Detection of bladder cancer

Abstract

The present invention provides a method for detecting the presence of or risk of bladder cancer in a female patient, the method comprising the steps of: detecting the presence of a biomarker panel in a sample isolated from a female patient, the biomarker panel comprising IL-13 and IL-12p70 and one or more biomarkers selected from the group consisting of BTA, midkine, PAI-1 / tPA, 8OHdG, CEA, CK18, Fibrillin, Creatinine, CXCL16, Cystatin B, Cystatin C, d-dimer, EGF, FAS, HAD, IL-1a, IL-1b, IL-4, IL-6, IL-7, IL-8, MCP-1, Microalbumin, MMP9 NGAL, MMP9 TIMP1, NGAL, NSE, Progranulin, TUP, TGFB1, Thrombomodulin, sTNFR1, TPA, VEGF and Triglyceride, and / or the concentration of albumin / microalbumin / protein to creatinine in a sample isolated from a female patient, expressed as an albumin:creatinine ratio; and assessing the result and comparing it to a normal control, wherein an increase in the presence of the biomarkers compared to the normal control indicates the presence or risk of cancer in the patient from which the sample was isolated.

Description

Technical Field

[0001] This invention relates to a method for detecting the presence or risk of bladder cancer in female patients. Background Technology

[0002] Bladder cancer is a leading cause of death worldwide. It occurs more than three times more often in men than women, but the mortality rate in women is twice that of men. Women with bladder cancer in England and Wales have nearly 20% lower survival rates at 1 and 5 years, and nearly 30% lower at 10 years, suggesting that female patients exhibit more severe disease. Multivariate analysis controlling for sex and race found that 39% of men with hematuria were referred to a urologist by their GP, compared to 17% of women with hematuria. Furthermore, men were more likely than women to have their hematuria fully assessed (22% vs. 12%) and less likely to have it incompletely assessed (55% vs. 69%).

[0003] The usefulness of a diagnostic test is measured by its sensitivity and specificity. The sensitivity of a test is the number of true positives (the number of individuals who test positive for a specific disease and have that disease), and the specificity is the number of true negatives (the number of individuals who test negative for a disease and do not have that disease). The most common symptom of bladder cancer is gross or microscopic hematuria, usually detected by a family physician, and observed in 85% of all bladder cancer patients. A simple urine test strip can be used to detect the presence of blood. While cancers without blood are rare, making the simple blood test strip highly sensitive, the test has poor specificity, with less than 5% of patients presenting with hematuria actually having bladder cancer. However, this 5% of patients presenting with hematuria are often diagnosed with an easily resectable superficial tumor.

[0004] Cystoscopy and cytology are the preferred methods for diagnosing bladder cancer. Cytological examination involves examining bladder epithelial cells in excreted urine. This method is highly specific and readily available for obtaining samples. However, it is less sensitive and subjective at low cell yields. Cytological evaluation is often combined with flexible cystoscopy. White light cystoscopy (WLC) allows direct visualization of the bladder and biopsy of suspicious areas. However, recent publications indicate that blue light cystoscopy (BLC) detects 34% more tumors (e.g., carcinoma in situ (CIS)) than WLC. Furthermore, cytology was negative in 20 / 53 patients with CIS lesions (37.7%) (Fradet et al., 2007; Witjes et al., 2010). Unfortunately, the false positive rate of BLC is higher than that of WLC (39% vs. 31%, respectively) (Fradet et al., 2007). The sensitivity and specificity of cystoscopy were 71% and 72%, respectively (National Collaborating Center for Cancer, Bladder Cancer: diagnosis and management; NICE Guideline 2, February 2015, Page 78).

[0005] Cystoscopy has several drawbacks: it is particularly expensive, causes patient discomfort, carries a risk of infection, and cannot be used for visualization of the upper urinary tract or for detecting small areas of bladder cancer recurrence (CIS). For example, the number of bladder cancer recurrences detected by cystoscopy increases when a urologist is informed of a positive urine test (cytology); however, this does not occur when the results are blinded (van der Aa et al., 2010).

[0006] In the art, efforts have been made to identify one or more biochemical biomarkers for bladder cancer that can identify patients with bladder cancer before cystoscopy. Currently, approximately 20% of patients have severe disease, resulting in poor prognosis. Therefore, efforts have been made in the art to identify proven biomarkers or groups of biomarkers that can be used as bladder cancer screening tools, particularly for low-risk, asymptomatic patients.

[0007] No single biomarker or group of biomarkers has yet reached the sensitivity and specificity required to reduce the frequency of cystoscopy needed for accurate diagnosis. Over the past decade, numerous bladder cancer markers, including bladder tumor antigen (BTA), nuclear matrix protein 22 (NMP22), telomerase, and fibrinogen degradation products (FDP), have been evaluated relative to the gold standard of urinary cytology, consistently yielding low specificity results. These markers are present in the urine of most patients with urinary tract diseases other than bladder cancer, as well as those with urinary tract infections (UTIs). NMP22 and BTA are FDA-approved for use as point-of-care assays. However, NMP22 requires immediate stability in urine, which is not always possible, and BTA can be confounded by blood present in the urine. New putative markers (such as survivin, hyaluronic acid, cytokeratin 8 and 18, and EGF)—which have been shown to induce matrix metalloproteinase 9 (MMP9) expression in certain bladder cancer cells—have been proposed as bladder cancer markers. However, none of the proposed biomarkers have yet achieved the high specificity of urine cytology or the high sensitivity of telomerase assays.

[0008] Therefore, in the field of bladder cancer diagnosis and treatment, existing biomarkers are unsatisfactory because they lack the sensitivity and specificity required for accurate diagnosis of bladder cancer or assessment of a patient's risk of developing it. As a result, clinicians are unable to properly assess whether further cystoscopy and cytological testing should be performed on patients, leading to high costs associated with the diagnosis and treatment of the disease.

[0009] Significant funds and resources should be allocated to providing cystoscopy for low-risk patients who could actually be treated in primary care, rather than increasing wait times for high-risk patients. Therefore, there is a need for accurate assessment tests that allow GPs to diagnose and rule out bladder cancer without sending patients for cystoscopy. Summary of the Invention

[0010] This invention is based on the understanding that there are significant differences in the biomarkers required for the diagnosis of bladder cancer in men and women. Therefore, this invention provides a specific set of biomarkers that can be used to diagnose bladder cancer in female subjects.

[0011] In a first aspect of the invention, a method for detecting bladder cancer or bladder cancer risk in female patients is provided, the method comprising the following steps:

[0012] (i) Detection of the presence of a biomarker group in samples isolated from female patients, the biomarker group comprising IL-13 and IL-12p70 and one or more biomarkers selected from: BTA, Midkine, PAI-1 / tPA, 8OHdG, CEA, CK18, Clusterin, Creatinine, CXCL16, Cystatin B, Cystatin C, d-dimer, E GF, FAS, HAD, IL-1a, IL-1b, IL-4, IL-6, IL-7, IL-8, MCP-1, microalbumin, MMP9NGAL, MMP9TIMP1, NGAL, NSE, progranulin, TUP, TGFB1, thrombomodulin, sTNFR1, TPA, VEGF and triglycerides, and / or the concentrations of albumin / microalbumin / protein to creatinine, expressed as albumin:creatinine ratio;

[0013] (ii) Assess the presence or risk of bladder cancer in female patients, wherein the presence of a biomarker that is elevated compared to a normal control indicates the presence or risk of cancer in female patients from which the sample was isolated.

[0014] In a second aspect of the invention, a solid carrier material is provided, comprising binding molecules attached thereto, the binding molecules having specific affinity for IL-13 and IL-12p70, respectively, wherein the binding molecules are each located at discrete positions on the carrier material.

[0015] In a third aspect of the invention, a method for detecting bladder cancer or bladder cancer risk in female patients is provided, the method comprising the following steps:

[0016] (i) Confirm that the female patient is not infected;

[0017] (ii) Detect the presence of one or more biomarkers in a sample isolated from the female patient, wherein the one or more biomarkers are selected from IL-13, IL12p70, BTA and intermediate factor;

[0018] (iii) Assess the presence or risk of bladder cancer in female patients, wherein the presence of a biomarker that is elevated compared to a normal control indicates the presence or risk of cancer in female patients from which the sample was isolated. Attached Figure Description

[0019] This invention is described with reference to the accompanying drawings, in which:

[0020] Figure 1The output shows the ROC curves for women (4 biomarkers) from SPSS analysis (HaBio);

[0021] Figure 2 The following is a display of the ROC curves for women (4 biomarkers + infection) output from SPSS analysis (HaBio); and

[0022] Figure 3 This displays a pyramid count of all cancer-infected individuals. Detailed Implementation

[0023] This invention is based on the discovery that certain biomarkers in female patients with bladder cancer enable more accurate diagnosis compared to existing diagnostic methods based on biomarkers used to diagnose men and women. The identification of specific biomarkers in samples isolated from female patients indicates susceptibility to or presence of cancer in these women, and surprisingly, these biomarkers are found to be significantly different between men and women.

[0024] As used herein, the term "biomarker" refers to a molecule present in a biological sample obtained from a patient, the concentration of which can indicate a pathological state. This article describes a variety of biomarkers that have been found to be used alone or in combination with other diagnostic methods for the diagnosis of bladder cancer, or as complementary biomarkers in combination with other biomarkers.

[0025] Diagnosis can be made based on the expression levels or concentrations of biomarkers in female patients isolated from the patient. The biomarkers of the present invention are typically identified in serum or urine samples from the patient. Preferably, the sample is a urine sample.

[0026] The biomarker group involved in this invention comprises IL-13 and IL-12p70, as well as one or more biomarkers selected from the following: BTA, metaphase factor, PAI-1 / tPA, 8OHdG, CEA, CK18, cytokinin, creatinine, CXCL16, cysteine ​​protease inhibitor B, cysteine ​​protease inhibitor C, d-dimer, EGF, FAS, HAD, IL-1a, IL-1b, IL-4, IL-6, IL-7, IL-8, MCP-1, microalbumin, MMP. 9NGAL, MMP9TIMP1, NGAL, NSE, granulin precursor, TUP, TGFB1, thrombomodulin, sTNFR1, TPA, VEGF, triglycerides, preferably BTA, interstitial factor, PAI-1 / tPA, cytokinin, IL-8, microalbumin, MMP9NGAL, NSE, cysteine ​​protease inhibitor C, d-dimer, IL-7, and / or the concentrations of albumin / microalbumin / protein to creatinine, expressed as albumin:creatinine ratio (ACR).

[0027] The biomarker set can be any combination listed in Table 2 or Table 3.

[0028] Preferably, the biomarker group is (i) BTA, IL-13 and IL12p70; (ii) mid-term factor, IL-13 and IL12p70; (iii) BTA, IL-13, IL12p70 and mid-term factor; or (iv) BTA, IL-13, IL12p70, mid-term factor and PAI-1 / tPA.

[0029] In some implementations, the concentrations of albumin and creatinine in a sample are expressed as an albumin:creatinine ratio. This can be calculated by measuring the concentrations of albumin and creatinine separately. Those skilled in the art will understand conventional methods for measuring albumin and creatinine concentrations; see the exemplary methods of the embodiments. When the kidneys are functioning normally, albumin is almost non-existent in urine.

[0030] In some embodiments, the patient may have hematuria and / or infection. For the avoidance of ambiguity, the term "hematuria" refers to the presence of red blood cells in the urine. Suitably, the infection can be bacterial or viral, preferably bacterial. Suitably, the method may also include a step of characterizing the patient's infection status. Characterizing infection means diagnosing the patient as having or not having an infection, and may include identifying the type of infection. Infection can be determined using a clinical-based diagnosis based on clinical history, biomarkers, dipstick analysis, or a UTI multiplex array (e.g., Randox urinary tract multiplex assay). By incorporating the initial infection test, the AUC can be increased, and the number of biomarkers used to diagnose bladder cancer can be reduced. In the context of this invention, the term "bladder cancer" should be understood to include urothelial carcinoma (UC), transitional cell carcinoma, squamous cell carcinoma of the bladder, and / or bladder adenocarcinoma. In some embodiments, the presence of hematuria and / or infection can further increase the elevated levels of biomarkers within a biomarker group compared to the absence of hematuria and / or infection in female bladder cancer patients.

[0031] Preferably, the biomarker is in the form of urine, i.e., it is identified in a urine sample.

[0032] In a preferred embodiment, biomarkers within a group can be identified in a sample isolated from a patient, and their concentrations in the sample can be determined sequentially or simultaneously. Biomarkers can be identified and their concentrations in the isolated sample determined using conventional methods known in the art, for example, by contacting the sample with a matrix having binding molecules specific to each biomarker included in the biomarker group. Preferably, at least two binding molecules are immobilized on the matrix, more preferably three, four, or more binding molecules, wherein each binding molecule is specific to a single biomarker, and a first probe is specific to IL-13, and a second probe is specific to IL-12p70. As used herein, the term "specific" means that the binding molecule binds only to one of the biomarkers of the present invention, while binding to other biomarkers of the present invention or other analytes in the analyzed biological sample is negligible. This ensures that the integrity of diagnostic assays using the biomarkers of the present invention and their results is not compromised by other binding events.

[0033] Biomarker concentrations can be measured using immunoassay-based methods. In this case, the binding molecule is preferably an antibody, such as a polyclonal or monoclonal antibody. As used herein, the term "antibody" includes any immunoglobulin or immunoglobulin-like molecule or fragments thereof, Fab fragments, ScFv fragments, and other antigen-binding fragments. The term "polyclonal antibody" refers to a heterogeneous population of antibodies that recognize multiple epitopes on a target / antigen. The term "monoclonal antibody" refers to a homogeneous population of antibodies (including antibody fragments) that recognize a single epitope on a target / antigen. Immunoassay techniques are also readily incorporated into portable or handheld devices for use outside of clinical settings. Quantitative immunoassays (e.g., Western blotting or ELISA) can be used to detect the amount of protein biomarkers. Preferred analytical methods include the use of multianalyte bioarrays, which enable the simultaneous detection and quantification of several proteins. 2D gel electrophoresis is also a technique that can be used for multianalyte analysis.

[0034] In a preferred embodiment, the binding molecules are immobilized on a solid support prepared for contact with a patient sample. The preferred solid support material is in the form of a biochip. Biochips are typically planar matrices, which may be based on, for example, minerals or polymers, but are preferably ceramics. The solid support can be manufactured according to methods disclosed, for example, in GB-A-2324866, the entire contents of which are incorporated herein by reference. The solid support can be screen-printed according to known methods disclosed, for example, in WO2017 / 085509. Preferably, the level of biomarkers in the sample can be determined using a Biochip Array Technology (BAT) system (available from Randox Laboratories Limited). More preferably, Evidence Evolution and Evidence Investigator devices (available from Randox Laboratories) can be used.

[0035] The solid carrier material contains binding molecules attached thereto, which have specific affinities for IL-13 and IL-12p70, respectively, wherein the binding molecules are each located at discrete positions on the carrier material. The solid carrier material may also contain one or more such binding molecules at discrete locations, each having a specific affinity for other biomarkers selected from: BTA, metaphase factor, PAI-1 / tPA, 8OHdG, CEA, CK18, cytokinin, creatinine, CXCL16, cysteine ​​protease inhibitor B, cysteine ​​protease inhibitor C, d-dimer, EGF, FAS, HAD, IL-1a, IL-1b, IL-4, IL-6, IL-7, IL-8, MCP-1, microalbumin, MMP9NGAL, MMP9TIMP1, NGAL, NSE, granulin precursor, TUP, TGFB1, thrombomodulin, sTNFR1, TPA, VEGF, and triglycerides, preferably BTA, metaphase factor, PAI-1 / tPA, cytokinin, IL-8, microalbumin, MMP9NGAL, NSE, cysteine ​​protease inhibitor C, d-dimer, and IL-7. For example, the binding molecules attached to the solid carrier material can have an affinity for the combination of biomarkers in Table 2 or Table 3, preferably (i) BTA, IL-13 and IL12p70; (ii) mid-term factor, IL-13 and IL12p70; (iii) BTA, IL-13, IL12p70 and mid-term factor; or (iv) BTA, IL-13, IL12p70, mid-term factor and PAI-1 / tPA.

[0036] The present invention also provides the use of the matrix in a method for detecting bladder cancer or bladder cancer risk in female patients.

[0037] This invention also provides a kit comprising probes for a biomarker set and optional reagents for measuring albumin and creatinine, said biomarker set comprising IL-13 and IL-12p70 and one or more biomarkers selected from: BTA, metaphase factor, PAI-1 / tPA, 8OHdG, CEA, CK18, cytokinin, creatinine, CXCL16, cysteine ​​protease inhibitor B, cysteine ​​protease inhibitor C, d-dimer, EGF, FAS, HAD, IL-1α, IL-1b, IL-4, IL-6, IL-7, IL-8, MCP-1, microalbumin, MMP9NGAL, MMP9TIMP1, NGAL, NSE, granulin precursor, TUP, TGFB1, thrombomodulin, sTNFR1, TPA, VEGF, and triglycerides, preferably BTA, interphase factor, PAI-1 / tPA, cluster protein, IL-8, microalbumin, MMP9NGAL, NSE, cysteine ​​protease inhibitor C, d-dimer, and IL-7. For example, the biomarker set can be a combination of those in Table 2 or Table 3, preferably (i) BTA, IL-13, and IL12p70; (ii) interphase factor, IL-13, and IL12p70; (iii) BTA, IL-13, IL12p70, and interphase factor; or (iv) BTA, IL-13, IL12p70, interphase factor, and PAI-1 / tPA. The kit can be used to detect bladder cancer or bladder cancer risk in female patients according to the first aspect of the invention.

[0038] The present invention also provides a method for detecting bladder cancer or bladder cancer risk in female patients, the method comprising the following steps:

[0039] (i) Confirm that the female patient is not infected;

[0040] (ii) Detecting the presence of one or more biomarkers in samples isolated from female patients, wherein the one or more biomarkers are selected from IL-13, IL12p70, BTA and intermediate factor;

[0041] (iii) Assess the presence or risk of bladder cancer in female patients, wherein the presence of a biomarker that is elevated compared to a normal control indicates the presence or risk of cancer in female patients from which the sample was isolated.

[0042] Suitablely, the one or more biomarkers are (i) IL-13+IL12p70; (ii) IL-13+BTA; (iii) IL-13+intermediate factor; (iv) IL12p70+BTA; (v) IL12p70+intermediate factor; or (vi) BTA+intermediate factor.

[0043] In the method of this invention, to diagnose bladder cancer or bladder cancer risk, elevated levels of biomarkers within the tested biomarker group are found compared to corresponding biomarkers in normal control samples. In some embodiments, the concentration of the biomarker is found to be significantly higher than in the control sample. The determination of "higher concentration" is relative and is relative to control subjects known not to have bladder cancer.

[0044] Control values ​​are derived from the concentrations of corresponding biomarkers in biological samples obtained from one or more individuals who do not have bladder cancer. These individuals can be, for example, healthy individuals or individuals with a disease other than bladder cancer. Alternatively, control values ​​may correspond to the concentrations of each biomarker in samples obtained from patients who previously had bladder cancer.

[0045] To avoid ambiguity, the term "corresponding biomarkers" refers to the concentrations of the same combination of biomarkers determined for a patient sample that are also used to determine control values. For example, if the concentrations of IL-13 and IL-12p70 in a patient sample are determined, then the concentrations of IL-13 and IL-12p70 in a control are also known.

[0046] In a preferred embodiment, each of the biomarker concentration values ​​for the female patient and controls is input into one or more statistical algorithms to produce an output value indicating whether the patient has bladder cancer. If the output value is less than the biomarker cut-off value, the patient is considered negative by the bladder cancer biochip test. If the output value is greater than the biomarker cut-off value, the patient is considered positive by the bladder cancer biochip test.

[0047] In a preferred embodiment, a Clinical Risk Score (CRS) is calculated for female patients, which is a cumulative score using, but not limited to, the following clinical and demographic measures: age, hematuria (visible hematuria vs. gross hematuria), smoking (pack years), BMI, blood pressure (controlled, normal, hypertensive), occupational risk score (FINJEM), social class (ONS code), comorbidities (such as diabetes, chronic kidney disease (CKD), etc.), medications (such as statins, antihypertensive drugs, etc.), specific medications (found to increase the risk of bladder cancer), pain relief, kidney transplant, kidney cancer, other cancers, pelvic radiotherapy, and UTI (with / without microbes).

[0048] Example scores used when calculating CRS in patients: age greater than 65 years equals 1 point; age less than 65 years equals 0 points; invisible hematuria (NVH) equals 1 point; gross hematuria equals 2 points. Therefore, when using age and hematuria as clinical risk scores, patients older than 65 years with gross hematuria have a cumulative score of 3 points.

[0049] In a preferred embodiment, biochip bladder cancer test data and CRS are combined to determine whether a patient falls into one of the following categories: low risk, intermediate risk, or high risk. This information will enable a GP to manage his / her patients in primary care and refer them for further testing when appropriate. For example, a patient with hematuria, a negative biochip test, and a low CRS will be monitored by their GP in primary care instead of being referred for cystoscopy. Patients with a negative biochip test and an intermediate CRS will be referred to urology for cystoscopy (non-urgent). Patients with a positive biochip test and a low CRS will be referred to urology for cystoscopy (non-urgent). Patients with a positive biochip test and an intermediate CRS will be marked as “red flagged” for emergency cystoscopy.

[0050]

[0051] Keywords: Y = Yes; N = No

[0052] The accuracy of the statistical methods used according to the present invention can be best described by their receiver operating characteristic (ROC). The ROC curve addresses not only the sensitivity and number of true positives of a test, but also its specificity and number of true negatives. Therefore, the sensitivity and specificity values ​​of a given combination of biomarkers are indicators of the accuracy of the assay. For example, if the sensitivity and specificity values ​​of a combination of biomarkers are 80%, then in 100 patients with bladder cancer, identifying the presence of that particular combination of biomarkers as a positive bladder cancer diagnosis would correctly identify 80, while in 100 patients without bladder cancer, 80 would accurately test negative for the disease.

[0053] ROC also provides a measure of the test's predictive ability in the form of the area under the curve (AUC). AUC is a measure of the probability that a condition can be correctly identified by a perceived measure. By convention, this area is always ≥0.5. Values ​​range between 1.0 (two sets of test values ​​are completely separated) and 0.5 (no significant difference in distribution between the two sets of test values). This area depends not only on specific parts of the graph, such as the point closest to the diagonal or the sensitivity at 90% specificity, but also on the entire graph. This is a quantitative, descriptive expression of how close the ROC graph is to a perfect graph (area = 1.0). Typically, tests with a sensitivity of about 80% or higher and a specificity of about 80% or higher are considered potentially applicable in the art, although these values ​​vary depending on clinical application. In a preferred embodiment, the AUC value for the biomarker group is at least 0.7, suitably at least 0.75, preferably at least 0.8, and more preferably at least 0.85.

[0054] It is well known in the art that normal or “background” concentrations of biomarkers can exhibit slight variations due to factors such as age, sex, or ethnic / geographical genotype. Therefore, the threshold values ​​used in the methods of this invention can also vary slightly depending on optimization for the target patient or population. Adjusting the threshold value will also allow the operator to increase sensitivity at the expense of specificity, and vice versa.

[0055] In one embodiment, the sensitivity and / or specificity of the algorithm are both at least 0.7. Preferably, the sensitivity of the algorithm is at least 0.75, more preferably at least 0.8, and / or the specificity is at least 0.75, more preferably at least 0.8.

[0056] In cases where two or more biomarkers are used in this invention, suitable mathematical or machine learning classification models, such as logistic regression equations, can be derived. Skilled statisticians will understand how such suitable models are derived; they may include other variables, such as the patient's age and sex. ROC curves can be used to evaluate the accuracy of the model, which can be used independently or in the algorithm to aid clinical decision-making. While logistic regression equations are a common mathematical / statistical procedure used in this context and are an option within the scope of this invention, other mathematical / statistical, decision tree, or machine learning procedures may also be used. Those skilled in the art will understand that it may be necessary to adjust the model generated for a given population to apply it to datasets obtained from different populations or patient groups.

[0057] The following embodiments illustrate the present invention with reference to the accompanying drawings.

[0058] Example

[0059] patient

[0060] A bladder cancer trial was conducted with 157 patients presenting with hematuria. After establishing the feasibility of the bladder cancer diagnostic algorithm in patients with hematuria, a large-scale hematuria biomarker study (HaBio) was designed and enrolled 675 patients.

[0061] Urine and serum collection

[0062] Collect urine samples (~50 ml) and serum samples (~10 ml) from all patients in sterile containers. Immediately aliquot the unfiltered and uncentrifuged urine samples and freeze at -80°C until analysis. Thaw the urine samples on ice and then centrifuge (1200 x g, 10 min, 4°C) to remove any particulate matter before analysis.

[0063] Biomarker measurement

[0064] All samples were tested in triplicate, and the results are expressed as mean ± SD (n = 3).

[0065] Biochip array technology (Randox Laboratories Ltd., Crumlin, Northern Ireland, UK) is used to simultaneously detect multiple analytes in a single patient sample (urine). This technology is based on the Randox biochip, a 9mm... 2A solid matrix supports an array of discrete test regions with immobilized antigen-specific antibodies. After activating the antibody with assay buffer, standards and samples are added and incubated at 37°C for 60 minutes, followed by 60 minutes at 370 rpm in a thermal shaker. Antibody conjugate (HRP) is then added and incubated at 370 rpm for 60 minutes in a thermal shaker. The chemiluminescence signal generated after the addition of luminol (at a 1:1 ratio with the conjugate) is detected and measured using digital imaging technology and compared with the chemiluminescence signal from a calibration curve to calculate the concentration of the analyte in the sample. The analytical sensitivities of the biochip are as follows: IL-2 4.8 pg / ml, IL-4 6.6 pg / ml, IL-6 1.2 pg / ml, IL-7 1.11 pg / ml, IL-8 7.9 pg / ml, IL12p70 2.61 pg / ml, IL-13 5.23 pg / ml, VEGF 14.6 pg / ml, TNFα 4.4 pg / ml, IL-1α 0.8 pg / ml, IL-1β 1.6 pg / ml, MCP-1 13.2 pg / ml, NSE 0.26 ng / ml, NGAL 17.8 ng / ml, sTNFRI 0.24 ng / ml, d-dimer 2.1 ng / ml, sTNFRII 0.2 ng / ml. The functional sensitivities for CEA and PSA (free and total) were 0.2, 0.02, and 0.045 ng / ml, respectively. Below-the-limit (LOD) / mean-detectable-dose (MDD) data—for any given test, when data are below the LOD / MDD, 90% of the LOD / MDD of that test is used for analysis (Papa L et al., 2012).

[0066] Commercial ELISA kits

[0067] The following markers were detected using commercially available ELISA kits according to the manufacturer's instructions: 8OHdG (CellBiolabs); BTA (Polymedco); CK18 (IDL); Pyrenin (R&D Systems; Quantikine ELISA Human Pyrenin, DCLU00); Creatinine (Randox Rx Daytona); CXCL16 (R&D Systems); Cysteine ​​Protease Inhibitor B (R&D Systems); Cysteine ​​Protease Inhibitor C (Randox Daytona Rx); FAS (RayBio); HAD (MyBioSource); Microalbumin (Randox Rx Daytona); Mid-term Factor (CellMid); MMP9NGAL (R&D Systems; Quantikine ELISA Human MMP-9 / NGAL Complex); MMP9TIMP1 (R&D Systems); PAI-1 / Tpa (AssayPro); Granulin Precursor (R&D Systems). Systems); TUP (Coomassie Brilliant Blue assay (Bradford Assay) A595nm); TGFB1 (R&D Systems); Thrombomodulin (R&D Systems) and TPA (Abcam).

[0068] Infect

[0069] Infection is diagnosed based on the following clinical criteria: patient clinical history, biomarkers, and test strip analysis. Infection can also be identified using UTI multiplex arrays (e.g., Randox urinary multiplex assay), which involve extracting DNA from a urine sample, followed by amplification (single-tube 28-fold PCR), hybridization, and detection.

[0070] Creatinine, osmolality, and TUP

[0071] Creatinine (μmol / L) measurements were determined using a quantitative in vitro diagnostic kit from Randox Laboratories (catalog number CR3814), and results were collected from a Daytona RX series clinical analyzer (Randox Laboratories Ltd). Creatinine measurement was linear up to 66,000 μmol / L with a sensitivity of 310 μmol / L.

[0072] use Microosmometer (Model 15) The osmotic pressure (mOsm) was measured using the Messtechnik (Berlin, Germany) instrument. In short, the osmoremeter was calibrated using three independent readings (0.1 ml) of distilled water and the 300 mOsm standard solution provided with the instrument. Calibration was confirmed by measuring the mOsm of a freshly prepared 0.9% NaCl solution (mean 286 ± 3 mOsm, n = 3). At the end of the analysis, the instrument calibration was also verified using the same 0.9% NaCl solution (mean 280.3 ± 0.58 mOsm, n = 3) to check for drift.

[0073] Using the Coomassie Brilliant Blue assay kit (A) 595 Total urinary protein levels (mg / ml) were determined using Bradford reagent (Pierce, Rockford, IL, USA) and BSA as standard solutions (1 mg / ml). Patient samples (10 μl / patient) were mixed with Bradford reagent (1 ml) and analyzed on an A1000 Hitachi spectrophotometer (model U-2800). 595 Readings were taken at nm. The levels in the urine samples were determined using a BSA calibration table (0-5 mg / ml, n=3).

[0074] Statistical analysis

[0075] Statistical analysis was performed using the Mann-Whitney U test (IBM SPSS v25) and R (Wilcoxon) to identify differentially expressed markers between controls and bladder cancer.

[0076] SPSS and R (stats, glmnet(Lasso), glmult) were used to identify helpful markers for the algorithm using binary logistic regression (based on Wald's forward and backward methods).

[0077] It is statistically significant at the p<0.05 level.

[0078] Examples are shown below (SPSS analysis (HaBio women), for combinations of 4 biomarkers; and 4 biomarkers + infection).

[0079] SPSS analysis (HaBio) – Female (4 biomarkers)

[0080] Classification table a

[0081]

[0082] a. The critical value is 250.

[0083] 0 = No cancer, 1 = Cancer present

[0084] Variables in the equation

[0085]

[0086] a. Variables entered in step 1: BTA, IL12p70, IL13, intermediate factor.

[0087] Area under the curve

[0088] Test outcome variable: Predicted probability

[0089]

[0090] a. Under nonparametric assumptions

[0091] b. Null assumption: True area = 0.5

[0092] The calculated ROC curve is as follows Figure 1 As shown.

[0093] SPSS analysis (HaBio) – Female (4 biomarkers + infection)

[0094] Classification table a

[0095]

[0096] a. The critical value is 250.

[0097] 0 = no cancer, 1 = cancer

[0098] Variables in the equation

[0099]

[0100] a. Variables entered in step 1: BTA, IL12p70, IL13, intermediate factor, infection.

[0101] Area under the curve

[0102] Test outcome variable: Predicted probability

[0103]

[0104] a. Under nonparametric assumptions

[0105] b. Null assumption: True area = 0.5

[0106] The calculated ROC curve is as follows Figure 2 As shown.

[0107] Co-infection status

[0108] Combining initial infection testing increases AUC and reduces the number of markers needed to diagnose bladder cancer.

[0109] biomarkers AUC without co-infection status AUC of co-infection status IL-13 0.686 0.830 IL12p70 0.632 0.796 BTA 0.754 0.868 Mid-term factors 0.694 0.863 IL-13+IL12p70 0.783 0.869 IL-13+BTA 0.830 0.902 IL-13+ intermediate factor 0.782 0.895 IL12p70+BTA 0.812 0.885 IL12p70+ intermediate factor 0.748 0.877 BTA+ Intermediate Factors 0.753 0.882

[0110] Results and discussion

[0111] Certain biomarkers were significantly higher in female bladder cancer patients, including BTA, interphase factor, PAI-1 / tPA, 8OHdG, CEA, CK18, cytokinin, creatinine, CXCL16, cysteine ​​protease inhibitor B, cysteine ​​protease inhibitor C, d-dimer, EGF, FAS, HAD, IL-1a, IL-1b, IL-4, IL-6, IL-7, IL-8, MCP-1, microalbumin, MMP9NGAL, MMP9TIMP1, NGAL, NSE, granulin precursor, TUP, TGFB1, thrombomodulin, sTNFR1, TPA, VEGF, and triglyceride 1 (p<0.050; Mann-Whitney test). The following biomarkers were most prominent: BTA, interphase factor, PAI-1 / tPA, cluster protein, IL-8, microalbumin, MMP9NGAL, NSE, cysteine ​​protease inhibitor C, d-dimer, and IL-7.

[0112] The algorithm used by the inventors is unexpected, and the biomarkers included in the algorithm are unpredictable.

[0113] Table 1 shows a summary of hypothesis tests using the Mann-Whitney U test. When a biomarker has a correlation of 0.7 or greater, it can be replaced by a related biomarker because the two biomarkers are correlated. A significance value less than 0.7 indicates that the two biomarkers are independent.

[0114] Figure 3 The data shows that hematuria in women may be due to infection (bacteria and / or virus) rather than cancer. The right half of the graph represents patients with infection, and the left half represents patients without infection; the lower half represents patients without cancer, and the upper half represents patients with cancer; therefore, approximately 2 out of 76 patients with infection had cancer. Testing women presenting with hematuria in the GP clinic for infection (using any method / test) can allow women with positive infections to return home for a course of antibiotics, thus reducing the NHS referral burden, in order to rule out bladder cancer and avoid cystoscopy. Figure 3Of the 184 patients, approximately 76 were sent home, while only 2 were incorrectly sent home (the 2 missed diagnoses will be referred due to antibiotic failure). The remaining approximately 108 uninfected patients will undergo biomarker testing.

[0115] The following is a list of abbreviations used in this manual:

[0116] 80HdG OxiSelect Oxidative DNA Damage

[0117] ACR Albumin: Creatinine Ratio

[0118] Area under the AUC curve

[0119] BLC (Blue Light Cystoscopy)

[0120] BMI (Body Mass Index)

[0121] BTA (Bladder Tumor Antigen)

[0122] CEA (Carcinoembryonic Antigen)

[0123] CIS carcinoma in situ

[0124] CK-18 Cytokeratin 18

[0125] CKD (Chronic Kidney Disease)

[0126] CRP (C-reactive protein)

[0127] CRS Clinical Risk Score

[0128] EGF (Epidermal Growth Factor)

[0129] FAS FAS protein

[0130] FDP fibrinogen degradation products

[0131] FINJEM Finnish Job Exposure Matrix

[0132] GP (General Practitioner)

[0133] HRP (Hordeum peroxidase)

[0134] IL-2 interleukin-2

[0135] IL-3 interleukin-3

[0136] IL-4 (Interleukin-4)

[0137] IL-6 interleukin-6

[0138] IL-7 interleukin-7

[0139] IL-8 (Interleukin-8)

[0140] IL-10 Interleukin-10

[0141] IL-12p70 (Interleukin-12p70)

[0142] IL-13 (Interleukin-13)

[0143] IL-18 Interleukin-18

[0144] IL-23 (Interleukin-23)

[0145] LOD detection limit

[0146] MCP (Monocyte Chemoattractant Protein)

[0147] MDD mean detectable dose

[0148] MMP9 matrix metalloprotein 9

[0149] MMP-9 / NGAL matrix metalloproteinase 9 / neutrophil gelatinase-associated lipotransferase complex

[0150] MMP9 / TIMP1 Matrix metalloproteinase 9 / tissue inhibitor of metalloproteinases 1

[0151] NGAL (Neurotrophic Gelatinase-Associated Lipotransferase)

[0152] NICE (National Institute for Clinical Excellence)

[0153] NMP22 nuclear matrix protein 22

[0154] NSE neuron-specific enolase

[0155] NVH (No Visible Hematuria)

[0156] ONS (Office for National Statistics)

[0157] PAI-1 / tPA plasminogen activator inhibitor 1 / tissue plasminogen activator

[0158] Point of Care (POC)

[0159] ROC acceptor operation curve

[0160] SD standard deviation

[0161] SDS-PAGE (Sodium Lauryl Sulfate-Polyacrylamide Gel Electrophoresis)

[0162] sTNFR1 (soluble tumor necrosis factor 1)

[0163] sTNFR2, a soluble tumor necrosis factor-2

[0164] TGFB1 Transforming growth factor β1

[0165] TM Thrombomonas

[0166] TPA (tissue plasminogen activator)

[0167] TPSA (Total Prostate-Specific Antigen)

[0168] Total Urinary Protein (TUP)

[0169] UTI (urinary tract infection)

[0170] VEGF (Vascular Endothelial Growth Factor)

[0171] WLC White Light Cystoscopy

[0172] Table 1 shows a summary of hypothesis tests using the Mann-Whitney U test.

[0173]

[0174]

[0175]

[0176] Table 2 shows the AUC, sensitivity, and specificity of biomarker combinations generated using GLMulti (using R) after Wald-based forward and backward binary logistic regression. "u" indicates that the biomarker is in urinary form, and "s" indicates that the biomarker is in serum form.

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184] Table 3 Biomarker Combinations

[0185]

[0186]

[0187]

[0188]

[0189]

[0190] References

[0191] Fradet Y et al., J Urol. 2007 Jul; 178(1):68-73; discussion 73.

[0192] Witjes JA et al., Eur Urol. 2010 Apr; 57(4):607-14.

[0193] National Collaborating Center for Cancer, Bladder Cancer: diagnosis and management; NICE Guidelines 2, February 2015, page 78.

[0194] Van der Aa MN et al., J Urol. 2010 Jan; 183(1):76-80.

[0195] Papa L et al., Ann Emerg Med. 2012 Jun; 59(6):471-83.

Claims

1. The use of binding molecules for a biomarker group in the preparation of a kit for detecting bladder cancer or bladder cancer risk in female patients. The biomarker group is selected from the following combinations of biomarkers: Where "u" indicates that the biomarker is in urine form, and "s" indicates that the biomarker is in serum form; The detection process includes the following steps: (i) Detecting the presence of a group of biomarkers in a sample isolated from a female patient; wherein the sample is a urine sample or a serum sample; (ii) Assess the presence or risk of bladder cancer in female patients, wherein the presence of a biomarker that is elevated compared to a normal control indicates the presence or risk of cancer in female patients from which the sample was isolated.

2. The use according to claim 1, wherein step (ii) further comprises a step of characterizing the infection status of the patient.

3. The use according to claim 1, wherein step (ii) includes inputting the measured concentration of the biomarker from step (i) into an algorithm such that the output of the algorithm indicates whether an individual has bladder cancer or is at risk of developing bladder cancer.

4. The use according to claim 3, wherein the sensitivity of the output of the algorithm is at least 0.

70.

5. The use according to claim 3, wherein the specificity of the algorithm's output is at least 0.

70.

6. The use according to claim 1, wherein the patient presents with hematuria.

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