Method and system for risk stratification and management of bladder cancer

Abstract

The present disclosure provides methods for risk stratification or management of recurrent bladder cancer or new bladder cancer in a patient by assessing urine D-dimer levels. In certain embodiments, the D-dimer may be determined by ELISA (Enzyme Linked Immunosorbent Assay) or LFA (Lateral Chromatography Assay). In the methods provided herein, when urine detection D-dimers are positive, biopsy (by cystoscopy) is performed on the patient to confirm the diagnosis. And if the urine detects that the D-dimer is negative, rechecking the patient in the subsequent follow-up visit. These methods provide an alternative to the majority of patients for unnecessary invasive or expensive diagnostic steps with poor diagnostic effects, thereby achieving savings for the patients and the entire medical industry and reducing the incidence of cystoscopy-related side effects.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 365,324, filed on May 25, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to systems and methods for evaluating peptides and fragments thereof in biological samples for bladder cancer management. Background Art

[0004] Bladder cancer is a common malignancy in women and the fourth most common malignancy in men. Bladder cancer ranges from non-invasive and usually non-aggressive tumors to invasive and aggressive tumors. The former will recur and require patients to undergo long-term invasive monitoring; the latter will result in high disease-related mortality. . Seventy-five (75)% of BC patients suffer from non-muscle invasive bladder cancer (NMIBC) and are treated by surgery or BCG therapy. It is worth noting that about 80% of these patients will relapse at least once, and about 30% will experience disease progression to muscle invasive bladder cancer (MIBC). Therefore, patients who are initially diagnosed with BC and treated are followed up every 3-6 months (and then once a year) to determine whether the patient has a bladder cancer recurrence. In these follow-ups, the techniques commonly used to detect possible recurrences are cystoscopy and cytology (and sometimes imaging). Unfortunately, the currently available recurrence detection methods (including these tests) are poor in diagnostic ability, expensive and / or invasive, and are accompanied by side effects. In fact, an intensive follow-up (surveillance) program to detect bladder cancer recurrence, including cystoscopy and urine cytology, is the most expensive follow-up program in oncology.

[0005] The disadvantages of cystoscopy, urine cytology and other tests include one or more of the following. Cystoscopy by biopsy or transurethral resection is necessary for histological confirmation. However, cystoscopy is invasive and relatively expensive. In addition, cystoscopy is often associated with complications including pain, urinary tract infection and hematuria. Urine cytology is also commonly used to diagnose and monitor BC. This non-invasive method involves examining cells collected from urine samples. Although urine cytology is non-invasive, it requires a dedicated urological pathologist and there is subjectivity when grading urothelial carcinoma on urine samples, so even between cytopathologists, considerable inter-observer and intra-observer differences will be generated. Although cytology has a fairly good specificity (about 86%), it shows a low sensitivity of about 48%, which drops to about 16% for low-grade tumors. Finally, neither of these two tests can provide instant detection. Other tests that are feasible for primary care institutions include urine test strips or microscopic urinalysis to detect hematuria, but these are not sensitive enough and have low specificity. Therefore, improved methods (eg, biomarkers) for non-invasive detection of BC are needed to support the selection of treatment options for patients. Summary of the invention

[0006] The present disclosure provides a method for risk stratification or management of recurrent bladder cancer or new bladder cancer in patients by assessing urine D-dimer levels. In certain embodiments, the D-dimer can be measured by ELISA (enzyme-linked immunosorbent assay) or LFA (lateral flow assay). In the method provided herein, when urine detection D-dimer is positive, a biopsy (by cystoscopy) is performed on the patient to confirm the diagnosis. If the urine detection D-dimer is negative, the patient will be reviewed in subsequent follow-up. These methods provide an alternative to unnecessary invasive or expensive and poor diagnostic steps for most patients, thereby achieving savings for the patient and the entire medical industry and reducing the incidence of cystoscopy-related side effects. In one embodiment, the product and method relate to a lateral flow test strip (LFT) for the instant detection of urine D-dimers, which can be used by patients to self-test urine for bladder cancer recurrence at home on a regular basis (e.g., monthly, every 2 weeks). In contrast, under current protocols, cancer recurrence may only be detected at the next follow-up visit, which may be 6 months or a year later. The products and methods described herein provide home monitoring for the occurrence or recurrence of bladder cancer. Recurrence of bladder cancer is detected earlier than currently detected, potentially leading to a reduction in patient morbidity and mortality.

[0007] Certain embodiments include a method for risk stratification of bladder cancer in a subject. One such method comprises the following steps: assessing the D-dimer level in a first urine sample obtained from the subject compared to a predetermined threshold level, and when the D-dimer level in the first urine sample is higher than the predetermined threshold, the subject is identified as having an increased risk of bladder cancer and identified as a candidate for invasive testing of bladder cancer. In addition, the method comprises the following steps: when the D-dimer level in the first urine sample is or is lower than the predetermined threshold, the subject is identified as having no increased risk of bladder cancer and identified as a candidate for not performing invasive testing of bladder cancer. In certain embodiments, the predetermined threshold is based on a receiver operating curve-area under the curve (ROC-AUC) analysis. In certain embodiments, the predetermined threshold for a positive test is about 90.3pg / ml. The D-dimer urine level in other diseases (such as urinary tract infection) can be determined. Certain embodiments relate to a method for risk stratification of bladder cancer recurrence in a subject who has already suffered from bladder cancer.

[0008] In certain embodiments, the method further comprises the following steps: (i) when the D-dimer level in the first urine sample is at or below a predetermined threshold, evaluating the D-dimer level in a second urine sample obtained from the subject compared to the predetermined threshold, wherein the second urine sample is obtained two or more weeks after the first urine sample is obtained from the subject; (ii) when the D-dimer level in the second urine sample is above the predetermined threshold, identifying the subject as having an increased risk of bladder cancer and as a candidate for invasive testing for bladder cancer; and (iii) when the D-dimer level in the second urine sample is at or below the predetermined threshold, identifying the subject as not having an increased risk of bladder cancer and as a candidate for not undergoing invasive testing for bladder cancer.

[0009] In some embodiments, the invasive test for bladder cancer comprises one or more of cystoscopy, biopsy, and transurethral resection. In some embodiments, the method further comprises the steps of identifying the subject as having an increased risk of bladder cancer as a candidate for urine cytology or nuclear matrix protein 22 analysis in a urine sample.

[0010] In certain embodiments, the method further comprises the steps of evaluating the urine sample obtained from the subject for an increased level of one or more of MMP-1, apolipoprotein A1, proteinase 3, and apolipoprotein L1 compared to a predetermined threshold level of MMP-1, apolipoprotein A1, proteinase 3, or apolipoprotein L1, and determining that the subject has an increased risk of bladder cancer based on the increased level of one or more of MMP-1, apolipoprotein A1, proteinase 3, and apolipoprotein L1 in the urine sample.

[0011] In certain embodiments, the method further comprises the steps of: assessing an increased level of one or more of apolipoprotein A1 and IL-8 in the urine sample obtained from the subject compared to a predetermined threshold level of apolipoprotein A1 or IL-8; and determining that the subject has an increased risk of bladder cancer based on the increased level of one or more of apolipoprotein A1 and IL-8 in the urine sample.

[0012] In certain embodiments, a method for identifying a subject for invasive testing of bladder cancer comprises the steps of evaluating an elevated D-dimer level in a urine sample obtained from the subject compared to a predetermined threshold level, determining that the subject is at risk for bladder cancer based on the elevated D-dimer level in the urine sample, and subjecting the subject to an invasive test for bladder cancer. In certain embodiments, the subject already has bladder cancer and is being evaluated for bladder cancer recurrence. In certain embodiments, the invasive test for bladder cancer comprises one or more of a cystoscopy, a biopsy, and a transurethral resection.

[0013] In certain embodiments, the method further comprises performing urine cytology or detecting the level of nuclear matrix protein 22 in the urine of a urine sample from a subject at increased risk of developing bladder cancer. In certain embodiments, the method further comprises the steps of evaluating the elevated level of one or more of MMP-1, apolipoprotein A1, proteinase 3, and apolipoprotein L1 in the urine sample obtained from the subject compared to a predetermined threshold level of MMP-1, apolipoprotein A1, proteinase 3, or apolipoprotein L1, and determining that the subject is at increased risk of developing bladder cancer based on the elevated level of one or more of MMP-1, apolipoprotein A1, proteinase 3, and apolipoprotein L1 in the urine sample. In certain embodiments, the method further comprises the steps of: assessing an increased level of one or more of apolipoprotein A1 and IL-8 in the urine sample obtained from the subject compared to a predetermined threshold level of apolipoprotein A1 or IL-8; and determining that the subject has an increased risk of bladder cancer based on the increased level of one or more of apolipoprotein A1 and IL-8 in the urine sample.

[0014] In certain embodiments, the method for treating bladder cancer in a subject comprises the following steps: (i) evaluating an elevated level of D-dimer in a urine sample obtained from the subject as compared to a predetermined threshold level by enzyme-linked immunosorbent assay (ELISA) or lateral flow assay (LFA) or vertical flow assay (VFA), (ii) identifying the subject as having an increased risk of bladder cancer when the D-dimer level in the urine sample is above the predetermined threshold, (iii) performing an invasive test for bladder cancer on the subject, (iv) determining that the subject has bladder cancer based on the results of the invasive test, and (v) treating the subject's bladder cancer by one or more of surgical resection, radiation, chemotherapy, and hormonal therapy. These treatment modalities may include immune checkpoint inhibitors, monoclonal antibodies, and intravesical or systemic chemotherapy, such as cisplatin, fluorouracil, mitomycin, gemcitabine, methotrexate, vinblastine, doxorubicin / adriamycin, and paclitaxel.

[0015] In some embodiments, the subject has bladder cancer and the treatment is for bladder cancer recurrence. The methods and products disclosed herein are used as prognostic markers. The assessment of urine D-dimer can also be used to track the response to treatment, following any previous treatment modalities. In some embodiments, the invasive test for bladder cancer includes one or more of the following: cystoscopy, biopsy, followed by pathological examination, transurethral resection, followed by pathological examination, urine cytology, and detection of nuclear matrix protein 22 levels in urine samples.

[0016] Certain embodiments include a product for detecting D-dimer levels in urine samples to risk stratify bladder cancer. The product is configured to be suitable for ELISA or LFA or VFA. In certain embodiments, the assay system includes a lateral flow test strip. In certain embodiments, the product can also be used to detect the level of one or more of MMP-1, apolipoprotein A1, proteinase 3, and apolipoprotein L1 in the urine sample. In certain embodiments, the product can be used to detect the level of apolipoprotein A1 or IL-8.

[0017] In certain embodiments, the assay system comprises a kit containing one or more of: a capture reagent and a detection reagent, each of which binds to a D-dimer; a signal generating element that can bind to the detection reagent; a wash buffer; a blocking buffer; information about a calibration curve for relating the signal to the D-dimer level; and / or instructions for use. In other embodiments, these same reagents can be configured to detect urine D-dimer using an LFA or VFA point-of-care test format for use in primary care settings, at home (i.e., self-testing by the patient), or in an outpatient setting, with a semi-quantitative or quantitative readout.

[0018] Aspects and advantages of these exemplary embodiments and other embodiments are discussed in detail herein. In addition, both the foregoing information and the following detailed description provide illustrative examples of various aspects and embodiments. The features of the various embodiments described herein are not mutually exclusive and may exist in various combinations and arrangements. This disclosure is not intended to identify the key features or essential features of the claimed subject matter, nor is it intended to be used as an auxiliary means for determining the scope of the claimed subject matter. The claimed subject matter is not limited to embodiments that address any or all of the shortcomings mentioned in the background technology section. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings are intended to further assist in understanding the embodiments of the present disclosure, have been incorporated into and constitute a part of this specification, and illustrate embodiments of the present disclosure and are used together with the detailed description to explain the principles of the embodiments discussed herein. No attempt is made to show the structural details of the present disclosure in more detail, except for a basic understanding of the embodiments discussed herein and the various ways of implementing them. As a matter of convention, the various features of the drawings discussed below are not necessarily drawn to scale. The sizes of the various features and elements in the drawings may be enlarged or reduced to more clearly illustrate the embodiments of the present disclosure.

[0020] Figure 1 is a schematic diagram of a bladder cancer risk stratification or treatment method according to certain embodiments of the present disclosure. .

[0021] Fig. 2A is a dot plot of urine D-dimer levels without creatinine normalization. The tested samples include 31 urology clinical controls (UC) and 37 bladder cancer patients (BC). Urine protein levels without creatinine normalization are shown. UC is represented by circles, and BC is represented by squares. Asterisks represent the significance level between groups: ****p<0.0001.

[0022] Figure 2B depicts a ROC-AUC plot generated for urine D-dimer in its ability to distinguish BC from UC without creatinine normalization. AUC values ​​and p-values ​​are listed. AUC close to 1 indicates that the protein has a higher discriminatory potential to distinguish the two groups.

[0023] Figure 3 A dot plot of urine D-dimer levels for a second validation cohort of a Chinese population containing 91 BC patients and 77 UC patients is depicted. UC subjects included 18 subjects with renal cancer, 50 subjects with renal calculi, and the remaining subjects had renal cysts, hamartomas, or other non-BC conditions. For each group, D-dimer values ​​normalized to creatinine are shown. UC is represented by circles, and BC is represented by squares. Asterisks indicate the significance level between groups: **p<0.01. DETAILED DESCRIPTION

[0024] The description may use the phrases "in certain embodiments," "in various embodiments," "in an embodiment," or "in an embodiment," which may each refer to one or more of the same or different embodiments. In addition, the terms "comprising," "including," "having," and the like as used with respect to the embodiments of the present disclosure are synonymous. The term "about" or "approximately" is defined as close to as understood by one of ordinary skill in the art. In a non-limiting embodiment, the term is defined as within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%. The term "reducing," "reduced," or any variation thereof when used in the claims and / or description includes any measurable reduction of one or more components in a mixture to achieve a desired result. The terms "wt. %, "vol. %," or "mol. %" refer to the weight, volume, or mole percentage of a component based on the total weight, total volume, or total moles of the material containing the component, respectively. In a non-limiting example, 10 grams of a component in 100 grams of a material is 10 wt. % of the component.

[0025] As used herein, a "subject" is an animal, such as a mammal, including a primate (such as a human, a non-human primate, such as a monkey). In certain aspects, the subject is a human who has, is suspected of having, is at risk of, or has had bladder cancer. The subject can be an adult subject or a pediatric subject.

[0026] As used herein, "diagnosis" refers to a method by which a person skilled in the art can assess or determine whether a patient suffers from a certain disease or has a probability (or possibility) of a risk of suffering from the disease. One such method includes using the result of a determination (e.g., an immunoassay) to detect the D-dimer level in a sample obtained from a subject, optionally together with other clinical characteristics, to obtain a diagnosis (occurrence or non-occurrence) of bladder cancer in a subject. "Diagnosis", "diagnosis" or making a diagnosis does not necessarily mean that the assessment of the disease is completely accurate. A skilled clinician can use biomarker results together with other clinical markers to diagnose. When the biomarker (e.g., D-dimer) level is located on one side of the predetermined diagnostic threshold (e.g., below the predetermined diagnostic threshold) relative to its measured level, it is determined that the subject has a greater possibility of developing a disease (e.g., bladder cancer) when the other side of the predetermined diagnostic threshold (e.g., above the threshold) is located.

[0027] As used herein, "risk stratification" refers to a method for estimating or determining an increased risk of a disease in a subject or population of subjects compared to a healthy or non-diseased population. The present disclosure provides a non-invasive method for risk stratifying bladder cancer in a subject. In certain embodiments, the subject already has bladder cancer, and the risk stratification is performed for bladder cancer recurrence.

[0028] MIBC is a more advanced stage of bladder cancer. MIBC is when the cancer has grown deep into the bladder wall (T2 stage and above). MIBC is usually treated with a combination of one or more of cystectomy, transurethral resection of bladder tumor (TURBT), chemotherapy and radiation. MIBC has a high recurrence rate. For example, in patients who undergo cystectomy, the recurrence rate can be 20-30% for T2 stage, 40% for T3, and greater than 50% for T4, and when lymph nodes are involved, the recurrence rate can usually be higher.

[0029] According to the TMN system (American Joint Committee on Cancer. Urinary Bladder. Amin MB et al., eds. AJCC Cancer Staging Manual. 8th ed. New York: Springer; 2017), primary tumors of bladder cancer can be classified as shown in Table 1.

[0030] Table 1: Primary tumor (T) classification of bladder cancer

[0031] TX The primary tumor cannot be assessed T0 No evidence of a primary tumor Ta Non-invasive papillary carcinoma Tis Carcinoma in situ: "flat tumor" T1 Tumor invades the lamina propria (subepithelial connective tissue) T2 Tumor invasion into the muscularis propria pT2a Tumor invades the superficial muscularis propria (inner half) pT2b Tumor invades the deep muscularis propria (outer half) T3 The tumor invades the tissues surrounding the bladder pT3a Through a microscope pT3b Macroscopic (extravesical mass) T4 Tumor invades any of the following: prostate stroma, seminal vesicles, uterus, vagina, pelvic wall, abdominal wall T4a Tumor invades prostate stroma, uterus, vagina T4b Tumor invades the pelvic and abdominal walls

[0032] D-dimer is a soluble degradation product of fibrin produced during the fibrinolysis process. The products and methods described herein relate to assessing the D-dimer level in a urine sample for risk stratification of bladder cancer. In certain embodiments, the D-dimer level in a urine sample is detected by immunoassay. Immunoassays generally involve contacting a sample with at least one antibody that specifically binds to D-dimer or another biomarker of interest. A signal is then generated to indicate the presence or content of a complex formed by the biomarker or its fragment in the sample in combination with the antibody. The signal is then associated with the presence or content of a biomarker (e.g., D-dimer) in the sample. Many methods and devices for the detection and analysis of biomarkers are well known. See, for example, The Immunoassay Handbook, David Wild, ed. Stockton Press, New York, 1994, which is hereby incorporated by reference in its entirety.

[0033] Any suitable immunoassay can be used, for example, enzyme-linked immunosorbent assay (ELISA), lateral flow assay (LFA), radioimmunoassay (RIA), and competitive binding assay. Assay devices and methods known in the art can utilize various sandwich, competitive or non-competitive assay formats of labeled molecules to generate signals related to the presence or amount of the biomarker of interest. Suitable assay formats also include chromatography, mass spectrometry, and protein "blotting". In addition, certain methods and devices (such as biosensors and optical immunoassays) can be used to determine the presence or amount of D-dimer without the need for labeled molecules.

[0034] In certain embodiments, the assay system is an enzyme-linked immunosorbent assay (ELISA) system. The ELISA, which can be used interchangeably with enzyme immunoassay (EIA), is a solid-phase-based assay technique designed to detect and quantify soluble substances (such as peptides, proteins, antibodies, and hormones). In ELISA, the antigen is fixed on a solid surface and then compounded with an antibody connected to a reporter enzyme. Detection is accomplished by measuring the activity of the reporter enzyme via incubation with an appropriate substrate to produce a measurable product. The most critical element of ELISA is the highly specific antibody-antigen interaction.

[0035] According to the method provided herein, any form of ELISA can be used. This form of ELISA includes direct, indirect or sandwich capture and detection methods. The key step is the fixation of the antigen of interest, which is completed indirectly by direct adsorption to the assay plate or by the capture antibody that has been attached to the plate. Then the antigen is detected directly (labeled primary antibody) or indirectly (such as labeled secondary antibody). The sandwich ELISA assay indirectly fixes and indirectly detects the presence of the target antigen, wherein the analyte to be measured is combined between two primary antibodies, and the two primary antibodies each detect different epitopes of the antigen-capture antibody and the detection antibody. The direct detection method uses a primary antibody labeled with a reporter enzyme or a label, and the primary antibody directly reacts with the antigen. Direct detection can be performed with the antigen directly fixed on the assay plate or with a capture assay format. Although direct detection is not widely used in ELISA, it is quite common for immunohistochemical staining of tissues and cells. The indirect detection method uses a labeled secondary antibody or a biotin-streptavidin complex for amplification, and is the most popular form of ELISA. The secondary antibody is specific to the primary antibody. In a sandwich ELISA, it is critical that the secondary antibody is specific only for detection by the primary antibody (and not the capture antibody), otherwise the assay will not be specific for the antigen. Typically, this is achieved by using capture and primary antibodies from different host species (e.g., mouse IgG and rabbit IgG, respectively).

[0036] In certain embodiments, the assay system is a lateral flow assay (LFA) system. A lateral flow assay system is an immunoassay format in which a test sample flows chromatographically along a hygroscopic or non-hygroscopic porous solid substrate. Lateral flow assays can be operated as competitive assays or sandwich format assays.

[0037] Lateral flow devices can be disposable, single-use test devices. For example, in certain embodiments, the LFA system includes a lateral flow test strip. The sample is applied to the test device in the application zone and passed through the substrate, wherein the sample encounters a line or zone pretreated with an antibody or antigen. The term "test zone" as used herein refers to a discrete position on the lateral flow test strip that is detected to generate a signal related to the presence or content of an analyte of interest (e.g., D-dimer). Detectable signals can be obtained by visual reading or by inserting a disposable test device into an analytical instrument (e.g., a reflectometer, a fluorometer, a transmission photometer) or any other instrument. The sample can be applied to the application zone without pretreatment, or can be premixed with one or more assay reagents before application. In the latter case, the antibody can be provided in a container separate from the disposable test device.

[0038] Subjects (e.g., patients) can use urine D-dimer instant detection lateral flow test strips (LFT) to perform urine self-tests for bladder cancer recurrence monthly (or even every 2 weeks) in the comfort of his / her home. This scheme based on home monitoring for bladder cancer recurrence has the advantage of detecting bladder cancer recurrence earlier than using invasive tests (e.g., cystoscopy) as first-line risk stratification, thereby leading to a reduction in patient morbidity and mortality. In certain embodiments, the product can also be used to detect the level of one or more of MMP-1, apolipoprotein A1, proteinase 3, and apolipoprotein L1 in the urine sample. In certain embodiments, the product can be used to detect the level of apolipoprotein A1 or IL-8.

[0039] In certain embodiments, the assay system comprises: a capture reagent and a detection reagent, each of which binds to D-dimer; a signal generating element that can bind to the detection reagent; a wash buffer; a blocking buffer; information on a calibration curve for correlating the signal with the D-dimer level; and / or instructions for use.

[0040] Certain embodiments include methods for risk stratifying bladder cancer in a subject. One such method includes the steps of detecting a D-dimer level in a first urine sample from the subject compared to a predetermined threshold level, and identifying the subject as having an increased risk of bladder cancer and as a candidate for invasive testing for bladder cancer when the D-dimer level in the first urine sample is above the predetermined threshold; and identifying the subject as not having an increased risk of bladder cancer and as a candidate for not undergoing invasive testing for bladder cancer when the D-dimer level in the first urine sample is at or below the predetermined threshold. Figure 1 In the present invention, an exemplary method 100 for risk stratification of bladder cancer is described. According to the method provided herein, a urine sample is obtained from a subject (e.g., a risk of developing bladder cancer, a risk of bladder cancer recurrence). At 102, the method measures the D-dimer level in the urine sample obtained from the subject. The method continues to 104, wherein it is determined that the D-dimer level in the sample is positive or negative. The D-dimer level in the sample can be compared with a predetermined threshold. When the D-dimer level in the sample is higher than a predetermined threshold, it can be considered that the urine D-dimer detection is "positive". When the D-dimer level in the sample is lower than a predetermined threshold, it can be considered that the urine D-dimer detection is "negative".

[0041] When the urine D-dimer test is positive, at 106, the subject is identified as having an increased likelihood (e.g., risk) of bladder cancer. At 108, the subject is further identified as a candidate for invasive or additional testing to confirm the diagnosis of bladder cancer. Invasive testing may include cystoscopy, biopsy, and resection, followed by pathological examination of samples obtained from the subject. Additional testing may include urine cytology and measurement of biomarkers (e.g., NMP22).

[0042] When the urine D-dimer test is negative, at 108, the subject is identified as having no increased likelihood (e.g., risk) of bladder cancer. At 112, the subject is further identified as a candidate for continued monitoring, e.g., without undergoing invasive testing. Continued monitoring can include follow-up clinic visits and examinations and repeat urine D-dimer tests two weeks or more (e.g., 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or more) after the first urine D-dimer test, e.g., steps 102, 104, 106, 110, 108, and / or 112.

[0043] Selecting a predetermined threshold, such as to compare with the D-dimer level in the sample at 104, involves, among other things, considering the probability of disease, the distribution of correct and incorrect diagnoses at different test thresholds, and the estimation of the results of treatment (or treatment failure) based on diagnosis. For example, when considering the administration of a specific therapy that is highly effective and has a low risk level, fewer tests are required because clinicians can accept greater diagnostic uncertainty. On the other hand, in cases where the treatment options are less effective and riskier, clinicians generally require a higher degree of diagnostic certainty. Therefore, a cost / benefit analysis involves selecting a diagnostic threshold.

[0044] Suitable threshold values ​​can be determined in various ways. One method is to set the percentile (e.g., 97.5 percentile) of a normal population. Another method can be to observe a series of samples from the same patient, in which previous "baseline" results are used to monitor the time variation of D dimer levels. And, another method involves setting a cutoff value to maximize the ROC Youden index. Youden index provides the maximum potential effectiveness of D dimer.

[0045] In certain embodiments of the methods provided herein, the subject already has bladder cancer, and the risk stratification is for bladder cancer recurrence.

[0046] In certain embodiments, the method further comprises the following steps: (i) when the D-dimer level in the first urine sample is at or below a predetermined threshold, evaluating the D-dimer level in a second urine sample obtained from the subject compared to the predetermined threshold, wherein the second urine sample is obtained two or more weeks after the first urine sample is obtained from the subject; (ii) when the D-dimer level in the second urine sample is above the predetermined threshold, identifying the subject as having an increased risk of bladder cancer and as a candidate for invasive testing for bladder cancer; and (iii) when the D-dimer level in the second urine sample is at or below the predetermined threshold, identifying the subject as not having an increased risk of bladder cancer and as a candidate for not undergoing invasive testing for bladder cancer.

[0047] In certain embodiments, the invasive testing for bladder cancer comprises one or more of a cystoscopy, a biopsy, and a transurethral resection.

[0048] In certain embodiments, D-dimer detection as provided herein is used in combination with other risk stratification or diagnostic methods for bladder cancer (e.g., bladder cancer treatment). For example, the method may further include identifying a subject identified as having an increased risk of bladder cancer as a candidate for additional testing, such as urine cytology or nuclear matrix protein 22 detection in a urine sample. In certain embodiments, the method further comprises the following steps: compared to a predetermined threshold level of MMP-1, apolipoprotein A1, proteinase 3 or apolipoprotein L1, assessing an increase in the level of one or more of MMP-1, apolipoprotein A1, proteinase 3 and apolipoprotein L1 in the urine sample obtained from the subject, and based on an increase in the level of one or more of MMP-1, apolipoprotein A1, proteinase 3 and apolipoprotein L1 in the urine sample, determining that the subject has an increased risk of bladder cancer. In certain embodiments, the method further comprises the steps of: assessing an increased level of one or more of apolipoprotein A1 and IL-8 in the urine sample obtained from the subject compared to a predetermined threshold level of apolipoprotein A1 or IL-8; and determining that the subject has an increased risk of bladder cancer based on the increased level of one or more of apolipoprotein A1 and IL-8 in the urine sample.

[0049] Cytology, nuclear matrix protein 22 (NMP22) kit, NMP22 BladderChek test, BTA-TRAK / Stat, cell search and UroVysion are FDA-approved for the initial diagnosis and monitoring of BC, and can be used in combination with the D-dimer test provided in this article. The NMP22 test evaluates the nuclear matrix proteins in urine with a wide range of sensitivity and specificity metrics. In a meta-analysis of 19 studies, the sensitivity and specificity of NMP22 were 56% (52-59%) and 88% (87-89%), respectively, with an AUC of 0.83. The average sensitivity of the NMP22 test to Ta, T1, ≥T2, Tis, G1, G2 and G3 diseases was 13.7%, 29.5%, 74.0%, 34.6%, 44.2%, 56.3% and 67.3%, respectively.

[0050] One study reported that the sensitivity of the NMP22 test and cytology for detecting BC recurrence was 78.8% and 44.2%, respectively, while the specificity was 69.6% and 83.7%, respectively (Hosseini et al. 2012 Urol J 9(1):367-72). Another study reported that the specificity of the NMP22 test and cytology was 77% and 96%, respectively, and combining the two tests increased the sensitivity to 91% (Kumar et al. 2006 J Clin Oncol 36(3):172-175). Others have analyzed results from a multicenter study comparing the two tests in 1272 subjects, 6% of whom had BC (Lotan et al. 2009 Br J Urol 103:1368-1374). In the study, although both NMP22 and cytology were independent predictors of BC, the area under the curve (AUC) for NMP22 was 76%, compared to 56.2% for cytology. However, the NMP22 test had a low PPV and a low positive post-test likelihood ratio (3.5 vs. 19) compared to cytology.

[0051] The risk stratification and detection methods provided herein can be combined with any treatment for bladder cancer to treat subjects identified as suffering from bladder cancer. As used herein, the term "treating" or "treatment" refers to a beneficial or desired result in the context of treating a disease, such as reducing at least one related sign, symptom, condition or complication in a subject. "Treatment" also refers to preventive treatment, such as preventing a disease or preventing at least one sign, symptom, condition or complication associated with a disease. "Treatment" can also mean extending the survival period compared to the expected survival period without treatment. Treatment of bladder cancer according to the method provided herein can include surgical resection, chemotherapy, radiation and hormone therapy.

[0052] In certain embodiments, a method for treating bladder cancer in a subject comprises the following steps: (i) evaluating a urine sample obtained from the subject for an elevated level of D-dimer compared to a predetermined threshold level by enzyme-linked immunosorbent assay (ELISA) or lateral flow assay (LFA); (ii) identifying the subject as having an increased risk of bladder cancer when the level of D-dimer in the urine sample is above the predetermined threshold; (iii) performing an invasive test for bladder cancer on the subject; (iv) determining that the subject has bladder cancer based on the results of the invasive test; and (v) treating the subject for bladder cancer by one or more of surgical resection, radiation, chemotherapy, and hormonal therapy. In certain embodiments, the subject already has bladder cancer and the treatment is for a recurrence of bladder cancer. In certain embodiments, the invasive test for bladder cancer comprises one or more of the following: cystoscopy, biopsy followed by pathology, transurethral resection followed by pathology, urine cytology, and evaluating the level of nuclear matrix protein 22 in a urine sample. In certain embodiments, the method further comprises the steps of assessing the urine sample obtained from the subject for an increased level of one or more of MMP-1, apolipoprotein A1, proteinase 3, and apolipoprotein L1 compared to a predetermined threshold level of MMP-1, apolipoprotein A1, proteinase 3, or apolipoprotein L1, and determining that the subject has an increased risk of bladder cancer based on the increased level of one or more of MMP-1, apolipoprotein A1, proteinase 3, and apolipoprotein L1 in the urine sample. In certain embodiments, the method further comprises the steps of assessing the urine sample obtained from the subject for an increased level of one or more of apolipoprotein A1 and IL-8 compared to a predetermined threshold level of apolipoprotein A1 or IL-8, and determining that the subject has an increased risk of bladder cancer based on the increased level of one or more of apolipoprotein A1 and IL-8 in the urine sample.

[0053] The method for risk stratification and detection of bladder cancer provided herein (using urine D-dimer to monitor bladder cancer (e.g., recurrence)) provides several advantages compared to current technology. In certain embodiments, compared to current diagnostic methods, the method described herein is more accurate, with a ROC AUC value of 96-97%. These methods are non-invasive, and painless, with almost no side effects. In certain embodiments, the method is easy to handle, and if measured as a lateral flow test for detecting urine D-dimer, rapid determination results can be obtained within 10 minutes. In certain embodiments, the method described herein requires minimal training or expertise, because patients can use lateral flow strips for self-detection, which is contrary to cystoscopy and cytology, which both require expensive instruments, specialized training expertise that is only available in urology referral centers. In certain embodiments, the method described herein can potentially help detect bladder cancer recurrence earlier, because patients can self-check their urine every week or two weeks, contrary to the current follow-up period (which ranges from 3 months to >1 year). Early detection of bladder cancer recurrence will translate into earlier therapy, leading to reduced morbidity and mortality.

[0054] Example

[0055] Example 1: Identification of D-dimer as a biomarker for bladder cancer

[0056] A comprehensive targeted proteomic analysis of 1300 specific proteins in bladder cancer was performed compared with the urine protein profile of urological clinical (UC) control patients. UC does not refer to urothelial carcinoma, but to urological clinical controls. The top 30 most discriminating proteins were further validated by ELISA. The independent validation cohort of ELISA consisted of 31 UC samples and 37 BC samples at different stages of the disease. Among them, 30 subjects (10 Ta samples, 10 Tis samples, 10 T1 samples) were classified as NMIBC, while 7 subjects (T2-T4 stage samples) were classified as MIBC. Urological clinical controls include patients who were tested for hematuria but were not found to have any urological cancer. Unless otherwise stated, all bladder cancer subjects included in this study suffered from urothelial carcinoma. The results of this validation are shown in Table 2. Among the 30 most discriminating proteins validated by ELISA, D-dimer is the urine protein with the best accuracy value for distinguishing BC patients from controls. Furthermore, urine D-dimer demonstrated sensitivity and specificity values ​​of 0.90 to 0.95, far exceeding current diagnostic platforms.

[0057] Table 2: Urinary protein markers validated by ELISA for detecting BC but not UC

[0058]

[0059] In Table 2, biomarker protein units (normalized to creatinine) are as follows: n = ng / mg, p = pg / mg. Statistical significance was analyzed by the Mann-Whitney U test comparing BC to UC. Sensitivity 0.8 The sensitivity at a fixed specificity value of 0.8 is depicted. Indicated are statistically significant p values ​​comparing BC to UC, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0060] Example 2: Bladder cancer diagnostic performance of urine D-dimer without creatinine normalization

[0061] To evaluate the discriminatory potential of urine D-dimer normalized to creatinine. Figures 2A and 2B As shown, urine D-dimer (normalized to creatinine) was discriminatory between UC and BC, with an AUC value of 0.97 ( Figures 2A and 2B ). The dot plot in FIG2A depicts urine D-dimer levels in the case of creatinine normalization. The samples tested included 31 UCs and 37 BCs. Non-creatinine normalized urine protein levels are shown. UCs are represented by circles and BCs are represented by squares. Asterisks indicate the significance level between the groups: ****p<0.0001. FIG2B depicts a ROC-AUC graph generated for urine D-dimer in terms of its ability to distinguish BC from UC without creatinine normalization. AUC values ​​and p values ​​are listed. If the AUC is close to 1, the protein has a higher potential for distinguishing the two groups.

[0062] Example 3: Bladder cancer diagnostic performance of urine D-dimer with creatinine normalization

[0063] The diagnostic performance of urine D-dimer was evaluated in a second cohort including Chinese BC patients. Figure 3 As shown, urine D-dimers in BC patients were significantly higher than those in UC controls. The dot plot depicts urine D-dimer levels in a second validation cohort of a Chinese population including BC patients (N=91) and UC (N=77). UC subjects included 18 subjects with renal cancer, 50 subjects with renal calculi, and the remaining subjects had renal cysts, hamartomas, etc. For each group, creatinine-normalized protein values ​​are shown. UC is represented by circles, and BC is represented by squares. Asterisks indicate the significance level between groups: **p<0.01.

[0064] According to the disclosed method, monitoring previously diagnosed with bladder cancer and the recurrence of bladder cancer in a subject for the treatment of bladder cancer. During a follow-up visit to a urology clinic, a urine sample is obtained from the subject, and the D-dimer level is measured (by ELISA or LFA). When urine detection D-dimer is positive (that is, the D-dimer level is higher than a predetermined threshold), the patient undergoes a bladder biopsy (by cystoscopy) to confirm the diagnosis of bladder cancer recurrence. When urine detection D-dimer is negative (that is, the D-dimer level is or lower than a predetermined threshold), a simple review will be performed to the patient in subsequent follow-ups. The method provided herein enables most patients to be free from unnecessary invasive / expensive procedures, which are generally characterized by diagnostic features lower than standard, thereby realizing overall savings for the patient and overall health care and also reducing the incidence of cystoscopy-related side effects. The early detection of bladder cancer can result in a reduction in patient morbidity and mortality.

[0065] In patients with hematuria, it will be helpful to identify who needs invasive cystoscopy assessment. Given that urine protein is easily measurable and suitable for instant detection, rapid urine testing may significantly affect triage and workflow in urology clinics. Similarly, in bladder cancer monitoring, reliable urine biomarkers can help determine whether cancer (such as carcinoma in situ) is missed or avoid cystoscopy in marker-negative patients. Similarly, urine biomarkers that can reliably distinguish MIBC from NMIBC can inform medical professionals who suffer from more aggressive diseases. When used as conventional instant detection (at home or in outpatient visits), these urine biomarkers can promote early identification of invasive diseases and design customized therapies.

[0066] As part of this work, >1000 urine proteins were screened for urine biomarker candidates in BC using aptamer-based screening. Systems biology analysis implicated molecular functions associated with extracellular matrix, collagen, integrin, heparin, and transmembrane tyrosine kinase signaling in BC susceptibility, with HNF4A and NFKB1 being key regulators. STEM analysis of dysregulated pathways implicated the functional roles of the immune system, complement, and interleukins in BC disease progression. Several urine proteins (D-dimer, apolipoprotein A1, MMP-1, properdin, calgranulin B) significantly distinguished BC from UC, with AUC values ​​ranging from 0.85 to 0.96 (p-value <0.0001). Urine D-dimer was able to distinguish BC from UC with 96% accuracy (sensitivity = 95%; specificity = 90%). As a single biomarker, urine D-dimer outperforms current FDA-approved biomarkers and competing biomarkers in the research literature as a sensitive biomarker for BC detection.

[0067] Embodiments of the present disclosure include multi-marker panels and methods for comparing BC and UC or for comparing MIBC and NMIBC. These multi-marker panels may include two or more of the following: urine D-dimer, MMP-1, apolipoprotein A1, proteinase 3, apolipoprotein L1, IL-8, ficolin-3, and properdin. Certain embodiments include multi-marker panels containing apolipoprotein A1, D-dimer, and IL-8. Certain embodiments include a 5-marker panel containing urine D-dimer, MMP-1, apolipoprotein A1, proteinase 3, and apolipoprotein L1. One such embodiment using the 5-marker panel to compare BC and UC had an AUC of 0.95, a sensitivity of 0.89, and a specificity of 0.87. Certain embodiments include a 5-marker panel containing urine IL-8, ficolin-3, apolipoprotein L1, properdin, and proteinase 3. One such embodiment using the 5-marker panel to compare MIBC to NMIBC had an AUC of 0.98, a sensitivity of 0.79, and a specificity of 0.95. Three proteins in this panel (IL-8, proteinase 3, apolipoprotein L1) were also ranked among the best performing individual markers based on their individual AUC values. Another embodiment comparing MIBC to NMIBC included a multi-marker panel containing two or more of the following eight urine proteins: urine apolipoprotein L1, complement C2, endothelial cell-specific molecule, fibronectin, IgA, IL-8, MMP-12, and proteinase 3. Their AUC values ​​ranged from 0.75 to 0.99. Urine IL-8 best distinguished the two groups (AUC = 0.99, sensitivity = 100%; specificity = 93%). As shown in Table 3, urine IgA also outperformed the other markers, with an AUC of 89%, a sensitivity of 86%, and a specificity of 87%. Of particular note, at 80% sensitivity, urinary IgA showed the highest specificity of 80% for MIBC, outperforming IL-8.

[0068] In Table 3, the ability of 30 ELISA-validated urine proteins to differentiate bladder cancer patients by their own disease stage. Thirty urine proteins were measured by ELISA using a UTSW cohort that included 7 MIBC subjects and 30 NMIBC subjects. ELISA results for MIBC vs. NMIBC are shown. Biomarker protein units (normalized to creatinine) are as follows: n = ng / mg, p = pg / mg. Specificity 0.8 Specificity at a fixed sensitivity of 0.8 is depicted. Indicated are statistically significant p values ​​determined by Mann-Whitney U test, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0069] Table 3: Results of 30 ELISAs for MIBC vs. NMIBC stage comparison.

[0070]

[0071] According to the aforementioned drawings, detailed description and examples, other purposes, features and advantages of the present disclosure will become apparent. However, it should be understood that while indicating specific embodiments of the present disclosure, the drawings, detailed description and examples are given only by way of illustration and are not intended to be limiting. In addition, it will become apparent to those skilled in the art that changes and modifications within the spirit and scope of the present disclosure will become apparent upon consideration based on the detailed description. In other embodiments, features from a specific embodiment may be combined with features from other embodiments. For example, features from one embodiment may be combined with features from any of the other embodiments. In other embodiments, additional features may be added to the specific embodiments described herein.

Claims

1. A non-invasive method for risk stratifying bladder cancer in a subject, the method comprising: (a) obtaining a first urine sample from the subject; (b) assessing the level of D-dimer in the first urine sample obtained from the subject compared to a predetermined threshold; (c) identifying the subject as having an increased risk for bladder cancer and as a candidate for invasive testing for bladder cancer when the D-dimer level in the first urine sample is above the predetermined threshold; and (d) identifying the subject as not having an increased risk for bladder cancer and as not being a candidate for invasive testing for bladder cancer when the D-dimer level in the first urine sample is at or below the predetermined threshold.

2. The method of claim 1, wherein the subject already has bladder cancer and the risk stratification is performed for bladder cancer recurrence.

3. The method according to claim 1 or 2, further comprising the following steps: (e) when the D-dimer level in the first urine sample is at or below the predetermined threshold, evaluating the D-dimer level in a second urine sample obtained from the subject compared to a predetermined threshold, wherein the second urine sample is obtained two or more weeks after the first urine sample is obtained from the subject; (f) identifying the subject as having an increased risk for bladder cancer and as a candidate for invasive testing for bladder cancer when the D-dimer level in the second urine sample is above the predetermined threshold; and (g) identifying the subject as not having an increased risk for bladder cancer and as not being a candidate for invasive testing for bladder cancer when the D-dimer level in the second urine sample is at or below the predetermined threshold.

4. The method of any one of claims 1 to 3, wherein the invasive testing for bladder cancer comprises one or more of cystoscopy, biopsy, and transurethral resection.

5. The method according to claim 1 or 2, further comprising assessing the urine sample obtained from the subject for an elevated level of one or more of MMP-1, apolipoprotein A1, proteinase 3, and apolipoprotein L1 compared to a predetermined threshold level of MMP-1, apolipoprotein A1, proteinase 3, or apolipoprotein L1; and The subject is determined to have an increased risk of bladder cancer based on the elevated level of one or more of MMP-1, apolipoprotein A1, proteinase 3, and apolipoprotein L1 in the urine sample.

6. A method for identifying a subject for invasive testing for bladder cancer, the method comprising: obtaining a first urine sample from the subject; assessing an elevated D-dimer level in a urine sample obtained from the subject compared to a predetermined threshold level; determining that the subject has an increased risk of bladder cancer based on the elevated level of the D-dimer in the urine sample; as well as The subject is subjected to invasive testing for bladder cancer.

7. The method of claim 6, wherein the subject already has bladder cancer and the evaluation is for bladder cancer recurrence.

8. The method of claim 6 or 7, wherein the invasive testing for bladder cancer comprises one or more of cystoscopy, biopsy, and transurethral resection.

9. The method according to any one of claims 6 to 8, further comprising performing urine cytology or assessing the level of nuclear matrix protein 22 in the urine sample obtained from the subject determined to be at increased risk for bladder cancer.

10. The method according to any one of claims 6 to 9, wherein the D-dimer level in the urine sample is not normalized with the creatinine level in the urine sample for comparison with the predetermined threshold value.

11. The method according to any one of claims 6 to 9, wherein the D-dimer level in the urine sample is normalized with the creatinine level in the urine sample for comparison with the predetermined threshold value.

12. A method for treating bladder cancer in a subject, the method comprising: (a) assessing an increased level of D-dimer in a urine sample obtained from the subject compared to a predetermined threshold level by enzyme-linked immunosorbent assay (ELISA) or lateral flow assay (LFA); (b) identifying the subject as having an increased risk of bladder cancer when the D-dimer level in the urine sample is above the predetermined threshold; (c) performing invasive testing for bladder cancer on the subject; (d) determining that the subject has bladder cancer based on the results of the invasive test; as well as (e) treating the bladder cancer in the subject by one or more of surgical resection, radiation, chemotherapy, and hormone therapy.

13. The method of claim 12, wherein the subject already has bladder cancer and the subject is treated for a recurrence of bladder cancer.

14. The method of claim 12 or 13, wherein the invasive testing for bladder cancer comprises one or more of cystoscopy, biopsy, and transurethral resection.

15. The method according to any one of claims 12 to 14, further comprising: assessing the urine sample obtained from the subject for an elevated level of one or more of MMP-1, apolipoprotein A1, proteinase 3, and apolipoprotein L1 compared to a predetermined threshold level of MMP-1, apolipoprotein A1, proteinase 3, or apolipoprotein L1; and The subject is determined to have an increased risk of bladder cancer based on the elevated level of one or more of MMP-1, apolipoprotein A1, proteinase 3, and apolipoprotein L1 in the urine sample.

16. An assay system for assessing D-dimer levels in a urine sample for use in risk stratifying bladder cancer.

17. The assay system according to claim 16, wherein the assay system is an enzyme-linked immunosorbent assay system.

18. The assay system of claim 16, comprising a lateral flow test strip.

19. The assay system according to any one of claims 16 to 18, comprising: A capture reagent and a detection reagent, each of which binds to D-dimer; a signal generating element, the signal generating element being capable of binding to the detection reagent; Wash buffer; Blocking buffer; a calibration curve for correlating the signal to the D-dimer level; and / or Instructions for use.

20. The assay system of claim 16, wherein the assay system is a vertical flow assay.

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