quantify the colour intensity.

In embodiments in which the device is a lateral flow or flow through device, the device may further comprise a control line. Colouring of the control line indicates successful completion of the test.

In embodiments in which the device is a lateral flow or flow through device, the device may further comprise a test line. The test line may comprise capture antibodies to the one or more biomarkers.

Colouring of the test line indicates the presence or absence of the biomarkers in the sample (as discussed above). Comparison of the coloured intensity of the control line and test line can be used to indicate the biomarker level in the sample and therefore whether the sample is from a subject in which prostate cancer is present. A reader, for example a lateral flow reader may be used to quantify the coloured intensity of the control and test lines.

In embodiments, the device may further comprise a housing. The substrate may be positioned within the housing.

The device can be used to detect biomarkers in urine, for example.

Preferably the device provides a test result in 60 minutes or less from test initiation. The device may provide a test result in 30 minutes or less, 20 minutes or less or 10 minutes or less from test initiation.

The device may be used in the method of the invention. Kit

The present invention also provides a kit for determining the presence of prostate cancer in a subject, the kit comprising: (i) an immunological capture device comprising a substrate carrying capture antibodies to one or more of the following biomarkers: 3hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid and Nheptanoylglycine; and a second antibody, wherein the second antibody is specific to the one or more biomarkers or to the capture antibodies.

The present invention also provides a kit for determining the presence of BPH in a subject, the kit comprising: (i) an immunological capture device comprising a substrate carrying capture antibodies to one or more of the following biomarkers: N-Isobuty1-2,4,8,10,12- tetradecapentaenamide, PE-N Me2(24: 1(15Z)/20:5(5Z,8Z,11Z,14Z, 17Z)), 1-Phosphatidyl-D-myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid; and (ii) a second antibody, wherein the second antibody is specific to the one or more biomarkers or to the capture antibodies.

The present invention also provides a kit for determining the presence of prostate cancer and/or BPH in a subject, the kit comprising: (I) an immunological capture device comprising a substrate carrying capture antibodies to one or more of the following biomarkers: -3- hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid, N- heptanoylglycine; and one or more of the following biomarkers N-Isobutyl- 2,4, 8, 10,12-tetrad ecapentaen am ide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-D-myo-inositol, LUrobilin, 18-Carboxy-dinar-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, NAcetyl-D-Glucosamine 6-Phosphate and cholic acid; and (ii) a second antibody, wherein the second antibody is specific to the one or more biomarkers or to the capture antibodies.

The device and second antibody of the kits are described above in relation to the immunological capture device of the present invention. The kits may be used in the methods of the invention. Uses

The present invention also provides the use of one or more biomarkers for detecting prostate cancer in a subject, the one or more biomarkers being selected from: 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid and N-heptanoylglycine.

The present invention also provides the use of one or more biomarkers for detecting BPH in a subject, the one or more biomarkers being selected from: N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-Dmyo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.

The described and illustrated embodiments are to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the scope of the inventions as defined in the claims are desired to be protected.

The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention as set out herein are also to be read as applicable to any other aspect or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each exemplary embodiment of the invention as interchangeable and combinable between different exemplary embodiments.

It should be understood that while the use of words such as "preferable", "preferably", "preferred" or "more preferred" in the description suggest that a feature so described may be desirable, it may nevertheless not be necessary and embodiments lacking such a feature may be contemplated as within the scope of the invention as defined in the appended claims. In relation to the claims, it is intended that when words such as "a," "an," or "at least one," are used to preface a feature there is no intention to limit the claim to only one such feature unless specifically stated to the contrary in the claim.

Detailed Description of the Invention

The present invention will now be further described with reference to the following figures which show: Figure 1: Potential biomarkers that distinguish between Prostate Cancer (P) and Symptom Controls (M), Partial Least Squares Discriminant Analysis (PLS-DA) of P and M metabolomes showing good separate clustering.

Figure 2: Potential biomarkers that distinguish between BPH (B) Symptom Controls (M), Partial least squared discriminant analysis (PLS-DA) of P and M showing good separate clustering.

Examples

Metabolomic approaches have been used to identify biochemical biomarkers of prostate cancer. The inventors have shown that urine metabolites can distinguish between patients with prostate cancer, controls and those with BPH.

Urine samples were used from 46 subjects with prostate cancer (PCa) (biopsy confirmed with Gleason and TNM scoring), 29 subjects with BPH (digital rectal exam, MRI or biopsy confirmed) and 29 aged matched male symptom controls. Symptom controls include patients with raised PSA, enlarged prostate, high grade prostatic intraepithelial neoplasia, urinary retention, kidney or bladder cancer, urinary tract infections, kidney stones and haematuria. Liquid samples used to identify biochemical markers were typically 100p1.

Metabolomic comparisons were initially undertaken of urine taken from PCa (P) patients versus symptom controls (M) (Figure 1). This indicated that the urine metabolomes of each group were significantly different (Figure 1). Partial least squares discrimiant analysis (PLsDA) of the P and M metabolomes showed good separate clustering. Further assessments identified 165 metabolites that differed significantly (P < 0.001) (corrected for false discovery rates [FDR]) between the patient groups. A metabolomic signature of fourmetabolites gives accuracies of 87.5% (CI 0.781-0.956). The top four metabolites had individual AUC values ranging between 0.82 and 0.79. The top performing metabolites are listed in Table 1.

Table 1: Prostate cancer Specific changes compared with the Male control group m.z Change (Left = AUC Fold P Value Metabolite control, Right = change cancer) (C/P) 494.249.:.',..,.... 0.832 1.9 1.54E-08 3-Hydroxyhexa " decanoylcarnitine N \ : 176.059 * 0.82 0.2 7.72E-05 L-Histidinol N \ -; 241.012. , A"..;\ 0.81 2.1 1.46E-06 alpha lipoic acid \ . ..\ \ ::...\. \, 3.58E-07 1.8 0.8 186.113 \^**.. \ \ \\ \\\\\\ N-heptanoylglycine.

Next, BPH (B) urine samples were compared with the symptom controls (M). PLS-DA indicated that the metabolomes were distinct (Figure 2). Further assessments identified 164 metabolites that differed significantly (P < 0.001) (corrected for FDR) between the patient groups. AUC -ROC assessment of the major sources of variation identified a signature of metabolites which distinguished between the groups with high accuracies of 90.7% (CI 0.731-1).These are listed in Table 2. These biomarkers can also distinguish between BPH and PCa.

Table 2: Benign Prostatic Hyperplasia Specific changes compared with the Male control group m.z Change (Left = AUC Fold change (BPH/Con) P Value Metabolite BPH, Right = male controls) 272.198 0.86345 1.9013 1.08E-07 N-Isobuty1-2,4,8,10,12-tetradecapentaenamide r 436.813 0.85517 1.5349 4.61E-07 PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)) / * 389.051 593.336 440.178 0.8469 1.617 2.74E-07 1-Phosphatidyl-D-myo-inositol 0.8331 1.9672 4.53E-07 L-Urobilin 0.8269 1.798 3.00E-08 18-Carboxy-dinor-LTE4 611.285 206.976 300.05 0.81793 1.722 1.49E-06 Urobilin/Urobilinogen 0.81655 1.5192 5.82E-05 5,6-Dihydrothymine 0.81655 0.39982 1 81E-05 N-Acetyl-D-Glucosamine 6-Phosphate 443.26 0.8131 1.8527 7.13E-06 Cholic acid

Conclusion

These diagnostic fingerprints demonstrated good levels of sensitivity and specificity for prostate cancer. The detection of prostate cancer in urine could represent an inexpensive point of case (POC) test that could guide people to follow up diagnostic assessments.

It will be appreciated that numerous modifications to the above-described methods, including smaller and alternative selections of the identified biomarkers, may be made without departing from the scope of the invention as defined in the appended claims.

References de Azambuja, E., et al., The landscape of medical oncology in Europe by 2020t. Annals of Oncology, 2014. 25(2): p. 525-528.

Rawla, P., Epidemiology of Prostate Cancer. World J Oncol, 2019. 10(2): p. 63-89.

Bostwick, D.G., et al., The association of benign prostatic hyperplasia and cancer of the prostate. Cancer, 1992. 70(1 Suppl): p. 291-301.

Alberts, A.R., I.G. Schoots, and M.J. Roobol, Prostate-specific antigen-based prostate cancer screening: Past and future. Int J Urol, 2015. 22(6): p. 524-32.

Albertsen, P.C., The Evolving Paradigm of Prostate Cancer Screening. JAMA Netw Open, 2019. 2(8): p. e198392.

Claims

Claims 1. A method for determining the presence of prostate cancer in a subject, the method comprising the steps of: (i) determining the level of one or more biomarkers in a sample from the subject; (h) comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether prostate cancer is present in the subject; wherein the one or more biomarkers are selected from: 3-hydroxyhexadecanoylcarnitine, Lhistidinol, alpha lipoic acid and N-heptanoylglycine.
2. The method according to claim 1 wherein when the one or more biomarkers is L-histidinol a biomarker level in the sample from the subject which is lower than the biomarker level in the control sample indicates that prostate cancer is present in the subject.
3. The method according to claim 1 or 2 wherein when the one or more biomarkers comprises 3-hydroxyhexadecanoylcarnitine, alpha lipoic acid and N-heptanoylglycine a biomarker level in the sample from the subject which is higher than the biomarker level in the control sample indicates that prostate cancer is present in the subject.
4. The method according to claim 1, wherein the biomarkers consist of 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid and N-heptanoylglycine.
5. The method according to any preceding claim, wherein the subject is a human.
6. The method according to any preceding claim, wherein the method further comprises providing a sample from the subject.
7. The method according to any preceding claim wherein the sample comprises a urine sample.
8. An immunological capture device for detecting prostate cancer in a subject, the device comprising a substrate carrying capture antibodies to one or more of the following biomarkers: 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid and N-heptanoylglycine
9. The immunological capture device according to claim 8, wherein the substrate carries capture antibodies to 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid and Nheptanoylglycine.
10. A kit for determining the presence of prostate cancer in a subject, the kit comprising: an immunological capture device comprising a substrate carrying capture antibodies to one or more of the following biomarkers: 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid and N-heptanoylglycine; and (ii) a second antibody, wherein the second antibody is specific to the one or more biomarkers or to the capture antibodies.
11. The kit according to claim 10 wherein the second antibody comprises a coloured particle covalently linked to the second antibody.
12. A method for determining the presence of BPH in a subject, the method comprising the steps of: determining the level of one or more biomarkers in a sample from the subject; (ii) comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether BPH is present in the subject; wherein the one or more biomarkers are selected from: N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,117,147,177)), 1-Phosphatidyl-Dmyo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.
13. The method according to claim 12 wherein when the one or more biomarkers is N-AcetylD-Glucosamine 6-Phosphate a biomarker level in the sample from the subject which is higher than the biomarker level in the control sample indicates that BPH is present in the subject.
14. The method according to claim 12 or 13 wherein when the one or more biomarkers comprises N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-D-myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, and cholic acid a biomarker level in the sample from the subject which is lower than the biomarker level in the control sample indicates that BPH is present in the subject.
15. The method according to claim 12, wherein the biomarkers consist of N-Isobutyl- 2,4,8, 10,12-tetradecapentaenamide, PE-NMe2(24: 1(15Z)/20:5(5Z,8Z,11Z,14Z, 17Z)), 1-Phosphatidyl-D-myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.
16. The method according to any of claims 12-15, wherein the subject is a human.
17. The method according to any of claims 12-16, wherein the method further comprises providing a sample from the subject.
18. The method according to any of claims 12-17 wherein the sample comprises a urine sample.
19. An immunological capture device for detecting BPH in a subject, the device comprising a substrate carrying capture antibodies to one or more of the following biomarkers: N-Isobuty1- 2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(157)/20:5(57,8Z,1 17,147,17Z)), 1-Phosphatidyl-D-myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.
20. The immunological capture device according to claim 19, wherein the substrate carries capture antibodies to N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-D-myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.
21. A kit for determining the presence of BPH in a subject, the kit comprising: (i) an immunological capture device comprising a substrate carrying capture antibodies to one or more of the following biomarkers: N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PENMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-D-myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.(ii) a second antibody, wherein the second antibody is specific to the one or more biomarkers or to the capture antibodies.
22. A method for determining whether a subject has prostate cancer or BPH, the method comprising the steps of: (i) determining the level of one or more biomarkers in a sample from the subject; (ii) comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether prostate cancer or BPH is present in the subject; wherein the one or more biomarkers that indicate prostate cancer are selected from: 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid and N-heptanoylglycine; and wherein the one or more biomarkers that indicate BPH are selected from: N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphafidyl-D-myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.
23. The method according to claim 22 wherein when the one or more biomarkers is L-histidinol a biomarker level in the sample from the subject which is lower than the biomarker level in the control sample indicates that prostate cancer is present in the subject.
24. The method according to claim 22 or 23 wherein when the one or more biomarkers comprises 3-hydroxyhexadecanoylcarnitine, alpha lipoic acid and N-heptanoylglycine a biomarker level in the sample from the subject which is higher than the biomarker level in the control sample indicates that prostate cancer is present in the subject.
25. The method according to any of claims 22-24 wherein when the one or more biomarkers is N-Acetyl-D-Glucosamine 6-Phosphate a biomarker level in the sample from the subject which is higher than the biomarker level in the control sample indicates that BPH is present in the subject.
26. The method according to any of claims 22-25 wherein when the one or more biomarkers comprises N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-D-myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, and cholic acid a biomarker level in the sample from the subject which is lower than the biomarker level in the control sample indicates that BPH is present in the subject.
27. The method according to claim 22, wherein the biomarkers consist of 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid, N-heptanoylglycine, N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-D-myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.
28. The method according to any of claims 22-27, wherein the subject is a human.
29. The method according to any of claims 22-28, wherein the method further comprises providing a sample from the subject.
30. The method according to any of claims 22-29 wherein the sample comprises a urine sample.
31. An immunological capture device for detecting prostate cancer in a subject, the device comprising a substrate carrying capture antibodies to one or more of the following biomarkers: 3-hydroxyhexadecanoylcarnitine, L-Histidinol, alpha lipoic acid and N-heptanoylglycine; and one or more of the following biomarkers: N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-D-myo-inositol, L-Urobi lin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.
32. The immunological capture device according to claim 31, wherein the substrate carries capture antibodies to 3-hydroxyhexadecanoylcarnitine, L-histidinol, alpha lipoic acid, N- heptanoylglycine, N-Isobuty1-2,4,8,10,12-tetradecapentaenamide, PE- NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-D-myo-inositol, L-Urobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, N-Acetyl-D-Glucosamine 6-Phosphate and cholic acid.
33. A kit for determining the presence of prostate cancer or BPH in a subject, the kit comprising: (i) (i) an immunological capture device comprising a substrate carrying capture antibodies to one or more of the following biomarkers: 3-hydroxyhexadecanoylcarnitine, L-Histidinol, alpha lipoic acid and Nheptanoylglycine; and one or more of the following biomarkers N-Isobuty1- 2,4,8,10,12-tetradecapentaenamide, PE-NMe2(24:1(15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), 1-Phosphatidyl-D-myo-inositol, LUrobilin, 18-Carboxy-dinor-LTE4, Urobilin/Urobilinogen, 5,6-Dihydrothymine, NAcetyl-D-Glucosamine 6-Phosphate and cholic acid; and (ii) a second antibody, wherein the second antibody is specific to the one or more biomarkers or to the capture antibodies.