Cancer biomarkers

Abstract

The present invention relates to a method for the early and efficient diagnosis of cancer, such as prostate cancer in an individual, as well as arrays and kits for use in such methods.

Description

[0001] CANCER BIOMARKERS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to novel methods for diagnosing cancer, such as prostate cancer, in an individual, as well as arrays and kits for use in such methods.

[0004] BACKGROUND OF THE INVENTION

[0005] Cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020 (data from WHO website). Many cancers can be cured if detected early enough. Early diagnosis is fundamental to providing effective treatment options and improving the overall 5-year survival rate. However, a major problem associated with early detection of cancer is that once symptoms are apparent, the cancer usually spreads to other organs and is at the stage where extensive chemotherapy or radiotherapy is needed to prolong lifespan. Although screening programs for different cancers exist, these are often expensive, time-consuming, and the procedures can be invasive and uncomfortable.

[0006] In particular, prostate cancer is a malignant tumour that originates in the prostate gland which is responsible for producing seminal fluid. It is one of the most common cancers affecting men, particularly those over the age of 50. Prostate cancer often develops slowly and may remain localized for years, but it can also grow aggressively and spread to other parts of the body. The disease is typically detected through prostate-specific antigen (PSA) blood tests, digital rectal exams (DRE), and biopsy procedures. Early detection and treatment options, such as surgery, radiation therapy, and hormone therapy, significantly improve patient outcomes.

[0007] Historically, individual preclinical and clinical studies have been conducted, outlining individual biomarkers in prostate cancer. However, the accuracy of early cancer detection using a single "gold standard" biomarker is low, owing to the fact that cancer is highly heterogeneous and that no single biomarker has a single functional role in cells. At present, the gold standard method for detecting prostate cancer is the measurement of Prostate-Specific Antigen (PSA) levels in blood. However, the biomarker, PSA, is also known to be elevated in patients with prostate inflammation and therefore, is not necessarily indicative of prostate cancer. Measurement of PSA levels and other current techniques, such as digital rectal exams (DRE), often lack specificity and sensitivity, leading to false positives, unnecessary biopsies, and missed early-stage cancers. The provision of a novel panel of specific biomarkers could, therefore, improve the accuracy, reliability and cost efficiency of such detection methods, resulting in a superior detection method to the current 'gold standard'.

[0008] Nucleic acids are often used in place of proteins in the cancer diagnostics space due to lower costs and facile high-throughput DNA / RNA sequencing technologies. The use of protein-based diagnostics provides a more dynamic and accurate diagnosis of both the presence and the stage of cancers. Such protein-based diagnostics may include analysis of the salivary proteome, which is currently an undermined method of diagnosis.

[0009] The inventors have surprisingly found a method for diagnosing prostate cancer comprising measuring the expression of a unique panel of a combination of biomarkers, particularly in saliva.

[0010] SUMMARY OF THE INVENTION

[0011] The inventors have surprisingly found that a unique panel of biomarkers of the present invention, is expected to provide an early and effective diagnostic method for prostate cancer, which is non-invasive, more accurate, cost efficient and convenient than current diagnostic methodologies. As will be evident from the in vivo data below, the unique combination of biomarkers of the present invention is expected to provide methods for the effective diagnosis of prostate cancer in an individual. The biomarkers have been surprisingly found to express significantly in saliva allowing for reliable yet accessible testing.

[0012] A first aspect of the present invention is a method for diagnosing or determining the presence of prostate cancer in an individual, the method comprising the steps of: i) providing a saliva test sample from the individual; and ii) measuring the expression, in the test sample, of one or more biomarkers selected from: C4B, C6, C7 , C9, CFB, CFH, CP, CSRP1, DPMI, EFEMP1, HINT2, IGKV3-15, LCN2, MUC1, MUC5AC, RARRES1, SCGB1D2, SERPINA1, SERPINA3 and SERPING1, wherein the expression in the saliva test sample of the one or more biomarkers is indicative of prostate cancer in the individual.

[0013] A second aspect of the invention is a method for diagnosing or determining the presence of cancer in an individual, the method comprising the steps of: i) providing a test sample from the individual; and ii) measuring the expression, in the test sample, of one or more biomarkers selected from: CFH, DPMI, IGKV3-15 and MUC5AC, wherein the expression in the test sample of the one or more biomarkers is indicative of cancer in the individual.

[0014] A third aspect of the invention is a method for diagnosing or determining the presence of prostate cancer in an individual, the method comprising the steps of: i) providing a test sample from the individual; and ii) measuring the expression, in the test sample, of one or more biomarkers selected from: C4B, C6, C9, CFB, CFH, DPMI, HINT2, IGKV3-15, and MUC5AC, wherein the expression in the test sample of the one or more biomarkers is indicative of prostate cancer in the individual. A fourth aspect of the invention is a method for diagnosing or determining the presence of a disease in an individual, the method comprising the steps of: i) providing a saliva test sample from the individual; and ii) measuring the expression, in the saliva test sample, of one or more biomarkers selected from: CFB, CSRP1, HINT2, IGKV3-15, and RARRES1, wherein the expression, in the saliva test sample, of the one or more biomarkers is indicative of a disease in the individual.

[0015] A fifth aspect of the invention is a method for diagnosing or determining the presence of cancer in an individual, the method comprising the steps of: i) providing a saliva test sample from the individual; and ii) measuring the expression, in the saliva test sample, of one or more biomarkers selected from: C4B, C6, C7, C9, CFH, CP, EFEMP1, MUC5AC, SCGB1D1, SERPINA1, SERPINA3 and SERPING1, wherein the expression, in the saliva test sample, of the one or more biomarkers is indicative of a cancer in the individual.

[0016] A sixth aspect of the invention is an array comprising an agent or agents for detecting the expression of one or more of the biomarkers selected from: C4B, C6, C7, C9, CFB, CFH, CP, CSRP1, DPMI, EFEMP1, HINT2, IGKV3-15, LCN2, MUC1, MUC5AC, RARRES1, SCGB1D2, SERPINA1, SERPINA3, and SERPING1.

[0017] A seventh aspect of the invention is use of one or more biomarkers selected from the group comprising C4B, C6, C7, C9, CFB, CFH, CP, CSRP1, DPMI, EFEMP1, HINT2, IGKV3-15, LCN2, MUC1, MUC5AC, RARRES1, SCGB1D2, SERPINA1, SERPINA3, and SERPING1 for diagnosing or determining the presence of cancer in an individual.

[0018] An eighth aspect of the invention is a kit, optionally wherein the kit is for diagnosing or determining the presence of cancer, the kit comprising: i) the array according to the sixth aspect of the invention, or components for making the same; and ii) instructions for performing the method as defined in any one of the first to the fifth aspects of the invention.

[0019] A ninth aspect of the invention is a method of treating cancer, preferably prostate cancer, in an individual comprising the steps of: i) diagnosing or determining the presence of cancer according to the method defined in any one of the first to the fifth aspects of the invention; and ii) providing the individual with cancer therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 shows the ROC curve for one of the biomarkers identified in the mass spectrometry analysis in prostate cancer patients vs healthy controls.

[0021] Figure 2 shows the ROC curve for three of the biomarkers identified in the mass spectrometry analysis in prostate cancer patients vs healthy controls.

[0022] Figures 3(a)-(h) show box-and-whisker plots that show the level of RNA expression in prostate cancer tissue (left group) compared to healthy tissue (right group), (a) corresponds to expression of the biomarker combination shown in Figure 2. (b) - (g) correspond to the expression of SERPINA3, SERPINA1, SCGB1D2, IGKV3-15, HINT2 and C4B respectively.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] In the present invention, and as demonstrated by the below in vivo data, methods of the invention have been shown to have utility for diagnosing prostate cancer in an individual and are therefore, expected to offer therapeutic options for early and efficient diagnosis of prostate cancer, as well as, for the treatment of prostate cancer.

[0025] The inventors have surprisingly identified a method for the early and effective diagnosis of prostate cancer, which is non-invasive, more accurate, cost efficient and convenient than current diagnostic methodologies. The method comprises measuring the expression of a unique panel of 20 biomarkers in samples and in particular, in saliva samples.

[0026] Unless indicated otherwise, all technical and scientific terms used herein will have their common meaning as understood by one of ordinary skills in the art to which this invention pertains.

[0027] The term "comprises" or "comprising" will take its usual meaning in the art, namely indicating that the component includes but is not limited to the relevant features (i.e. including, among other things). As such, the term "comprises" will include references to the component consisting essentially of (such as consisting of) the relevant features. The term "consists of" or "consisting of" will take its usual meaning in the art, namely indicating that the component includes and is limited to the relevant features.

[0028] The term "biomarker" as used herein refers to biological molecules (or components or fragments thereof) that provide information that is useful in the diagnosis of cancer and in particular, prostate cancer. The biomarker may be a nucleic acid molecule, for example an mRNA or cDNA molecule, a protein, peptide or polypeptide.

[0029] The term "biomarker signature" as used herein refers to the combination of biomarkers that are measured in the sample that are useful in the diagnosis of cancer. The term "measuring the expression" as used herein refers to measuring the presence and / or amount of a particular biomarker. We also include measuring whether a particular biomarker is up- or down-regulated compared to an expected expression level or a control expression level.

[0030] The terms "patient", "subject" and "individual" are used interchangeably and refer to a subject of diagnosis with the methods of the invention. Preferably, the individual is human. Suitably, the individual has cancer, preferably prostate cancer.

[0031] In a preferred embodiment, the one or more biomarkers is two or more biomarkers, preferably three or more biomarkers and most preferably, four or more biomarkers. A combination of biomarkers provides the advantage that it increases the accuracy and reliability of the diagnosis of the cancer. For example, should a particular individual (e.g., an individual with cancer) exhibit weak expression levels or an unreadable result, of one biomarker, the diagnosis may still be determined by the other biomarker(s) measured.

[0032] In a preferred embodiment, the one or more biomarkers according to the second aspect of the invention is MUC5AC and / or IGKV3-15.

[0033] In a preferred embodiment, the one or more biomarkers is selected from C4B, C7, C9, CP, EFEMP1, SERPINA1, SERPINA3 and SCGB1D2, preferably wherein two or more biomarkers, most preferably wherein three or more biomarkers are selected from: C4B, C7, C9, CP, EFEMP1, SERPINA1, SERPINA3 and SCGB1D2.

[0034] In a preferred embodiment, the one or more biomarkers is selected from C4B, C7, C9, EFEMP1, SERPINA1, and SERPINA3, preferably wherein two or more biomarkers, most preferably wherein three or more biomarkers are selected from: C4B, C7, C9, EFEMP1, SERPINA1, and SERPINA3.

[0035] In an embodiment of the invention, the method further comprises the steps of: iii) providing one or more (negative) control samples from an individual not afflicted with cancer, such as prostate cancer; and iv) determining a biomarker signature of the one or more control samples by measuring the expression in the control sample of the one or more biomarkers measured in step ii), wherein the cancer, such as prostate cancer, is identified in the event that the expression in the saliva test sample or test sample of the one or more biomarkers measured in step ii) is different from the expression in the control sample of the one or more biomarkers measured in step iv).

[0036] Preferably, the negative control sample is a test sample derived from normal prostate tissue from the individual; or from a healthy individual; or a pool of healthy individuals. Preferably, the negative control sample is a saliva test sample from a healthy individual; or from a healthy individual; or a pool of healthy individuals.

[0037] The phrase "is different from the expression in the negative control sample" refers to the situation where the biomarker is detected in the test sample or saliva test sample but is not detected in the negative control sample(s), and vice versa. We also include the situation where the biomarker in question is upregulated or downregulated in the test sample or saliva test sample compared to the same biomarker in the control sample. By "upregulated or downregulated" we include where the amount of the biomarker in the test sample differs from the amount of the biomarker in the control sample by at least ±5%, for example, at least ±6%, ±7%, ±8%, ±9%, ±10%, ±11%, ±12%, ±13%, ±14%, ±15%, ±16%, ± 17%, ±18%, ±19%,

[0038] ±20%, ±21%, ±22%, ±23%, ±24%, ±25%, ±26%, ±27%, ±28%, ±29%, ±30%, ±31%,

[0039] ±32%, ±33%, ±34%, ±35%, ±36%, ±37%, ±38%, ±39%, ±40%, ±41%, ±42%, ±43%,

[0040] ±44%, ±45%, ±41%, ±42%, ±43%, ±44%, ±55%, ±60%, ±65%, ±66%, ±67%, ±68%, ±69%, ±70%, ±71%, ±72%, ±73%, ±74%, ±75%, ±76%, ±77%, ±78%, ±79%, ±80%, ±81%, ±82%, ±83%, ±84%, ±85%, ±86%, ±87%, ±88%, ±89%, ±90%, ±91%, ±92%,

[0041] ±93%, ±94%, ±95%, ±96%, ±97%, ±98%, ±99%, ±100%, ±125%, ± 150%, ± 175%, ±200%, ±225%, ±250%, ±275%, ±300%, ±350%, ±400%, ±500% or at least ±1000% of the one or more control sample(s) (e.g., the negative control sample).

[0042] In an embodiment of the present invention, the expression in the test sample or saliva test sample differs from the mean expression in the control samples by at least >1 standard deviation from the mean presence or amount in the control samples, for example, >1.5, >2, >3, >4, >5, >6, >7, >8, >9, >10, >11, >12, >13, >14 or >15 standard deviations from the mean presence or amount in the control samples. Any suitable means may be used for determining standard deviation, however, in one embodiment, standard deviation is determined using the direct method (i.e., the square root of [the sum the squares of the samples minus the mean, divided by the number of samples]). In additional or alternative embodiments, other statistical methods that are well known in the art can be used to determine whether there is a difference between the expression of a biomarker in the test sample or saliva test sample compared to a control sample. Such methods may include but are not limited to the following: Student t-test, Mann- Whitney U test, one-way analysis of variance (ANOVA), Kruskal-Wallis test, Limma test.

[0043] The expression "corresponds to the expression in the control sample" refers to the presence and / or amount is identical to that of a positive control sample; or closer to that of one or more positive control sample than to one or more negative control sample (or to predefined reference values representing the same). Preferably the presence and / or amount is within ±40% of that of the one or more control sample (or mean of the control samples), for example, within ±39%, ±38%, ±37%, ±36%, ±35%, ±34%, ±33%, ±32%, ±31%, ±30%, ±29%, ±28%, ±27%, ±26%, ±25%, ±24%, ±23%, ±22%, ±21%, ±20%, ± 19%, ±18%, ±17%, ±16%, ±15%, ± 14%, ±13%, ±12%, ±11%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.05% or within 0% of the one or more control sample (e.g., the positive control sample).

[0044] In an embodiment of the present invention, the difference in the expression in the test sample or saliva test sample is <5 standard deviation from the mean presence or amount in the control samples, for example, <4.5, <4, <3.5, <3, <2.5, <2, <1.5, <1.4, < 1.3, <1.2, <1.1, <1, <0.9, <0.8, <0.7, <0.6, <0.5, <0.4, <0.3, <0.2, <0.1 or 0 standard deviations from the from the mean presence or amount in the control samples, provided that the standard deviation ranges for differing and corresponding biomarker expressions do not overlap (e.g., abut, but no not overlap).

[0045] Alternatively, the phrase "corresponds to the expression in the control sample" refers to the presence or amount in the test sample or saliva test sample correlates with the amount in the control sample in a statistically significant manner. By "correlates with the amount in the control sample in a statistically significant manner" we mean or include that the presence or amount in the test sample or saliva test sample correlates with the that of the control sample with a p-value of <0.05, for example, <0.04, <0.03, <0.02, <0.01, <0.005, <0.004, <0.003, <0.002, <0.001, <0.0005 or <0.0001.

[0046] Suitably, an increase in the amount of C4B expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample.

[0047] Suitably, an increase in the amount of C6 expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample.

[0048] Suitably, an increase in the amount of C7 expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample.

[0049] Suitably, an increase in the amount of C9 expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample.

[0050] Suitably, an increase in the amount of CFB expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample.

[0051] Suitably, an increase in the amount of CFH expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample. Suitably, an increase in the amount of CP expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample.

[0052] Suitably, an increase in the amount of CSRP1 expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample.

[0053] Suitably, a decrease in the amount of DPMI expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the decrease is as compared to a negative control sample.

[0054] Suitably, an increase in the amount of EFEMP1 expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample.

[0055] Suitably, an increase in the amount of HINT2 expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample.

[0056] Suitably, an increase in the amount of IGKV3-15 expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample.

[0057] Suitably, an increase in the amount of LCN2 expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample.

[0058] Suitably, an increase in the amount of MUC1 expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample.

[0059] Suitably, an increase in the amount of MUC5AC expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample.

[0060] Suitably, an increase in the amount of RARRES1 expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample. Suitably, an increase in the amount of SCGB1D2 expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample.

[0061] Suitably, an increase in the amount of SERPINA1 expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample.

[0062] Suitably, an increase in the amount of SERPINA3 expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample.

[0063] Suitably, an increase in the amount of SERPING1 expression measured in step ii) is indicative of prostate cancer in the individual. Suitably, the increase is as compared to a negative control sample.

[0064] In an embodiment of the present invention, the method of the invention may further comprise or consist of the steps of: v) providing one or more (positive) control samples from an individual afflicted with cancer, such as prostate cancer; and vi) determining a biomarker signature of the control sample by measuring the expression in the control sample of the one or more biomarkers measured in step ii); wherein the cancer, such as prostate cancer, is identified in the event that the expression in the test sample or saliva test sample of the one or more biomarkers measured in step ii) corresponds to the expression in the control sample of the one or more biomarkers measured in step vi).

[0065] The term "diagnosis" as used herein has its normal meaning in the art and refers to determining the presence or absence of a disease state in an individual. For example, determining whether an individual is or is not suffering from cancer, such as prostate cancer.

[0066] The term "treatment" or "treating" as used herein, refers to therapeutic (curative) treatment including amelioration. Treatment also includes stopping the disease from developing or slowing further progression of the disease. For example, treatment may include preventing symptoms from worsening.

[0067] The term "prevention" as used herein, refers to prophylaxis treatment i.e., action taken to prevent disease.

[0068] The term "cancer" as used herein has its normal meaning in the art and refers to a disease in which abnormal cells undergo uncontrolled proliferation. These cells evade the regulatory mechanisms that normally govern cell growth, division, and programmed cell death, leading to the formation of tumours. Tumours may be benign or malignant, with malignant tumours possessing the capacity to invade adjacent tissues and metastasize to distant sites, thereby disrupting normal physiological functions and presenting significant health risks.

[0069] The term "prostate cancer" as used herein has its normal meaning in the art and refers to any type of cancer that forms in the tissues of the prostate. Prostate cancer usually forms in males and is one of the most common types of cancer.

[0070] The term "early prostate cancer" or "early-stage prostate cancer" has its normal meaning in the art and refers to prostate cancer comprising or consisting of Stage 0, Stage 1 and / or Stage 2 prostate cancers, for example as determined by the American Joint Committee on Cancer (AJCC) TNM or Gleason score system.

[0071] The term "prostate specific antigen" or "PSA" has its normal meaning in the art and refers to a protein produced by the prostate gland and found in the blood. PSA levels may be higher than normal in men who have prostate cancer, benign prostatic hyperplasia (BPH) or infection or inflammation of the prostate gland. A PSA score of less than 10 ng / ml generally may indicate the presence of slow growing Stage 1 cancer, while a PSA score of between 15 ng / ml and 20 ng / ml generally may indicate late stage cancer.

[0072] Suitably, the Gleason score system (a grading system for prostate cancer) may be used to determine the stage and properties of the cancer, by seeing how closely the cancer cells look like normal cells when viewed under a microscope. The scoring is from 2 to 10, wherein a score of 6 or less indicates that the cancer is of slow growth and not aggressive and higher scores than 6 indicate a faster growing cancer that is more likely to spread. Gleason scores may also be considered in combination with PSA, for example, if the prostate specific antigen (PSA) score is less than 10 ng / ml and the Gleason score is 6 or less, Stage 1 cancer is likely to grow slowly.

[0073] In the context of prostate cancer, term "adenocarcinoma" as used herein has its normal meaning in the art and refers to a type of prostate cancer that forms and develops in the gland cells that line the prostate (acinar adenocarcinoma) and the tubes of the prostate gland (ductal adenocarcinoma). Adenocarcinomas are the most common type of prostate cancer.

[0074] In the context of prostate cancer, the term "squamous cell carcinoma" as used herein has its normal meaning in the art and refers to a type of prostate cancer which develops from flat cells that cover the prostate. They are less common and tend to grow and spread more quickly than adenocarcinoma of the prostate. In the context of prostate cancer, the term "transitional cell carcinoma" as used herein has its normal meaning in the art and refers to a type of prostate cancer that starts in the cells that line the urethra, starting in the bladder and spreading into the prostate.

[0075] In the context of prostate cancer, the term "small cell prostate cancer" as used herein has its normal meaning in the art and refers to a type of prostate cancer that develops from neuroendocrine cells of the prostate. These types of cells do not make prostate specific antigen (PSA). Therefore, the level of PSA is often normal, or only slightly higher than normal. Small cell prostate cancers grow much quicker than other types of prostate cancer, wherein most patients with small cell prostate cancer have advanced cancer by the time they are diagnosed i.e., wherein the cancer has already spread to other parts of the body such as the bones.

[0076] In a preferred embodiment, the methods of the invention permit the diagnosis of prostate cancer and / or early prostate cancer.

[0077] In some embodiments of the present invention, the methods of the invention are able to diagnose prostate cancer at an early stage, as the methods can be carried out on a sample from an individual either when they are showing no symptoms (i.e., asymptomatic) or when they are displaying minor symptoms. Therefore, one advantage of the invention described herein is that it can allow diagnosis of prostate cancer at a much earlier stage than traditional diagnostic methods, allowing patients to seek prompt treatment that is key to long term survival.

[0078] In some embodiments of the present invention, the methods of the invention permit the diagnosis of prostate cancer, wherein the individual has adenocarcinoma of the prostate, transitional cell carcinoma of the prostate, squamous cell carcinoma of the prostate and / or small cell prostate cancer.

[0079] In some embodiments of the present invention, the prostate cancer to be diagnosed is early- stage prostate cancer, such as Stage 0, Stage 1 or Stage 2, or is advanced prostate cancer, such as Stage 3 or Stage 4.

[0080] The term "test sample" refers to a sample to be tested in the invention, such as a sample that had been taken or derived from an individual to be tested, wherein the sample comprises endogenous proteins, peptides and / or nucleic acid molecules.

[0081] Suitably, the test sample is a bodily fluid sample, preferably blood (i.e., unfractionated blood that can be separated into serum or plasma for testing) or saliva and most preferably, the test sample is a saliva sample. Saliva as a diagnostic fluid is particularly beneficial as it allows the use of minimally invasive and inexpensive procedures, which is largely independent of age (from children to elderly patients). Furthermore, the samples are easy to handle and store, and provide real-time diagnostic values. While nucleic acids have been used more frequently than proteins in the cancer diagnostics space, proteins provide a more dynamic and accurate readout of the presence and stage of cancer. However, identifying the biomarkers present in saliva is challenging due to several factors, such as low concentration of biomarkers in the saliva compared to other diagnostic fluids (such as blood); a complex composition of biomarkers and other proteins, enzymes, nucleic acids which can interfere with the detection of specific biomarkers; and environmental contaminants such as food particles, bacteria, and other external contaminants. As such, the present invention provides a particularly beneficial advantage over the prior art, as the identified biomarkers have been found to be reliably identifiable in saliva samples.

[0082] Preferably, the sample to be tested is provided from an individual that is a mammal, preferably a human, preferably a male human.

[0083] The term "positive control sample" refers to samples derived from an individual with confirmed prostate cancer or a pool of prostate cancer samples. In the case where the positive control is a pool of prostate cancer samples, the amount of the biomarker may be an average value of the amount of the biomarker measured in each of the prostate cancer samples.

[0084] It will be appreciated by persons skilled in the art that, in addition to measuring the biomarkers in a sample from an individual to be tested, the methods of the invention may also comprise measuring those same biomarkers in one or more control samples.

[0085] Differential expression (up-regulation or down-regulation) of biomarkers, or lack thereof, can be determined by any suitable means known to a skilled person. Differential expression is determined to a p value of a least less than 0.05 (p = < 0.05), for example, at least <0.04, <0.03, <0.02, <0.01, <0.009, <0.005, <0.001, <0.0001, <0.00001 or at least <0.000001.

[0086] It will be appreciated by persons skilled in the art that differential expression may relate to a single biomarker or to multiple biomarkers considered in combination (i.e., as a biomarker signature). Thus, a p-value may be associated with a single biomarker or with a group of biomarkers. Indeed, proteins having a differential expression p-value of greater than 0.05 when considered individually may nevertheless still be useful as biomarkers in accordance with the invention when their expression levels are considered in combination with one or more other biomarkers. Therefore, in some embodiments the classification of step ii) may be achieved by comparing the expression of biomarkers in the test sample or saliva test sample to those in the one or more positive and / or negative control sample(s).

[0087] In an embodiment of the present invention, the presence and / or amount in the test sample or saliva test sample of the one or more biomarkers measured in step ii) may be compared against predetermined reference values representative of the measurements in steps iv) and / or vi), i.e., reference negative and / or positive control values.

[0088] As detailed above, the methods of the invention may also comprise measuring, in one or more negative or positive control samples, the presence and / or amount of the one or more biomarkers measured in the test sample or saliva test sample in step ii).

[0089] In a preferred embodiment of the first aspect of the invention, the method is repeated on the individual. Thus, steps i) and ii) may be repeated using a sample from the same individual taken at different time to the original sample tested (or the previous method repetition). Such repeated testing may enable disease progression to be assessed, for example to determine the efficacy of a selected treatment regime and (if appropriate) to select an alternative regime to be adopted.

[0090] Thus, in an embodiment of the present invention, the method is repeated using a test sample or saliva test sample taken between 1 day to 100 weeks to the previous test sample(s) used, for example, between 1 week to 100 weeks, 1 week to 90 weeks, 1 week to 80 weeks, 1 week to 70 weeks, 1 week to 60 weeks, 1 week to 50 weeks, 1 week to 40 weeks, 1 week to 30 weeks, 1 week to 20 weeks, 1 week to 10 weeks, 1 week to 9 weeks, 1 week to 8 weeks, 1 week to 7 weeks, 1 week to 6 weeks, 1 week to 5 weeks, 1 week to 4 weeks, 1 week to 3 weeks, or 1 week to 2 weeks.

[0091] In an embodiment of the present invention, the method may be repeated using a test sample or saliva test sample taken every period from the group consisting of: 1 day, 2 days, 3 day, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 35 weeks, 40 weeks, 45 weeks, 50 weeks, 55 weeks, 60 weeks, 65 weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104, weeks, 105 weeks, 110 weeks, 115 weeks, 120 weeks, 125 weeks and 130 weeks.

[0092] In an embodiment of the present invention, the method may be repeated at least once, for example, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times or 25 times. In an embodiment of the present invention, the method is repeated continuously.

[0093] In an embodiment of the present invention, the method is repeated during therapy, immediately following the completion of therapy, and twice per year for the first five years that the patients are in remission.

[0094] In an embodiment of the present invention, the method is repeated until cancer, such as prostate cancer, is diagnosed in the individual using the methods of the present invention and / or conventional clinical methods (i.e., until confirmation of the diagnosis is made).

[0095] As exemplified in the accompanying Example, the profiles of certain genes in a test sample or saliva test sample may be indicative of cancer, particularly prostate cancer, in an individual. For example, the relative expression of certain genetic biomarkers in a single test sample or saliva test sample may be indicative of the presence of cancer, particularly prostate cancer, in an individual.

[0096] In some embodiments, steps ii), iv) and / or step vi) is performed using one or more first binding agents capable of binding to a biomarker.

[0097] In some embodiments of the method of the invention, steps ii), iv) and / or vi) comprises measuring the expression of a nucleic acid, protein and / or peptide molecule encoding the one or more biomarkers. Preferably, steps ii), iv), and / or vi) comprises measuring the expression of a protein molecule encoding the one or more biomarkers.

[0098] The nucleic acid molecule may be a gene expression intermediate or derivative thereof, such as an mRNA molecule or a cDNA molecule. In some preferred embodiments, the nucleic acid molecule is an mRNA molecule. By "mRNA molecule" we include pre-mRNA and mature mRNA.

[0099] In one embodiment, steps ii), iv) and / or vi) is performed using one or more binding agents, each individually capable of binding selectively to a nucleic acid, protein and / or peptide molecule encoding one of the biomarkers.

[0100] Conveniently, the one or more binding moieties may each comprise or consist of a nucleic acid molecule, such as DNA, RNA, peptide nucleic acid (PNA), locked nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA) or phosphorodiamidate morpholino oligomer (PMO).

[0101] Advantageously, the one or more binding moieties may be 5 to 100 nucleotides in length. For example, they may be 15 to 35 nucleotides in length. It will be appreciated that the nucleic acid-based binding moieties may comprise a detectable moiety. The detectable moiety may be selected from the group consisting of: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety (for example, a radioactive atom); or an enzymatic moiety.

[0102] In an embodiment of the present invention, the detectable moiety may comprise or consist of a radioactive atom, for example selected from the group consisting of technetium-99m, iodine- 123, iodine-125, iodine-131, indium-ill, fluorine-19, carbon-13, nitrogen-15, oxygen-17, phosphorus-32, sulphur-35, deuterium, tritium, rhenium-186, rhenium-188 and yttrium-90.

[0103] In an embodiment of the present invention, the detectable moiety of the binding moiety may be a fluorescent moiety.

[0104] In some embodiments of the present invention, measuring the expression of the one or more biomarkers in steps ii), iv) and / or vi) may be performed using a method selected from the group consisting of Southern hybridisation, Northern hybridisation, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative reverse transcriptase PCR (RT-qPCR), nanoarray, microarray, autoradiography, in situ hybridisation, mass spectrometry and / or western blotting. In addition, measurement of mRNA may be useful for particular sample types that are more difficult to extract proteins from for analysis.

[0105] Preferably, the expression of the biomarkers in the test sample is determined using RT-PCR, mass spectrometry and / or western blotting. In some embodiments, the expression of the biomarkers in the test sample or saliva test sample is determined using mass spectrometry.

[0106] In some embodiments, when nucleic acid biomarkers are detected, measurement of the nucleic acid is carried out using another transcriptomics-based technique. These include techniques generally known in the art for detecting nucleic acids (e.g., mRNA) in a sample. They may include, but are not limited to, the following:

[0107] Chromogenic in situ hybridization (CISH);

[0108] Fluorescence in situ hybridization (FISH);

[0109] RNA sequencing;

[0110] RNA microarrays;

[0111] Digital RT-PCR;

[0112] Northern blot;

[0113] Digital colour-coded nucleic acid barcode (nCounter®) technology;

[0114] - RT-PCR-ELISA. In an embodiment of the present invention, expression of the one or more biomarker(s) is determined using an RIMA or DNA microarray.

[0115] In an embodiment of the methods of the invention, step i) comprises providing a sample from an individual to be tested and step ii) comprises measuring in the sample the expression of the protein, peptide and / or polypeptide of the one or more biomarker(s). Thus, a biomarker signature for the sample may be determined at the protein level.

[0116] Therefore, in an embodiment of the present invention, steps ii), iv), and / or vi) comprises measuring the expression of the protein, peptide and / or polypeptide of the one or more biomarkers.

[0117] In one preferred embodiment, steps ii), iv) and / or vi) is performed using one or more first binding agent capable of binding to a protein, peptide and / or polypeptide biomarker.

[0118] Suitable binding agents (also referred to as binding molecules) can be selected from a library, based on their ability to bind a given target molecule, as discussed below.

[0119] In a preferred embodiment, at least one type of the binding agents, and more typically all of the types, may comprise or consist of an antibody or antigen-binding fragment of the same, or a variant thereof.

[0120] Methods for the production and use of antibodies are well known in the art, for example see Antibodies: A Laboratory Manual, 1988, Harlow & Lane, Cold Spring Harbor Press, ISBN-13: 978-0879693145, Using Antibodies: A Laboratory Manual, 1998, Harlow & Lane, Cold Spring Harbor Press, ISBN-13: 978-0879695446 and Making and Using Antibodies: A Practical Handbook, 2006, Howard & Kaser, CRC Press, ISBN-13: 978-0849335280 (the disclosures of which are incorporated herein by reference).

[0121] Thus, a fragment may contain one or more of the variable heavy (VH) or variable light (VL) domains. For example, the term antibody fragment includes Fab-like molecules (Better et al., (1988) Science 240, 1041); Fv molecules (Skerra et al., (1988) Science 240, 1038); singlechain Fv (scFv) molecules where the VH and VL partner domains are linked via a flexible oligopeptide (Bird et al., (1988) Science 242, 423; and single domain antibodies (dAbs) comprising isolated V domains (Ward et al., (1989) Nature 341, 544).

[0122] For example, the binding agent(s) may be whole antibodies or scFv molecules.

[0123] The term "antibody variant" includes any synthetic antibodies, recombinant antibodies or antibody hybrids, such as but not limited to, a single-chain antibody molecule produced by phage-display of immunoglobulin light and / or heavy chain variable and / or constant regions, or other immuno-interactive molecule capable of binding to an antigen in an immunoassay format that is known to those skilled in the art.

[0124] A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter & Milstein (1991) Nature 349, 293-299.

[0125] Molecular libraries such as antibody libraries (Clackson et al, 1991, Nature 352, 624-628; Marks et al., 1991, J Mol Biol 222(3): 581-97), peptide libraries (Smith, 1985, Science 228(4705): 1315-7), expressed cDNA libraries (Santi et al., (2000) J Mol Biol 296(2): 497-508), libraries on other scaffolds than the antibody framework such as affibodies (Gunneriusson et al., 1999, Appl Environ Microbiol 65(9): 4134-40) or libraries based on aptamers (Kenan et al., 1999, Methods Mol Biol 118, 217-31) may be used as a source from which binding molecules that are specific for a given motif are selected for use in the methods of the invention.

[0126] Conveniently, the binding agent(s) may be immobilised on a surface (e.g., on a multiwell plate or array).

[0127] In an embodiment of the methods of the invention, determining the expression of the protein, peptide and / or polypeptide biomarkers is performed using an assay comprising a second binding agent capable of binding to the one or more biomarkers, the second binding agent comprising a detectable moiety. For example, an immobilised (first) binding agent may initially be used to 'trap' the protein biomarker on to the surface of a microarray, and then a second binding agent may be used to detect the 'trapped' protein.

[0128] The second binding agent may be as described above in relation to the (first) binding agent, such as an antibody or antigen-binding fragment thereof.

[0129] It will be appreciated by the skilled person that the one or more biomarkers (e.g., proteins) in the test sample or saliva test sample may be labelled with a detectable moiety, prior to performing step ii). Likewise, the one or more biomarkers in the control sample(s) may be labelled with a detectable moiety.

[0130] In an embodiment of the present invention, the first and / or second binding agents may be labelled with a detectable moiety.

[0131] In an embodiment of the present invention, the one or more biomarkers in the test sample, saliva test sample and / or control sample(s) are labelled either directly or indirectly with a detectable moiety. The term "directly labelled" refers to the biomarkers that are bound by a binding agent that is itself or is coupled to a detectable moiety (e.g., as used during a direct ELISA). By "indirectly labelled" we include that the biomarkers are bound by a first binding agent that is not directly labelled, and a second binding agent is used that is itself, or is coupled to, a detectable moiety (e.g., as used during an indirect ELISA).

[0132] Suitable detectable moieties are well known in the art. For example, the detectable moiety may be selected from the group comprising : a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety; an enzymatic moiety.

[0133] Thus, the detectable moiety may be a fluorescent and / or luminescent and / or chemiluminescent moiety which, when exposed to specific conditions, may be detected. For example, a fluorescent moiety may need to be exposed to radiation (i.e., light) at a specific wavelength and intensity to cause excitation of the fluorescent moiety, thereby enabling it to emit detectable fluorescence at a specific wavelength that may be detected.

[0134] In an embodiment of the present invention, the detectable moiety may be an enzyme which is capable of converting a (preferably undetectable) substrate into a detectable product that can be visualised and / or detected. Examples of suitable enzymes are discussed in more detail below in relation to, for example, ELISA assays.

[0135] In an embodiment of the present invention, the detectable moiety may be a radioactive atom which is useful in imaging. Suitable radioactive atoms include99mTc and123I for scintigraphic studies. Other readily detectable moieties include, for example, spin labels for magnetic resonance imaging (MRI) such as123I again,131I,niIn,19F,13C,15N,17O, gadolinium, manganese or iron. Clearly, the agent to be detected (such as, for example, the one or more biomarkers in the test sample and / or control sample described herein and / or an antibody molecule for use in detecting a selected protein) must have sufficient of the appropriate atomic isotopes in order for the detectable moiety to be readily detectable.

[0136] Preferred assays for detecting proteins include immunohistochemistry (IHC), enzyme linked immunosorbent assays (ELISA), radioimmunoassay (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and / or polyclonal antibodies. Exemplary sandwich assays are described by David et al., in US Patent Nos. 4,376,110 and 4,486,530, hereby incorporated by reference.

[0137] In some embodiments, the assay is an ELISA (Enzyme Linked Immunosorbent Assay) which typically involves the use of enzymes giving a coloured reaction product, usually in solid phase assays. Enzymes such as horseradish peroxidase and phosphatase have been widely employed. A way of amplifying the phosphatase reaction is to use NADP as a substrate to generate NAD which now acts as a coenzyme for a second enzyme system. Pyrophosphatase from Escherichia coli provides a good conjugate because the enzyme is not present in tissues, is stable and gives a good reaction colour. Chemiluminescent systems based on enzymes such as luciferase can also be used.

[0138] ELISA methods are well known in the art, for example see The ELISA Guidebook (Methods in Molecular Biology), 2000, Crowther, Humana Press, ISBN-13: 978-0896037281 (the disclosures of which are incorporated by reference).

[0139] In one embodiment, the detectable moiety is fluorescent moiety (for example an Alexa Fluor dye, e.g., Alexa647).

[0140] In some embodiments, the individual is selected from the group comprising: a primate (for example, a human; a monkey; an ape); a rodent (for example, a mouse, a rat, a hamster, a guinea pig, a gerbil, a rabbit); a canine (for example, a dog); a feline (for example, a cat); an equine (for example, a horse); a bovine (for example, a cow); or a porcine (for example, a pig).

[0141] In a preferred embodiment, the individual is a human. In a further preferred embodiment, the individual is a human who is suspected of having cancer, preferably prostate cancer.

[0142] In some embodiments, the test sample is selected from the group comprising: a biopsy, preferably a core needle biopsy, fine needle biopsy or bronchoscopy sample; a tissue sample; an organ sample; and a bodily fluid sample, preferably a blood, saliva or pleural fluid sample. It will be appreciated that the test sample, saliva test sample and / or any control samples should be from the same species.

[0143] Suitably, the tissue sample is a prostate tissue sample.

[0144] In some embodiments, the biopsy can be analysed using the methods of the present invention either with or without purification of cells from the biopsy sample. The test sample can be taken specifically for the purpose of performing the methods of the present invention, or, in alternative embodiments, the methods of the invention can be carried out on historical samples that have been appropriately stored.

[0145] In some embodiments, the test sample is a prostate tissue sample. In an alternative or additional embodiment, the test sample is a sample comprising or consisting of prostate cells, for example epithelial cells or stromal cells. Preferably, the test sample comprises one or more cancer cells, preferably prostate cancer cells. The methods of this invention are suitable for testing a sample from any individual who has, or is suspected of having, cancer, such as prostate cancer. For example, the individual may be from one of the following groups:

[0146] Individuals with previously diagnosed cancer (of any type or stage);

[0147] Individuals with suspected cancer;

[0148] Individuals with normal, healthy and health-conscious individuals with a family history of cancers, such as prostate cancer;

[0149] Individuals over the age of 50 who are at higher risk of developing cancers, such as prostate cancer;

[0150] Individuals with symptoms suggestive of or consistent with cancer (e.g., trouble urinating, decreased force in the stream of urine, blood in the urine, blood in the semen, bone pain, erectile dysfunction, unintentional or unexplained weight loss, pain in passing urine, fatigue).

[0151] In an embodiment of the present invention, the expression of the biomarkers in the test sample, saliva test sample and / or control samples is determined using mass spectrometry, an affinitybased method, a transcriptomics-based method, ELISA or flow cytometry.

[0152] In a preferred embodiment, the expression of the biomarkers in the test sample, saliva test sample and / or control samples is determined by mass spectrometry.

[0153] Arrays perse are well known in the art. Typically, they are formed of a linear or two-dimensional structure having spaced apart (i.e., discrete) regions ("spots"), each having a finite area, formed on the surface of a solid support. An array can also be a bead structure where each bead can be identified by a molecular code or colour code or identified in a continuous flow. Analysis can also be performed sequentially where the sample is passed over a series of spots each adsorbing the class of molecules from the solution. The solid support is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs, silicon chips, microplates, polyvinylidene difluoride (PVDF) membrane, nitrocellulose membrane, nylon membrane, other porous membrane, non-porous membrane (e.g. plastic, polymer, perspex, silicon, amongst others), a plurality of polymeric pins, or a plurality of microtitre wells, or any other surface suitable for immobilising proteins, polynucleotides and other suitable molecules and / or conducting an immunoassay. The binding processes are well known in the art and generally consist of cross-linking covalently binding or physically adsorbing a protein molecule, polynucleotide or the like to the solid support. By using well-known techniques, such as contact or non-contact printing, masking or photolithography, the location of each spot can be defined. For reviews see Jenkins, R.E., Pennington, S.R. (2001, Proteomics, 2,13-29) and Lal etal., (2002, Drug Discov Today 15;7(18 Suppl):S143-9). In some embodiments, the affinity-based method is an array. Once suitable binding molecules (discussed above) have been identified and isolated, the skilled person can manufacture an array using methods well known in the art of molecular biology.

[0154] In one preferred embodiment, an array is provided for diagnosing or determining the presence of cancer in an individual comprising an agent or agents for detecting the presence and / or amount of one or more of the biomarkers selected from C4B, C6, C7, C9, CFB, CFH, CP, CSRP1, DPMI, EFEMP1, HINT2, IGKV3-15, LCN2, MUC1, MUC5AC, RARRES1, SCGB1D2, SERPINA1, SERPINA3, and SERPING1. Preferably, the cancer is prostate cancer. This embodiment of the invention may have any of the preferred features described above.

[0155] Suitably, the array is configured for use with saliva test samples.

[0156] Suitably, the array is a lateral flow device or plate-based assay, such as an antibody plate-based assay. Preferably, the array is an antibody plate-based assay or lateral flow device. Lateral flow devices are particularly advantageous as they allow individuals to undertake the testing at home. Currently available home test kits for the diagnosis of cancers either require blood or stool testing and are 'mail-in' tests that must be sent to specific labs. Therefore, such lateral flow devices allowing individuals to do the test at home are not currently available on the market.

[0157] In some embodiments of the present invention, the lateral flow device comprises a test sample receiving region, a capture membrane test region comprising an immobilized detectable moiety and an indicator conjugate.

[0158] In some embodiments of the present invention, the lateral flow device further comprises a buffer solution in which the test sample moves through the regions through capillary action, wherein if test sample binds to the detectable moiety, a colour change will occur.

[0159] In further embodiments, the lateral flow device comprises a reservoir region positioned downstream of the capture membrane for absorbing an excess of fluid.

[0160] Typically, the array is a microarray. By "microarray" we include the meaning of an array of regions having a density of discrete regions of at least about 100 / cm2, and preferably at least about 1000 / cm2. The regions in a microarray have typical dimensions, e.g., diameters, in the range of between about 10-250 pm, and are separated from other regions in the array by about the same distance. The array may also be a nanoarray.

[0161] Accordingly, in some embodiments, the lateral flow device comprises conjugate-based gold and silver nanoparticles which are highly sensitive compared to other nanoparticles. Gold nanoparticles (AuNPs) possess distinct physical and chemical attributes that make them excellent scaffolds for the fabrication of novel chemical and biological sensors. AuNPs offer a suitable platform for multi-functionalization with a wide range of organic or biological ligands for the selective binding and detection of small molecules and biological targets. Accordingly, in a preferred embodiment, the lateral flow device comprises conjugate-based gold nanoparticles for the effective detection of multiple proteins on the same test.

[0162] Once suitable binding molecules (as discussed elsewhere herein) have been identified and isolated, the skilled person can manufacture an array using methods well known in the art of molecular biology.

[0163] In an embodiment of the present invention, the present invention includes wherein in the event that the individual is diagnosed with cancer, such as prostate cancer, the method or use further comprises a step of providing the individual with a cancer therapy, such as prostate cancer therapy.

[0164] Thus, a related aspect of the invention provides a method of treating cancer, such as prostate cancer, in an individual comprising the steps of:

[0165] (i) diagnosing cancer, such as prostate cancer, according to the method described in any of the aspects of the invention described elsewhere herein; and

[0166] (ii) providing the individual with cancer therapy, such as prostate cancer therapy.

[0167] The cancer therapy may be selected from the group comprising: surgery, chemotherapy, radiotherapy, immunotherapy, chemoimmunotherapy, and combinations thereof, adoptive cell therapies, gene therapies, cancer vaccines, and oncolytic virus therapies. The skilled person will be aware of the most appropriate treatments in order to provide an individual with cancer treatment.

[0168] A further aspect of the invention provides a kit for diagnosing or determining the presence of cancer, such as prostate cancer, the kit comprising:

[0169] (i) an array according to the invention elsewhere described herein, or components for making the same; and

[0170] (ii) instructions for performing the method as elsewhere herein (e.g., in any of the aspects of the invention described herein).

[0171] A further aspect of the invention provides a use of one or more binding agents to a biomarker as described herein in the preparation of a kit for diagnosing or determining a cancer-associated disease state in an individual. Thus, multiple different binding agents may be used, each targeted to a different biomarker, in the preparation of such as kit. In one embodiment, the binding agent is an antibody or antigen-binding fragment thereof (e.g., scFv) or a nucleic acid binding molecule, as described herein.

[0172] In some embodiments, the invention provides the use of one or more biomarkers selected from the group comprising C4B, C6, C7, C9, CFB, CFH, CP, CSRP1, DPMI, EFEMP1, HINT2, IGKV3-15, LCN2, MUC1, MUC5AC, RARRES1, SCGB1D2, SERPINA1, SERPINA3, and SERPING1 for diagnosing or determining the presence of cancer in an individual. Preferably, the cancer is prostate cancer. This embodiment of the invention may have any of the preferred features described above and elsewhere herein.

[0173] The following examples illustrate the invention.

[0174] EXAMPLES

[0175] Abbreviations

[0176] Abbreviations as used herein will be known to those skilled in the art. In particular, the following abbreviations may be used herein:

[0177] Example 1 - Proteome Expression in Prostate Cancer Patients

[0178] Saliva Collection

[0179] Prior to sample collection:

[0180] • any consumption of nicotine and prescription / over-the-counter medications within the prior 12 hours was documented;

[0181] • vigorous physical activity and the presence of oral diseases or injury was documented;

[0182] • food and fluid (apart from water) ingestion or chewing gum for at least 2 hours before collection was avoided;

[0183] • brushing teeth 2 hours before collection was avoided; and

[0184] • mouths were rinsed with water to remove food residue and saliva was collected at least 10 minutes after the rinsing to avoid sample dilution. Materials:

[0185] 1. Saliva Collection Aid (Salimetrics, Item No. 5016.04 or 5016.04B)

[0186] 2. Cryovials (Salimetrics, Item No. 5004.01-06)

[0187] 3. Bar-Coded labels (Salimetrics, Item No. 5009.07). Clear and comprehensible labeling was necessary for proper sample identification and handling. Permanent markers or bar- coded labels were used for long-term storage.

[0188] 4. Cryostorage box (Salimetrics, Item No. 5023.05)

[0189] 5. An enzyme inhibitor cocktail consisting of pefabloc, leupeptin, and aprotinin (Roche Diagnostics, Mannheim, Germany) at 25, 0.5, and 0.25 mg, respectively, in 5 mL of PBS with 50 mg of bovine serum albumin and 20 mg of EDTA was to prevent protein degradation in sample.

[0190] Sample Collection:

[0191] 15 early-stage to late stage prostate cancer subjects and 15 healthy controls were provided for the following sample collection. All 30 subjects mimicked chewing on food by slowly moving the jaws in a chewing motion and allowing oral fluid to pool in the mouth.

[0192] The specimen was gently forced through a short plastic drinking straw into a vial. Up to 500 pl of saliva was collected into the tube (the typical volume of 1 donation is 200-500 pl). To avoid problems with analyte retention or the introduction of contaminants, validated polypropylene vials were used for collection (SalivaBio 2 mL cryovials, Salimetrics Item No. 5004.01). Vials were sealed tightly, to be able to withstand temperatures down to -80°C and were externally threaded to allow for use of the Saliva Collection Aid (SCA - Salimetrics Item No. 5016.02) to effectively guide drool directly into the cryovial. 1 mL cold PBS with protease inhibitors (as mentioned above) was added and mixed by vortexing. The sample was kept cold and worked on ice. The sample was transferred to a 1.5 mL reaction tube. The sample was centrifuged at 1500 x g for 15 minutes.

[0193] If the samples appear viscous, they were centrifuged at a higher speed such as 2500 x g or the clot was broken up with a pipette tip and re-centrifuged. Assays were performed using only clear saliva, avoiding the pellet formed at the bottom of the tube. When pipetting viscous solutions such as saliva, greater accuracy was obtained by aspirating slowly to avoid the formation of bubbles. Samples were aliquoted after careful mixing (if needed), or immediately stored at -80 °C up to a year.

[0194] Cell Lysis (optional):

[0195] 100 pl IX RIPA lysis buffer (Sigma, cat #20-188, with protease inhibitors added as above) was added. The sample was incubated at 4 °C end-over-end for 30 minutes. The sample was centrifuged at full speed (14,000 rpm) for 10 min at 4°C using a tabletop centrifuge. The supernatant was removed, and usual sample storage steps were applied. Mass Spectrometry of Salivary Proteome

[0196] Protein Extraction: For PTM experiments, inhibitors were added to a lysis buffer (1% protease inhibitor cocktail, 1% phosphatase inhibitor for phosphorylation, 50 pM PR-619. 3 pM TSA and 50 mM NAM for acetylation). The remaining debris was removed by centrifugation at 12,000 g at 4°C for 10 min. Finally, the supernatant was collected, and the protein concentration was determined with BCA kit according to the manufacturer's instructions (ThermoFisher Scientific).

[0197] Trypsin Digestion: The protein sample was added with 1 volume of pre-cooled acetone, vortexed to mix, 4 volumes of pre-cooled acetone were added and precipitated at -20°C for 2 h. The precipitate was washed 2-3 times with the pre-cooled acetone. The protein sample was then redissolved in 200 mM TEAB and ultrasonically dispersed. Trypsin was added at 1 :50 trypsin- to-protein mass ratio for the first digestion overnight. The sample was reduced with 5 mM dithiothreitol for 30 min at 56°C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. Finally, the peptides were desalted by Strata X SPE column.

[0198] Mass Spectrometer: The tryptic peptides were dissolved in solvent A (0.1% formic acid in water), directly loaded onto a home-made reversed- phase analytical column (15-cm length, 100 pm i.d.). The mobile phase consisted of solvent A and solvent B (0.1% formic acid, 80% acetonitrile / in water). Peptides were separated with the following gradient: 0-1.6 min, 4%- 22.5%B; 1.6-2.0 min, 22.5%-35%B; 2.0-2.6 min, 35%-55%B; 2.6-2.7 min, 55%-99%B; 2.7- 6.8 min, 99%B; 6.8-7.6 min, 99%B, and all at a constant flow rate of 300 nl / min on a Vanquish Neo UPLC system (ThermoFisher Scientific). The separated peptides were analyzed in Orbitrap Astral with a nano-electrospray ion source. The electrospray voltage applied was 1900 V. Precursors were analyzed at the Orbitrap detector, and the fragments were analyzed at the Astral detector. The full MS scan resolution was set to 240000 for a scan range of 380-980 m / z]. The MS / MS scan was fixed first mass as 150.0 m / z] at a resolution of 80000. The HCD fragmentation was performed at a normalized collision energy (NCE) of 25%. Automatic gain control (AGC) target was set at 500%, with a maximum injection time of 3 ms.

[0199] Database Search: The DIA data were processed using DIA-NN search engine (v.1.8). Tandem mass spectra were searched against Homo_sapiens_9606_SP_20231220.fasta (20429 entries) concatenated with reverse decoy database. Trypsin / P was specified as cleavage enzyme allowing up to 1 missing cleavage. Excision on N-term Met and carbamidomethyl on cysteine (Cys) were specified as fixed modification. FDR was adjusted to < 1%.

[0200] Analysis: Peptides with less than 20% coverage and with missing values in more than 50% of the samples in each group (prostate cancer and healthy control) were filtered out to ensure the robustness of the proposed biomarkers. Missing values were imputed using single imputation for principal component analysis to visually detect outliers. Imputed values were not used for differential expression analysis.

[0201] The expression was Iog2-transformed and compared between cancer and normal values using limma package with default settings. P-values of <0.05 were considered particularly significant and Benjamini-Hochberg correction for multiple comparisons were used. All tests were two- sided. Multivariable logistic regression was used to build a model differentiating cancer from normal samples using the best proteins selected from the differential analysis. 95% confidence intervals for the area under the ROC curve was computes using 2000 stratified bootstrap replicates, as implemented in R package pROC.

[0202] Adjusted p-values were used to account for the increased risk of false positives when multiple tests were performed. Adjusted p values are typically used in bioinformatics analyses where there are a lot of features (genes, proteins) compared.

[0203] Results

[0204] The differential expression analysis identified 20 genes that were differentially expressed in the saliva samples of 15 prostate cancer patients when compared to those of the 15 healthy controls as shown by the adjusted p-values of <0.16 for all 20 genes. It was, therefore, determined that these 20 proteins would be useful for the detection and diagnosis of prostate cancer in individuals and in particular, by measuring the expression of these proteins in the saliva of patients. As shown in Table 1 below, 8 proteins in the saliva samples showed particularly significant differential expression in the prostate cancer saliva samples with adjusted p-values of <0.05.

[0205] Table 1. Differential Expression Analysis - Prostate Cancer vs Healthy Control

[0206] A: Adj. P-value <0.05; B: Adj. P-value 0.05-0.16

[0207] Figure 1 shows the logistic regression of prostate cancer patients vs healthy controls based on a single protein from Table 1 with an adjusted p-value of <0.05. Figure 2 shows the logistic regression of the 15 prostate patients vs 15 healthy controls based on three of the proteins from Table 1 with adjusted p-values of <0.05. Area under the ROC curve (AUC) represents the accuracy of salivary biomarkers identified in the mass spectrometry analysis of 15 prostate cancer patients vs 15 healthy controls.

[0208] The ROC curve of Figure 1 shows an ROC score of 0.9 with a 95% confidence interval based on one of the identified proteins with an adj. p-value of <0.05, and the ROC curve of Figure 2 shows an improved ROC score of 0.92 with a 95% confidence interval based on three of the identified proteins with adj. p-values of <0.05. In comparison, the gold standard biomarker for screening prostate cancer, PSA, has at best, an ROC score of 0.73. The inventors have, therefore, identified a unique panel of biomarkers in human saliva, that may be used individually, or in combinations, for the detection and diagnosis of prostate cancer, whereby even a single identified biomarker provides more accurate and reliable results than the current gold standard methodology for diagnosing prostate cancer in patients.

[0209] Upregulation of biomarkers in prostate cancer tissue

[0210] The inventors have analysed the RNA expression profile of the biomarkers described above in prostate cancer tissue compared to health tissue. Box-and-whisker graphs in Figure 3 were generated using GEPIA2 and demonstrate the genes identified as upregulated in saliva at the protein level are also upregulated at the RNA level in prostate cancer tissue compared to normal prostate tissue, based on publicly available TCGA and GTEx datasets. This provides further evidence that the biomarkers identified are elevated in prostate cancer.

[0211] CLAUSES

[0212] Particular embodiments of the invention are illustrated by clauses below. 1. A method for diagnosing or determining the presence of prostate cancer in an individual, the method comprising the steps of: i) providing a saliva test sample from the individual; and ii) measuring the expression, in the test sample, of one or more biomarkers selected from: C4B, C6, C7, C9, CFB, CFH, CP, CSRP1, DPMI, EFEMP1, HINT2, IGKV3-15, LCN2, MUC1, MUC5AC, RARRES1, SCGB1D2, SERPINA1, SERPINA3, and SERPING1. wherein the expression in the saliva test sample of the one or more biomarkers is indicative of prostate cancer in the individual.

[0213] 2. A method for diagnosing or determining the presence of cancer in an individual, the method comprising the steps of: of: i) providing a test sample from the individual; and ii) measuring the expression, in the test sample, of one or more biomarkers selected from: CFH, DPMI, MUC5AC and IGKV3-15, wherein the expression in the test sample of the one or more biomarkers is indicative of cancer in the individual.

[0214] 3. The method according to clause 2, wherein the one or more biomarkers is MUC5AC and / or IGKV3-15.

[0215] 4. A method for diagnosing or determining the presence of prostate cancer in an individual, the method comprising the steps of: i) providing a test sample from the individual; and ii) measuring the expression, in the test sample, of one or more biomarkers selected from: C4B, C6, C9, CFB, CFH, DPMI, HINT2, IGKV3-15, and MUC5AC. wherein the expression in the test sample of the one or more biomarkers is indicative of prostate cancer in the individual.

[0216] 5. A method for diagnosing or determining the presence of a disease (preferably cancer) in an individual, the method comprising the steps of: i) providing a saliva test sample from the individual; and ii) measuring the expression, in the saliva test sample, of one or more biomarkers selected from: CFB, CSRP1, HINT2, IGKV3-15, and RARRES1. wherein the expression, in the saliva test sample, of the one or more biomarkers is indicative of a disease in the individual.

[0217] 6. A method for diagnosing or determining the presence of cancer in an individual, the method comprising the steps of: i) providing a saliva test sample from the individual; and ii) measuring the expression, in the saliva test sample, of one or more biomarkers selected from: C4B, C6, C7, C9, CFH, CP, EFEMP1, MUC5AC, SCGB1D1, SERPINA1, SERPINA3, SERPING1, wherein the expression, in the saliva test sample, of the one or more biomarkers is indicative of a cancer in the individual.

[0218] 7. The method according to any one of the preceding clauses, wherein the one or more biomarkers is two or more biomarkers.

[0219] 8. The method according to any one of the preceding clauses, wherein the one or more biomarkers is three or more biomarkers, most preferably four or more biomarkers.

[0220] 9. The method according to clause 1 or clause 6, wherein the one or more biomarkers is selected from: C4B, C7, C9, EFEMP1, SERPINA1, and SERPINA3, preferably wherein two or more biomarkers, most preferably wherein three or more biomarkers are selected from: C4B, C7, C9, EFEMP1, SERPINA1, and SERPINA3.

[0221] 10. The method according to any one of the preceding clauses, wherein the method is for diagnosis of prostate cancer, preferably early prostate cancer.

[0222] 11. The method according to any one of the preceding clauses, wherein the method is for diagnosis of prostate cancer and wherein the individual has adenocarcinoma of the prostate, transitional cell carcinoma of the prostate, squamous cell carcinoma of the prostate and / or small cell prostate cancer.

[0223] 12. The method according to clause 11, wherein the prostate cancer in the individual is early- stage prostate cancer, such as Stage 0, Stage 1 or Stage 2, or is advanced prostate cancer, such as Stage 3 or Stage 4.

[0224] 13. The method according to any one of the preceding clauses, further comprising the steps of: iii) providing one or more (negative) control samples from an individual not afflicted with cancer; and iv) determining a biomarker signature of the one or more control samples by measuring the expression in the control sample of the one or more biomarkers measured in step ii), wherein the cancer is identified in the event that the expression in the saliva test sample or test sample of the one or more biomarkers measured in step ii) is different from the expression in the control sample of the one or more biomarkers measured in step iv). 14. The method according to any one of the preceding clauses, wherein step ii) comprises measuring the expression of a nucleic acid, protein and / or peptide molecule encoding the one or more biomarkers, preferably step ii) comprises measuring the expression of a protein and / or peptide molecule encoding the one or more biomarkers.

[0225] 15. The method according to clause 14, wherein the nucleic acid molecule is an mRNA molecule or a cDNA molecule.

[0226] 16. The method according to any one of the preceding clauses, wherein step ii) is performed using RT-qPCR, mass spectrometry and / or western blotting, preferably mass spectrometry.

[0227] 17. The method according to any one of the preceding clauses, wherein the test sample comprises one or more prostate cancer cells and is optionally selected from the group comprising : a biopsy (such as a core needle biopsy; fine needle biopsy; bronchoscopy sample); a tissue sample; an organ sample; and a bodily fluid sample (such as blood, saliva or pleural fluid).

[0228] 18. The method according to any one of the preceding clauses, wherein the expression of the biomarkers in the saliva test sample or test sample is determined using mass spectrometry, an affinity-based method, a transcriptomics-based method, ELISA or flow cytometry.

[0229] 19. An array comprising an agent or agents for detecting the expression of one or more of the biomarkers selected from: C4B, C6, C7, C9, CFB, CFH, CP, CSRP1, DPMI, EFEMP1, HINT2, IGKV3-15, LCN2, MUC1, MUC5AC, RARRES1, SCGB1D2, SERPINA1, SERPINA3, and SERPING1.

[0230] 20. Use of one or more biomarkers selected from the group comprising C4B, C6, C7, C9, CFB, CFH, CP, CSRP1, DPMI, EFEMP1, HINT2, IGKV3-15, LCN2, MUC1, MUC5AC, RARRES1, SCGB1D2, SERPINA1, SERPINA3, and SERPING1 for diagnosing or determining the presence of cancer in an individual.

[0231] 21. The array or use according to clause 19 or 20 for use in diagnosing or determining the presence of cancer in an individual, preferably wherein the cancer is prostate cancer.

[0232] 22. The array or use according to any one of clauses 19 to 21 wherein the one or more biomarkers are selected from: C4B, C7, C9, EFEMP1, SERPINA1, and SERPINA3.

[0233] 23. The array or use according to any one of clauses 19 to 22 wherein the one or more biomarkers are selected from those listed in any one of clauses 1 to 6 or 9. 24. The array or use according to any one of clauses 19 to 23 wherein the one or more biomarkers are two or more biomarkers, preferably three or more biomarkers.

[0234] 25. The array according to any one of clauses 19 to 24 wherein the array is a lateral flow device or plate-based assay, such as an antibody plate-based assay.

[0235] 26. The array according to any one of clauses 19 to 25 wherein the array is configured for use with saliva test samples and / or is for use with saliva test samples.

[0236] 27. The method according to any one of clauses 1 to 18 or use according to clauses 20 to 24, wherein in the event that the individual is diagnosed with prostate cancer, the method further comprises a step of providing the individual with a prostate cancer therapy.

[0237] 28. The method or use according to clause 27, wherein the prostate cancer therapy is selected from the group comprising: surgery, chemotherapy, radiotherapy, immunotherapy, chemoimmunotherapy, thermochemotherapy and combinations thereof, adoptive cell therapies, gene therapies, cancer vaccines, and oncolytic virus therapies.

[0238] 29. A kit, optionally wherein the kit is for diagnosing or determining the presence of cancer, the kit comprising: iii) the array according to any one of clauses 19 to 26, or components for making the same; and iv) instructions for performing the method as defined in any one of clauses 1 to 18.

[0239] 30. A method of treating cancer, preferably prostate cancer, in an individual comprising the steps of: v) diagnosing the cancer according to the method defined in any one of clauses 1- 18; and vi) providing the individual with cancer therapy.

[0240] 31. The method, array or use according to any one of the preceding clauses wherein the individual is selected from the group comprising: a primate (for example, a human; a monkey; an ape); a rodent (for example, a mouse, a rat, a hamster, a guinea pig, a gerbil, a rabbit); a canine (for example, a dog); a feline (for example, a cat); an equine (for example, a horse); a bovine (for example, a cow); or a porcine (for example, a pig), preferably wherein the individual is a human.

[0241] 32. The method, array or use according to any one of the preceding clauses wherein the one or more biomarkers comprise C4B. 33. The method, array or use according to any one of the preceding clauses wherein the one or more biomarkers comprise C7.

[0242] 34. The method, array or use according to any one of the preceding clauses wherein the one or more biomarkers comprise C9.

[0243] 35. The method, array or use according to any one of the preceding clauses wherein the one or more biomarkers comprise CP.

[0244] 36. The method, array or use according to any one of the preceding clauses wherein the one or more biomarkers comprise EFEMP1.

[0245] 37. The method, array or use according to any one of the preceding clauses wherein the one or more biomarkers comprise SERPINA1.

[0246] 38. The method, array or use according to any one of the preceding clauses wherein the one or more biomarkers comprise SERPINA3.

[0247] 39. The method, array or use according to any one of the preceding clauses wherein the one or more biomarkers comprise SCGB1D2.

Claims

CLAIMS1. A method for diagnosing or determining the presence of prostate cancer in an individual, the method comprising the steps of: i) providing a saliva test sample from the individual; and ii) measuring the expression, in the test sample, of one or more biomarkers selected from: C4B, C6, C7, C9, CFB, CFH, CP, CSRP1, DPMI, EFEMP1, HINT2, IGKV3-15, LCN2, MUC1, MUC5AC, RARRES1, SCGB1D2, SERPINA1, SERPINA3, and SERPING1, wherein the expression in the saliva test sample of the one or more biomarkers is indicative of prostate cancer in the individual.

2. The method according to claim 1, wherein the one or more biomarkers is two or more biomarkers.

3. The method according to claim 1 or claim 2, wherein the one or more biomarkers is three or more biomarkers, most preferably four or more biomarkers.

4. The method according to any one of claims 1 to 3, wherein the one or more biomarkers, the two or more biomarkers, the three or more biomarkers or the four or more biomarkers are selected from: C4B, C7, C9, EFEMP1, SERPINA1, and SERPINA3.

5. The method according to any one of the preceding claims, wherein the method is for diagnosis of early prostate cancer.

6. The method according to any one of the preceding claims, wherein the method is for diagnosis of prostate cancer and wherein the individual has adenocarcinoma of the prostate, transitional cell carcinoma of the prostate, squamous cell carcinoma of the prostate and / or small cell prostate cancer.

7. The method according to any one of the preceding claims, wherein the prostate cancer in the individual is early-stage prostate cancer, such as Stage 0, Stage 1 or Stage 2, or is advanced prostate cancer, such as Stage 3 or Stage 4.

8. The method according to any one of the preceding claims, further comprising the steps of: iii) providing one or more (negative) control samples from an individual not afflicted with prostate cancer; and iv) determining a biomarker signature of the one or more control samples by measuring the expression in the control sample of the one or more biomarkers measured in step ii), wherein the prostate cancer is identified in the event that the expression in thesaliva test sample or test sample of the one or more biomarkers measured in step ii) is different from the expression in the control sample of the one or more biomarkers measured in step iv).

9. The method according to any one of the preceding claims, wherein step ii) comprises measuring the expression of a nucleic acid, protein and / or peptide molecule encoding the one or more biomarkers, preferably step ii) comprises measuring the expression of a protein and / or peptide molecule encoding the one or more biomarkers.

10. The method according to claim 9, wherein the nucleic acid molecule is an mRNA molecule or a cDNA molecule.

11. The method according to any one of the preceding claims, wherein step ii) is performed using RT-qPCR, mass spectrometry and / or western blotting, preferably mass spectrometry.

12. The method according to any one of the preceding claims, wherein the saliva test sample comprises one or more prostate cancer cells.

13. The method according to any one of the preceding claims, wherein the expression of the biomarkers in the saliva test sample is determined using mass spectrometry, an affinity-based method, a transcriptomics-based method, ELISA or flow cytometry.

14. An array for use in diagnosing or determining the presence of prostate cancer in an individual, the array comprising an agent or agents for detecting the expression of one or more of the biomarkers selected from: C4B, C6, C7, C9, CFB, CFH, CP, CSRP1, DPMI, EFEMP1, HINT2, IGKV3-15, LCN2, MUC1, MUC5AC, RARRES1, SCGB1D2, SERPINA1, SERPINA3, and SERPING1, optionally wherein the array is configured for receiving a saliva test sample.

15. Use of one or more biomarkers selected from the group comprising C4B, C6, C7, C9, CFB, CFH, CP, CSRP1, DPMI, EFEMP1, HINT2, IGKV3-15, LCN2, MUC1, MUC5AC, RARRES1, SCGB1D2, SERPINA1, SERPINA3, and SERPING1 for diagnosing or determining the presence of prostate cancer in an individual.

16. The array or use according to claim 14 or claim 15 wherein the one or more biomarkers are selected from: C4B, C7, C9, EFEMP1, SERPINA1, and SERPINA3.

17. The array or use according to any one of claims 14 to 16 wherein the one or more biomarkers are two or more biomarkers, preferably three or more biomarkers, such as four or more biomarkers.

18. The array according to any one of claims 14 to 17 wherein the array is a lateral flow device or plate-based assay, such as an antibody plate-based assay.

19. A kit for diagnosing or determining the presence of prostate cancer, the kit comprising: i) the array according to any one of claims 14 to 18, or components for making the same; and ii) instructions for performing the method as defined in any one of claims 1 to 13.

20. The method, array or use according to any one of the preceding claims wherein the individual is selected from the group comprising: a primate (for example, a human; a monkey; an ape); a rodent (for example, a mouse, a rat, a hamster, a guinea pig, a gerbil, a rabbit); a canine (for example, a dog); a feline (for example, a cat); an equine (for example, a horse); a bovine (for example, a cow); or a porcine (for example, a pig), preferably wherein the individual is a human.

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