Evaluation of hematuria and other urinary symptoms

Abstract

Methods for evaluating subjects with hematuria and / or other suspicious urinary tract symptoms indicative of malignancy are provided, and these methods are particularly useful for detecting primary bladder cancer in these subjects, or alternatively, for ruling out bladder cancer (particularly high-grade bladder cancer). The provided methods are based on the analysis of DNA methylation markers in DNA from cells of a urine sample from the subject.

Description

[Technical Field]

[0001] The present invention relates to the evaluation of subjects with hematuria and other suspicious urinary tract symptoms that may indicate malignant disease by analyzing DNA methylation markers in DNA derived from cells of a urine sample from the subject. [Background technology]

[0002] Hematuria and other lower urinary tract symptoms (LUTS) are common, accounting for an estimated 20% of urological evaluations. Urological etiologies of hematuria include malignancy, infection, inflammation, stone disease, benign prostatic hyperplasia (BPH), and congenital or acquired anatomical abnormalities. Renal disease and anticoagulant therapy can also cause hematuria. Hematuria can also be confused with urinary pigmentation from gynecological bleeding sources, myoglobinuria, or the ingestion of certain foods and drugs. Hematuria can be defined as microscopic hematuria (subvisible) and gross hematuria (hematuria). Suspicion of microscopic hematuria is often raised by a positive urine dipstick test, and gross hematuria is often reported by the patient. Suspicion of hematuria must be confirmed by urine microscopy. Hematuria is typically defined as the detection of 3 or more red blood cells in a high-power field (RBC / HPF), with the definition of microscopic versus macroscopic hematuria set at a cutoff of 25 RBC / HPF.

[0003] According to the 2020 AUA / SUFU (American Urological Association and Society of Urodynamics, Female Pelvic Medicine & Urogenital Reconstruction) guidelines on microscopic hematuria, the evaluation of hematuria should involve multiple medical disciplines. The detection of benign causes of hematuria, such as urinary tract infection or medical renal disease, does not exclude the presence of additional malignant etiologies, which may still require urological attention and further workup. The prevalence of bladder cancer was found to be 0.4% in the low-risk group, 1.0% in the intermediate-risk group, and 6.3% in the high-risk group.

[0004] Urological workup is tailored to a patient's risk of malignancy and includes cystoscopy, which uses an endoscope (cystoscope) to visually inspect the bladder, as well as imaging methods such as ultrasound and computed tomography (CT). Evaluation procedures are associated with various risks and patient discomfort; for example, cystoscopy is invasive, uncomfortable, and associated with the risk of infection, and imaging procedures may involve exposure to radiation. From a healthcare system perspective, evaluation procedures are laborious, expensive, and require specially trained personnel. The high prevalence of hematuria not associated with cancer can result in numerous unnecessary evaluation procedures.

[0005] Urine cytology is recommended for the initial evaluation of patients with gross hematuria in combination with cystoscopy and CT, but not for patients with microscopic hematuria, and is not recommended as a stand-alone test that can confirm or rule out cancer. This is likely due to two aspects: first, its relatively high specificity (approximately 85–90%) yields a favorable PPV in the gross hematuria population, but in the microscopic hematuria population, many individuals may still receive false-positive results, meaning that follow-up procedures are actually unnecessary in many individuals. Second, the sensitivity of cytology is moderate (50–60%) for high-grade disease, and therefore the test cannot reliably rule out high-grade disease. Therefore, cytology is not recommended as a stand-alone test that can direct the next steps in patient evaluation, such as cystoscopy and imaging.

[0006] There is underdiagnosis of hematuria and inconsistencies in care, with 23% of at-risk patients reported to undergo any type of imaging and 13% undergoing cystoscopy. Women and African Americans tend to have lower rates of hematuria evaluation. As a result, approximately 25% of bladder cancers are diagnosed at an invasive stage (AUA / SUO NMIBC guidelines). Delayed diagnosis of bladder cancer has been suggested to contribute to a 34% increased risk of cancer-specific mortality and a 15% increased risk of all-cause mortality.

[0007] Therefore, a bladder cancer biomarker that can be assayed quickly and non-invasively, preferably in the community, at the time of early detection of hematuria, has very high specificity and therefore a very low false positive rate, and also has high sensitivity for high-grade cancers, would be highly beneficial in identifying patients who should be promptly referred for urological evaluation. Such a biomarker would be useful in reliably ruling out high-grade bladder cancer in subjects with suspected hematuria and / or lower urinary tract symptoms, reducing the number of unnecessary evaluation tests.

[0008] There is a need for improved methods for evaluating subjects with hematuria and other lower urinary tract symptoms that can effectively identify patients at risk of having bladder cancer, particularly high-grade bladder cancer, and refer them for urological evaluation before progression to invasive disease. Summary of the Invention

[0009] The present invention provides methods for evaluating subjects with hematuria and / or other lower urinary tract symptoms associated with suspected malignancy, and these methods are particularly useful for detecting or alternatively ruling out primary bladder cancer in these subjects. The present invention is particularly useful with respect to high-grade bladder cancer.

[0010] The method of the present invention is non-invasive and utilizes a set of DNA methylation markers that can be detected in cells present in urine samples. More specifically, the method of the present invention utilizes a set of DNA methylation markers previously identified as being highly methylated in bladder cancer DNA compared to normal DNA. These markers are disclosed herein to have exceptionally high specificity for bladder cancer in a specific subject population, namely, subjects presenting with hematuria and / or additional lower urinary tract symptoms associated with suspected malignancy. As exemplified below, the set of markers demonstrated a specificity of over 98%. The set of markers was tested in two groups of subjects with suspicious lower urinary tract symptoms but no evidence of bladder cancer. Notably, the set of markers produced zero false-positive results in one of the groups and only three false-positive results in the other group, resulting in a specificity of 98% to 100%. Furthermore, the set of markers was tested in a group of subjects with histologically confirmed primary bladder cancer and was found to have an especially high sensitivity of 87.5% in detecting high-grade primary bladder cancer. Such high specificity in subjects with hematuria and / or other suspicious lower urinary tract symptoms, along with the marker's high sensitivity, is useful for identifying primary bladder cancer in this patient population, or alternatively, for reliably excluding high-grade bladder cancer, optimizing referral of hematuric patients for urological evaluation and timely cancer detection.

[0011] Using the disclosed markers, selected from a defined set of markers, high-grade bladder cancer can be detected or ruled out already during the initial evaluation of a subject with suspicious urinary symptoms, as the assay is non-invasive, reliable, and easy to operate. The ability to detect or rule out high-grade bladder cancer already during the initial evaluation stage is beneficial both for the timing of evaluation (as soon as possible after detection of suspicious symptoms) and also for access to evaluation for underserved or underrepresented populations, such as women and African Americans.

[0012] According to one aspect, the present invention provides a method for determining the presence or absence of primary bladder cancer in a human subject having at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS), the method comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from a subject; (b) calculating a subject score based on the methylation value; (c) detecting that the calculated score is above a predetermined cutoff, thereby determining a positive probability for the presence of primary bladder cancer in the subject.

[0013] According to another aspect, the present invention provides a method for evaluating a human subject having at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS), the method comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from a subject; (b) calculating a subject score based on the methylation value; (c) detecting that the calculated score is above a predetermined cutoff, thereby determining a positive probability for the presence of bladder cancer in the subject; and (d) requiring a cystoscopy for subjects with a score above a predetermined cutoff.

[0014] According to a further aspect, the present invention provides a method for ruling out bladder cancer in a human subject having at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS), the method comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from a subject; (b) calculating a subject score based on the methylation value; (c) detecting that the calculated score is below a predetermined cutoff, thereby ruling out bladder cancer in the subject; and (d) avoiding cystoscopy in the subject.

[0015] According to a further aspect, the present invention provides a method for assessing a human subject having at least one condition selected from hematuria and lower urinary tract symptoms (LUTS), the method comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from a subject; (b) calculating a subject score based on the methylation value; (c) detecting that the calculated score is below a predetermined cutoff, thereby ruling out bladder cancer in the subject; and (d) performing on the subject at least one assay selected from the group consisting of serum creatinine measurement, glomerular filtration rate measurement, blood calcium measurement, blood uric acid measurement, urine pH measurement, urine calcium measurement, urine culture, uroflowmetry, post-void residual volume test, renal ultrasound, abdominal ultrasound, and prostate ultrasound to assess the presence of a non-malignant genitourinary condition in the subject, wherein each assay represents a separate embodiment of the present invention.

[0016] In some embodiments, the non-malignant genitourinary condition is selected from the group consisting of a urinary tract infection, a kidney infection, kidney stones, bladder stones, benign prostatic hyperplasia (BPH), kidney disease or injury, and a congenital or acquired anatomical abnormality.

[0017] According to a further aspect, the present invention provides a method for assessing a human subject having hematuria, the method comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from a subject; (b) calculating a subject score based on the methylation value; (c) detecting that the calculated score is below a predetermined cutoff; and (d) administering a repeat urine test to the subject within 6 to 12 months.

[0018] In some embodiments, the subject has one or more lower urinary tract symptoms (LUTS).

[0019] According to a further aspect, the present invention provides a method for assessing the presence or absence of high-grade primary bladder cancer in a human subject having at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS), the method comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from a subject; (b) calculating a subject score based on the methylation value; (c) detecting that the calculated score is above a predetermined cutoff, thereby determining a positive probability for the presence of high-grade bladder cancer in the subject.

[0020] According to another aspect, the present invention provides a method for ruling out high-grade primary bladder cancer in a human subject having at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS), the method comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from a subject; (b) calculating a subject score based on the methylation value; (c) detecting that the calculated score is below a predetermined cutoff, thereby ruling out high-grade bladder cancer in the subject; and (d) avoiding cystoscopy in the subject.

[0021] In some embodiments, the method further comprises assessing the subject for the presence or absence of a non-malignant genitourinary condition.

[0022] In some embodiments, the assessment of the presence or absence of a non-malignant genitourinary pathology comprises at least one assay selected from the group consisting of serum creatinine measurement, glomerular filtration rate measurement, blood calcium measurement, blood uric acid measurement, urine pH measurement, urine calcium measurement, urine culture, uroflowmetry, post-void residual volume test, renal ultrasound, abdominal ultrasound, and prostate ultrasound.

[0023] According to a further aspect, the present invention provides a method for managing a human subject having at least one condition selected from hematuria and lower urinary tract symptoms (LUTS), the method comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from a subject; (b) calculating a subject score based on the methylation value; (c) detecting that the calculated score is above a predetermined cutoff; and (d) performing confirmatory bladder cancer testing on subjects with a score above a predetermined cutoff, including at least one of standard cystoscopy, enhanced cystoscopy, urine cytology, physical examination, biopsy, imaging using at least one of computed tomography (CT), ultrasound, and magnetic resonance imaging (MRI), and transurethral resection of bladder tumor (TURBT). Each option represents a separate embodiment of the present invention.

[0024] In some specific embodiments, the confirmatory bladder cancer test is cystoscopy. Thus, in some embodiments, provided herein is a method for managing a human subject having at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS), the method comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from the subject; (b) calculating a score for the subject based on the methylation value; (c) detecting that the calculated score is above a predetermined cutoff; and (d) performing a cystoscopy on subjects having a score above the predetermined cutoff.

[0025] In some embodiments where positive or suspicious finding(s) are identified by cystoscopy, the method of managing the subject further comprises performing at least one of a biopsy and a surgical resection of the finding(s) (TURBT) on the subject. Each option represents a separate embodiment of the present invention. The specimen(s) are then examined by a pathologist to definitively confirm or rule out the presence of a bladder tumor. A pathological diagnosis typically further includes characterizing the type, stage, and grade of bladder cancer. Patients with muscle-invasive tumors may require further workup to assess the extent of tumor spread, including ultrasound (US), CT, PET-CT, and MRI. Each option represents a separate embodiment of the present invention.

[0026] In some embodiments, the method of managing a subject further comprises characterizing the type, stage, and grade of bladder cancer in a subject identified with the presence of bladder cancer.

[0027] In some embodiments, the method of managing a subject further comprises administering a treatment to treat bladder cancer to a subject in whom the presence of bladder cancer has been confirmed and the type, stage, and grade of bladder cancer has been characterized. In some embodiments, the treatment comprises one or more of transurethral resection of bladder tumor (TURBT), cystectomy (partial or radical), lymphadenectomy, chemotherapy (intravesical or systemic), radiation therapy, immunotherapy (intravesical or systemic), and targeted therapy. Intravesical chemotherapy may comprise one or more of mitomycin, gemcitabine, epirubicin, and valrubicin. Intravesical chemotherapy may be administered directly, as hyperthermic intravesical chemotherapy, as microwave-induced thermotherapy, or as electrophysiological drug administration. Systemic immunotherapy may include one or more of platinum-based (e.g., cisplatin, carboplatin), fluorouracil (5-FU), mitomycin, gemcitabine, methotrexate, vinblastine, doxorubicin, docetaxel, paclitaxel, and vinflunine. Intravesical immunotherapy may include one or more of Bacillus Calmette-Guerin (BCG), interferon-α, and nadofalagen filadenovec. Systemic immunotherapy may include one or more of immune checkpoint inhibitors such as PD-1 and PD-L1 inhibitors (e.g., avelumab, nivolumab, atezolizumab, and pembrolizumab), and antibody-drug conjugates such as enfortumab vedotin and sacituzumab govitecan. Targeted therapy may include a fibroblast growth factor receptor (FGFR) inhibitor, such as erdafitinib. Treatment may further include supportive care. Various treatment options may be combined (e.g., chemoradiotherapy, combined intravenous infusion therapy). Each treatment option and combination represents a separate embodiment of the present invention.

[0028] The type of treatment is determined by a skilled physician(s) according to tumor characteristics, including tumor type, stage, and grade. The type of treatment is typically also determined based on additional factors, such as patient characteristics.

[0029] According to another aspect, there is provided herein a method for managing a human subject having at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS), the method comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from a subject; (b) calculating a subject score based on the methylation value; (c) detecting that the calculated score is below a predetermined cutoff, thereby ruling out bladder cancer in the subject and avoiding cystoscopy; and (d) performing at least one assay on the subject selected from the group consisting of serum creatinine measurement, glomerular filtration rate measurement, blood calcium measurement, blood uric acid measurement, urine pH measurement, urine calcium measurement, urine culture, uroflowmetry, post-void residual volume test, renal ultrasound, abdominal ultrasound, and prostate ultrasound to assess the presence of a non-malignant genitourinary condition.

[0030] According to a further aspect, there is provided herein a method for managing a human subject with hematuria, the method comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from a subject; (b) calculating a subject score based on the methylation value; (c) detecting that the calculated score is below a predetermined cutoff; and (d) administering a urine test to the subject within 6 to 12 months.

[0031] In some embodiments, lower urinary tract symptoms (LUTS) include at least one symptom selected from urinary frequency, urgency, urge incontinence, nocturia, altered urinary flow, hesitancy to urinate, straining and dribbling, and residual urine, with each option representing a separate embodiment of the present invention.

[0032] In some embodiments, the hematuria is microscopic hematuria. In other embodiments, the hematuria is gross hematuria.

[0033] In some embodiments, the at least one marker locus comprises one or more (preferably a plurality) marker loci selected from the group consisting of SEQ ID NOs: 1-15.

[0034] In some embodiments, at least one marker locus comprises the locus set forth in SEQ ID NO: 1. In some embodiments, at least one marker locus further comprises the locus set forth in SEQ ID NO: 5. In some embodiments, at least one marker locus comprises the locus set forth in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 1 ..., and further comprises at least one additional marker locus selected from the group of loci set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. In further embodiments, the at least one additional marker locus comprises the locus set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

[0035] In some embodiments, the methylation value is determined using one or more analytical methods selected from the group consisting of DNA sequencing, real-time PCR, and array hybridization.

[0036] In some embodiments, determining the methylation value comprises, for a plurality of potential methylation sites in the plurality of marker loci, quantifying methylated read counts at the potential methylation sites, and calculating the methylation value based on the number of methylated read counts.

[0037] In some embodiments, determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 and calculating a score comprises: (i) digesting DNA from cells of a urine sample from the subject with at least one methylation-sensitive restriction endonuclease that recognizes a sequence within the at least one marker locus that is highly methylated in cancer DNA compared to non-cancer DNA to obtain restriction endonuclease-treated DNA; (ii) co-amplifying at least one marker locus and a control locus from the restriction endonuclease-treated DNA, thereby generating an amplification product for each locus; (iii) determining the signal intensity of each generated amplification product; and (iv) comparing the ratio between the signal intensities of the amplification products for each of the at least one marker locus and the control locus to at least one reference ratio selected from a cancer reference ratio and a non-cancer reference ratio to calculate a score.

[0038] In some embodiments, step (i) is carried out using a single methylation-sensitive restriction endonuclease. In some embodiments, the methylation-sensitive restriction endonuclease is HhaI. In additional embodiments, the methylation-sensitive restriction endonuclease is HinP1I.

[0039] In some embodiments, the control locus is a locus that does not contain a nucleotide sequence recognized by a methylation-sensitive restriction endonuclease.

[0040] In some embodiments, the methylation-sensitive restriction endonuclease is selected from HhaI and HinP1I, and the control locus is the locus set forth in SEQ ID NO:16.

[0041] In some embodiments, step (ii) is performed using real-time PCR. In some embodiments, step (ii) comprises adding a fluorescent probe to assist in detection of the amplification products of the at least one marker locus and the control locus. In some embodiments, determining the methylation values ​​for the plurality of marker loci comprises determining a ΔCq for each marker locus and calculating a single composite methylation value based on the ΔCq determined for each marker locus. In some embodiments, the ratio between the signal intensities of the amplification products of each of the at least one marker locus and the control locus determines a quantification cycle (Cq) for each locus, and is calculated by multiplying the ΔCq by 2. (Cq対照遺伝子座-Cq制限遺伝子座) It is calculated by calculating

[0042] These and further aspects and features of the present invention will become apparent from the following detailed description, examples and claims. DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention relates to the evaluation of subjects with hematuria and other suspicious lower urinary tract symptoms (LUTS) that may be indicative of malignant disease by analyzing DNA methylation markers in DNA derived from cells of a urine sample from the subject.

[0044] Advantageously, DNA methylation markers are suitable for methylation analysis, which involves enzymatic digestion of DNA with at least one methylation-sensitive restriction enzyme, followed by real-time PCR of the methylation markers and an internal reference locus, and then accurate quantification of the ratio between the signal obtained from each marker locus and the signal from the internal reference locus. Thus, in some embodiments, methylation analysis according to the present invention does not require assessment of absolute methylation levels at the analyzed genomic loci, but rather requires calculation of a signal ratio (reflecting the methylation ratio) between the analyzed genomic loci and an internal reference locus in the same sample. This contrasts with conventional methods utilizing methylation analysis to distinguish tumor-derived DNA from normal DNA, which require determining the actual methylation level at a particular genomic locus. Thus, embodiments of the present invention eliminate the need for standard curves and / or additional laborious steps involved in determining the methylation level itself, thereby providing a simple and cost-effective procedure. An additional advantage over known approaches for analyzing methylation is provided by the signal ratio obtained according to some embodiments of the present invention, which is calculated between loci amplified in the same reaction mixture (i.e., under the same reaction conditions). This makes them immune to various "noise" factors, such as variations in template DNA concentration, PCR conditions, the presence of inhibitors, etc. Such noise is inherent in methods based on quantifying the methylation level of a locus by comparing signals from separate amplification reactions.

[0045] Methylation in the human genome occurs in the form of 5-methylcytosine and is limited to cytosine residues that are part of CG sequences, also known as CpG dinucleotides (cytosine residues that are part of other sequences are unmethylated). Some CpG dinucleotides in the human genome are methylated, while others are unmethylated. Furthermore, methylation is cell- and tissue-specific; therefore, a particular CpG dinucleotide may be methylated in certain cells and unmethylated in other cells, or methylated in certain tissues and unmethylated in other tissues. DNA methylation is a key regulator of gene transcription. Aberrant DNA methylation patterns, both hypermethylated and hypomethylated compared to normal tissues, are associated with numerous human malignancies.

[0046] The term "bladder cancer" refers to cancer that arises in the bladder and includes transitional cell carcinoma (TCC, also called urothelial cell carcinoma), squamous cell carcinoma, adenocarcinoma, small cell carcinoma, and sarcoma. Each option represents a separate embodiment of the invention. As used herein, "bladder cancer" does not include cancers that arise in other parts of the urinary system, such as upper tract urothelial carcinoma (UTUC).

[0047] Most bladder cancers are transitional cell carcinomas that arise from the epithelial cells of the bladder lining (urothelium). Other types of bladder cancer include squamous cell carcinoma, adenocarcinoma, sarcoma, and small cell carcinoma.

[0048] TCC, which arises from the epithelial cells of the bladder's inner lining (urothelium), is the most common type of bladder cancer, accounting for over 90% of cases. TCC typically includes two subtypes: papillary carcinoma, in which the tumor grows in long, finger-like projections from the inner surface of the bladder toward the hollow center, and squamous carcinoma, in which the tumor does not grow toward the hollow part of the bladder. Squamous cell carcinoma and adenocarcinoma are less common types of bladder cancer, while small cell carcinoma and sarcoma are relatively rare.

[0049] Bladder cancer can typically be further classified as either non-invasive, where the cancer cells are limited to the lining of the transitional epithelium, or invasive, where the cancer cells grow deeper into the lamina propria of the bladder or even into the muscle layer. Bladder cancer can also be described as superficial or non-muscle-invasive. These terms include both non-invasive tumors as well as any invasive tumor that has not grown into the main muscle layer of the bladder.

[0050] One common classification of bladder cancer is the T category, which describes how far the main tumor has grown into (or beyond) the wall of the bladder. Typically, bladder cancer begins in the urothelium, and as the cancer grows, it may invade other layers of the bladder and thus become more aggressive. The T category classification includes the following terms: -T0: No evidence of primary tumor -Ta: Papillary carcinoma in situ -Tis or CIS: Squamous carcinoma in situ (carcinoma in situ) - T1: The tumor has grown through the layer of cells lining the bladder into the connective tissue below. The tumor has not grown into the muscle layer of the bladder. -T2: The tumor has grown into the muscle layer. T2a: The tumor has grown only into the inner half of the muscle layer. T2b: The tumor has grown into the outer half of the muscle layer. -T3: The tumor has grown through the muscle layer of the bladder into the layer of fatty tissue that surrounds it. o T3a: Spread into fatty tissue can only be seen using a microscope. T3b: Spread into fatty tissue is large enough to be seen on an imaging test or to be seen or felt by the surgeon. - T4: The tumor has spread beyond the fatty tissue to nearby organs or structures. The tumor may grow into any of the following: the stroma (main tissue) of the prostate, seminal vesicles, uterus, vagina, pelvic wall, or abdominal wall. T4a: The tumor has spread to the stroma of the prostate gland (men) or to the uterus and / or vagina (women). T4b: The tumor has spread to the pelvic or abdominal wall.

[0051] Bladder cancer is also commonly described by its grade (G), which describes the similarity of cancer cells to healthy cells when viewed under a microscope. Healthy tissue usually contains various types of cells grouped together. If the cancer looks similar to healthy tissue but contains a different group of cells, it is called a differentiated or low-grade tumor. If the cancerous tissue looks very different from healthy tissue, it is called a poorly differentiated or high-grade tumor.

[0052] Urological surgeons may also grade tumors based on their likelihood of recurrence or progression (grow and spread) to plan treatment based on grade, using the following categories: - Papilloma (or benign papillary urothelial neoplasm of low malignant potential (PUNLMP)) - May recur but has a low risk of progression. -Low grade: More likely to recur and progress than PUNLMP. - High grade: Most likely to recur and progress.

[0053] Bladder cancer can sometimes affect many areas of the bladder simultaneously. If more than one tumor is found, the letter m is added to the appropriate T category. If the cancer is advanced, lymph node involvement and metastasis are also typically evaluated to complete the full staging based on the Tumor, Node, Metastasis (TNM) staging system, where T describes the size of the tumor and the spread of the cancer to nearby tissues, N describes the spread of the cancer to nearby lymph nodes, and M describes metastasis.

[0054] As used herein, the term "subject" is interchangeable with "individual" and refers to a human subject. The subject has hematuria and / or at least one lower urinary symptom that may be indicative of bladder malignancy, including at least one of urinary frequency, urgency, urge incontinence, nocturia, altered urinary flow, hesitancy to urinate, straining and dribbling, and residual urine. Hematuria can be microscopic hematuria, defined as ≥3 red blood cells per high-power field (RBC / HPF) upon microscopic evaluation of a single, properly collected urine specimen, or gross hematuria, the presence of visible blood in the urine (also referred to as gross hematuria). Gross hematuria is also defined as ≥25 RBC / HPF. Each option represents a separate embodiment of the invention.

[0055] The subject may be suspected of having bladder cancer. In some embodiments, the subject may be at risk of developing bladder cancer, for example, based on genetic predisposition and / or family history. The subject evaluated using the methods of the present invention is a subject with no history of bladder cancer or other urothelial cancer (the subject has not been previously diagnosed with bladder cancer or upper tract urothelial cancer).

[0056] Urine sample collection and processing The DNA analyzed by the methods and systems of the present invention is cellular DNA obtained from cells present in urine. The present invention can be practiced by processing a urine sample or cells previously collected from the urine sample. In some embodiments, the urine sample is processed to collect cells, and then DNA is extracted from the collected cells. Cell collection can be performed using centrifugation or other methods of capturing cells, such as the use of a filtration device that collects cells without the need for centrifugation, such as the filtration device described in Andersson et al., 2015, PLoS ONE, 10(7):e0131889.

[0057] In some embodiments, a urine sample may be collected from a subject using a conventional collection container or tube. Preferably, at least 10 mL of urine is collected.

[0058] In some embodiments, the method of the present invention can include extracting genomic DNA from urine sample.This can be achieved according to the method known in the art.Exemplary procedures are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012.

[0059] In some embodiments, the urine sample to be analyzed is centrifuged to produce a cell pellet, and DNA is then extracted from the cell pellet, which can be accomplished, for example, using an appropriate DNA extraction buffer, as described in the "Examples" section below.

[0060] Preferably, the DNA sample on which methylation analysis is performed is substantially free of single-stranded DNA (ssDNA). As used herein, "substantially free of ssDNA" or "substantially devoid of ssDNA" refers to a DNA sample in which less than 7% of the DNA is ssDNA, preferably less than 5% of the DNA is ssDNA, and more preferably less than 1% of the DNA is ssDNA (i.e., at least 99% of the DNA is double-stranded). In some embodiments, the DNA sample contains less than 0.1% ssDNA. In some embodiments, the DNA sample contains less than 0.01% ssDNA. In some embodiments, the DNA sample is completely free of ssDNA (no ssDNA). Extraction of DNA to obtain a DNA sample substantially free of ssDNA is described, for example, in International Publication No. WO 2020 / 188561, assigned to the same applicant as the present invention.

[0061] In some embodiments, all of the extracted DNA is used in accordance with the present invention. In some embodiments, the DNA is not quantified prior to methylation analysis in accordance with the present invention.

[0062] The methods of the invention may be performed on a urine sample (from which DNA is then extracted), or on cells already obtained from a urine sample (from which DNA is then extracted), or on DNA already extracted from or from cells therein.

[0063] Methylation levels As used herein, a "methylation value" is a numerical value representing the level of methylation at a particular genomic locus in a DNA sample. Because methylation can be analyzed and measured in a variety of ways, a "methylation value" according to the present invention can be expressed in a variety of numerical values. For example, a methylation value can be a methylation level expressed as a ratio or percentage of DNA molecules that are methylated at a marker locus out of the total number of DNA molecules containing the marker locus in a sample. As a further example, a methylation value can be a methylation level expressed as the copy number of methylated DNA molecules at a marker locus (e.g., read counts obtained after sequencing, as described in more detail below). As yet another example, a methylation value can be a methylation level expressed as the intensity of a signal obtained from the marker locus, e.g., a fluorescent signal obtained using a detectable fluorescent label / probe. The methylation value can be normalized to a reference locus and / or a reference DNA sample. In some specific embodiments, the methylation value is a methylation ratio between a marker locus and a control locus, expressed as the ratio between the signals obtained for these loci after methylation-sensitive enzyme digestion and PCR amplification of a DNA sample, as described in more detail below.

[0064] In some embodiments, the methods of the invention comprise determining a methylation value for a marker locus set forth in SEQ ID NO: 1, and optionally for at least one additional marker locus. In some embodiments, the methods of the invention comprise determining a methylation value for a marker locus set forth in SEQ ID NO: 2, and optionally for at least one additional marker locus. In some embodiments, the methods of the invention comprise determining a methylation value for a marker locus set forth in SEQ ID NO: 3, and optionally for at least one additional marker locus. In some embodiments, the methods of the invention comprise determining a methylation value for a marker locus set forth in SEQ ID NO: 4, and optionally for at least one additional marker locus. In some embodiments, the methods of the invention comprise determining a methylation value for a marker locus set forth in SEQ ID NO: 5, and optionally for at least one additional marker locus. In some embodiments, the methods of the invention comprise determining a methylation value for a marker locus set forth in SEQ ID NO: 6, and optionally for at least one additional marker locus. In some embodiments, the methods of the invention comprise determining a methylation value for a marker locus set forth in SEQ ID NO: 7, and optionally for at least one additional marker locus. In some embodiments, the methods of the invention comprise determining a methylation value for a marker locus set forth in SEQ ID NO: 8, and optionally for at least one additional marker locus. In some embodiments, the methods of the invention comprise determining a methylation value for a marker locus set forth in SEQ ID NO: 9, and optionally for at least one additional marker locus. In some embodiments, the methods of the invention comprise determining a methylation value for a marker locus set forth in SEQ ID NO: 10, and optionally for at least one additional marker locus.In some embodiments, the methods of the invention comprise determining a methylation value for a marker locus set forth in SEQ ID NO: 11, and optionally for at least one additional marker locus. In some embodiments, the methods of the invention comprise determining a methylation value for a marker locus set forth in SEQ ID NO: 12, and optionally for at least one additional marker locus. In some embodiments, the methods of the invention comprise determining a methylation value for a marker locus set forth in SEQ ID NO: 13, and optionally for at least one additional marker locus. In some embodiments, the methods of the invention comprise determining a methylation value for a marker locus set forth in SEQ ID NO: 14, and optionally for at least one additional marker locus. In some embodiments, the methods of the invention comprise determining a methylation value for a marker locus set forth in SEQ ID NO: 15, and optionally for at least one additional marker locus.

[0065] As described herein, each marker locus of the present invention comprises multiple differentially methylated CG dinucleotides located within the restriction site(s) of a methylation-sensitive restriction endonuclease. In some embodiments, when a marker locus is analyzed using methylation-sensitive enzymatic digestion of DNA, the methylation value of the marker locus is based on the CG dinucleotides within the restriction site(s) of the methylation-sensitive restriction endonuclease(s) used in the assay.

[0066] The following sections describe methylation analysis according to some embodiments of the present invention, which is based on methylation-sensitive enzymatic digestion of a DNA sample followed by quantitative PCR amplification and analysis of the amplified products, or methylation-sensitive enzymatic digestion of a DNA sample followed by high-throughput sequencing (next-generation sequencing). Those skilled in the art will understand that other methods for analyzing the methylation of marker loci and obtaining methylation value(s) may be used with the present invention.

[0067] Methylation analysis using methylation-sensitive enzyme digestion and PCR amplification A.DNA digestion In some embodiments, following extraction, the DNA is subjected to digestion with at least one methylation-sensitive restriction endonuclease, e.g., one, two, or three methylation-sensitive restriction endonucleases, each number of endonucleases used in the assay representing a separate embodiment of the present invention.

[0068] In some embodiments, the entire DNA extracted from the urine sample is used in the digestion step. In some embodiments, the DNA is not quantified before undergoing digestion. In other embodiments, the DNA may be quantified before digestion.

[0069] "Restriction endonucleases," which are used interchangeably herein with "restriction enzymes," refer to enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites. Restriction sites are usually 4-8 nucleotides in length and are typically palindromic (i.e., a sequence in one direction, e.g., reading 5' to 3', on one strand is identical to a sequence in the same direction (5' to 3') on the complementary strand).

[0070] A "methylation-sensitive" restriction endonuclease is one that cleaves its recognition sequence only if it is unmethylated (methylated sites remain intact). Thus, the extent of digestion of a DNA sample by a methylation-sensitive restriction endonuclease depends on the methylation level; higher methylation levels are protected from cleavage and therefore result in less digestion.

[0071] Examples of methylation-sensitive restriction endonucleases that can be used in accordance with the present invention include AciI, AfeI, ApaI, AscI, AvaI, AvaII, BanI, BbeI, BcgI, BfuCI, BsaAI, BsaHI, BsaI, BseYI, BsiEI, BslI, BsmAI, BsmFI, BsrBI, BsrFI, BssHII, BssKI, BstUI, Cac8I, DpnI, EciI, FauI, Fnu4HI, FspI, HaeII, HgaI, HgaII ... I, HhaI, HinfI, HinPII, HpaII, HpyI66ii, HpyI88iii, Hpy99I, HpyCH4IV, KasI, MmeI, MspAII, MwoI, NheI, NlaIV, PleI, PmlI, PspOMI, SacII, Sau3AI, Sau96I, ScrFI, SfoI, SgrAI, SmaI, TfiI, TscI, TseI, and TspMI, and their "HF" high fidelity variants, where available. Each option represents a separate embodiment of the present invention.

[0072] In general, embodiments that can be performed with methylation-sensitive restriction endonuclease(s) can instead be performed with methylation-dependent restriction endonucleases, with downstream steps adjusted accordingly.

[0073] In some embodiments, DNA extracted from a urine sample may be subjected to digestion with a single methylation-sensitive restriction endonuclease. In some specific embodiments, the methylation-sensitive restriction endonuclease may be HhaI. In additional specific embodiments, the methylation-sensitive restriction endonuclease may be HinP1I. In other embodiments, DNA extracted from a urine sample may be subjected to digestion with multiple methylation-sensitive restriction endonucleases. As used herein, "multiple" refers to "at least two."

[0074] In some embodiments, DNA digestion can be carried out until complete digestion is achieved. In some embodiments, the methylation-sensitive restriction endonuclease can be HhaI, and complete digestion can be achieved after 1-2 hours of incubation with the enzyme at 37°C.

[0075] In some embodiments, DNA digestion can be carried out until complete digestion is achieved. In some embodiments, the methylation-sensitive restriction endonuclease can be HinP1I, and complete digestion can be achieved after 1-2 hours of incubation with the enzyme at 37°C.

[0076] B. Amplification of genomic loci As used herein, the terms "genomic locus" or "locus" are interchangeable and refer to a DNA sequence at a specific location on a chromosome. A specific location can be identified by the location of the molecule, i.e., the number of starting and ending base pairs on the chromosome. Variants of a DNA sequence at a given genomic location are called alleles. Alleles of a locus are located at the same site on homologous chromosomes. A locus includes gene sequences as well as other genetic elements, such as intergenic sequences.

[0077] A "marker locus," as disclosed herein, refers to a genomic locus that is differentially methylated between sources of DNA, and therefore analysis of its methylation provides an indication as to the source of the DNA.

[0078] "Control locus" and "internal reference locus" are used interchangeably herein to describe a locus whose digestion by the restriction enzyme applied in the digestion step is independent of the presence or absence of methylation. In some embodiments, a control locus is a locus that exhibits the same digestion and amplification profile in bladder cancer and healthy tissue. In some embodiments, a control locus is a locus that lacks a recognition sequence for the restriction enzyme applied in the digestion step, such that the sequence of the control locus remains intact regardless of the methylation state when the DNA sample is digested. Advantageously, a control locus is an internal locus, i.e., a locus within the analyzed DNA sample, thus eliminating the need for an external / additional control sample.

[0079] Marker loci for use in accordance with the present invention include any one or more of the loci set forth in SEQ ID NOs: 1-15 (numbering according to the hg18 build of the human genome), as follows: SEQ ID NO: 1 corresponds to positions 65676359 to 65676418 on chromosome 17 (KCNJ2 gene); SEQ ID NO: 2 corresponds to positions 21958446 to 21958585 on chromosome 9 (CDKN2A gene); SEQ ID NO: 3 corresponds to positions 336844 to 336903 on chromosome 6 (IRF4 gene); SEQ ID NO: 4 corresponds to positions 33319507 to 33319636 on chromosome 21 (Olig2 gene); SEQ ID NO: 5 corresponds to positions 166502151 to 166502220 on chromosome 6 (intergenic region); SEQ ID NO: 6 corresponds to positions 896902 to 897031 on chromosome 18 (ADCYAP1 gene); SEQ ID NO: 7 corresponds to positions 32747873 to 32748022 on chromosome 5 (NPR3 gene); SEQ ID NO: 8 corresponds to positions 27949195 to 27949264 on chromosome 6 (intergenic region); SEQ ID NO: 9 corresponds to positions 27191603 to 27191672 on chromosome 7 (HOXA9 gene); SEQ ID NO: 10 corresponds to positions 170170302 to 170170361 on chromosome 16 (intergenic region); SEQ ID NO: 11 corresponds to positions 30797737 to 30797876 on chromosome 15 (intergenic region); SEQ ID NO: 12 corresponds to positions 7936767 to 7936866 on chromosome 1 (intergenic region); SEQ ID NO: 13 corresponds to positions 170077565 to 170077634 on chromosome 1 (DNM3 gene); SEQ ID NO: 14 corresponds to positions 1727592 to 1727661 on chromosome 2 (PXDN gene), and SEQ ID NO: 15 corresponds to positions 72919092 to 72919231 on chromosome 8 (MSC gene).

[0080] The marker loci set forth in SEQ ID NOS: 1-15, previously disclosed in commonly assigned International Publication No. WO 2017 / 006317, are differentially methylated between cancerous and normal bladder tissues. More specifically, these loci are increased in bladder cancer tissues compared to normal tissues.

[0081] Each of these loci contains a CG dinucleotide that is more methylated in DNA from cancerous bladder tissue compared to DNA from normal, non-cancerous bladder tissue. Advantageously, the differentially methylated CG dinucleotide is located within a recognition site for a methylation-sensitive restriction enzyme.

[0082] In some embodiments, each of these loci may contain at least one restriction site for a methylation-sensitive restriction enzyme, within which CG dinucleotides are more methylated in bladder cancer cells than in normal cells, meaning that more cells in cancerous tissues contain methylation at this position compared to normal tissues. In some embodiments, each of these loci may contain at least one HhaI restriction site (GCGC). In some embodiments, each of these loci may contain at least one HinP1I restriction site. Methylation-sensitive restriction enzymes cleave their recognition sequences only if they are unmethylated. Thus, a DNA sample with a high proportion of DNA molecules in which the CG dinucleotide at the restriction site is methylated will experience less extensive digestion than a DNA sample with a high proportion of DNA molecules in which the CG dinucleotide is unmethylated. Based on the methods disclosed herein, DNA digestion by methylation-sensitive restriction enzymes is less extensive for DNA from urine samples of bladder cancer patients than for DNA from normal individuals (healthy individuals). The differences in digestion efficiency result in different amplification patterns in the subsequent amplification and quantification steps, allowing differentiation of DNA from cancerous and normal bladder tissue.

[0083] In some embodiments, each of the loci set forth in SEQ ID NOs: 1-15 can contain an additional CG dinucleotide, the methylation status of which is not relevant or impactful to the assay - only the methylation in the recognition sequence of the restriction enzyme (e.g., HhaI or HinP1I) is relevant.

[0084] In some embodiments, the control locus is set forth in SEQ ID NO: 16, which corresponds to positions 121380854 to 121380913 on chromosome 7 (an intergenic region). In some embodiments, the control locus, also referred to as an internal reference locus, does not contain a recognition sequence for a restriction enzyme. In some embodiments, the sequence of the control locus remains intact (regardless of its methylation state) when a DNA sample is digested with a methylation-sensitive restriction enzyme.

[0085] In some embodiments, the sequence of the control locus exhibits the same digestion and amplification profile in bladder cancer tissue and healthy bladder tissue.

[0086] In some embodiments, the control locus comprises the locus set forth in SEQ ID NO: 16, and the amplification pattern of the control locus after digestion with a methylation-sensitive restriction enzyme is not affected by methylation.

[0087] In some embodiments, the methods of the invention comprise amplifying at least one marker locus and at least one control locus after digestion of the DNA sample.

[0088] As used herein, "at least one (marker / control) locus" can encompass a single locus or multiple distinct loci.

[0089] In some embodiments, the methods of the invention comprise amplifying the marker locus and control locus set forth in SEQ ID NO:1, and optionally at least one additional marker locus. In some embodiments, the methods of the invention comprise amplifying the marker locus and control locus set forth in SEQ ID NO:2, and optionally at least one additional marker locus. In some embodiments, the methods of the invention comprise amplifying the marker locus and control locus set forth in SEQ ID NO:3, and optionally at least one additional marker locus. In some embodiments, the methods of the invention comprise amplifying the marker locus and control locus set forth in SEQ ID NO:4, and optionally at least one additional marker locus. In some embodiments, the methods of the invention comprise amplifying the marker locus and control locus set forth in SEQ ID NO:5, and optionally at least one additional marker locus. In some embodiments, the methods of the invention comprise amplifying the marker locus and control locus set forth in SEQ ID NO:6, and optionally at least one additional marker locus. In some embodiments, the methods of the invention comprise amplifying the marker locus and control locus set forth in SEQ ID NO:7, and optionally at least one additional marker locus. In some embodiments, the methods of the invention comprise amplifying the marker locus and control locus set forth in SEQ ID NO: 8, and optionally at least one additional marker locus. In some embodiments, the methods of the invention comprise amplifying the marker locus and control locus set forth in SEQ ID NO: 9, and optionally at least one additional marker locus. In some embodiments, the methods of the invention comprise amplifying the marker locus and control locus set forth in SEQ ID NO: 10, and optionally at least one additional marker locus. In some embodiments, the methods of the invention comprise amplifying the marker locus and control locus set forth in SEQ ID NO: 11, and optionally at least one additional marker locus.In some embodiments, the methods of the invention comprise amplifying the marker locus and control locus set forth in SEQ ID NO: 12, and optionally at least one additional marker locus. In some embodiments, the methods of the invention comprise amplifying the marker locus and control locus set forth in SEQ ID NO: 13, and optionally at least one additional marker locus. In some embodiments, the methods of the invention comprise amplifying the marker locus and control locus set forth in SEQ ID NO: 14, and optionally at least one additional marker locus. In some embodiments, the methods of the invention comprise amplifying the marker locus and control locus set forth in SEQ ID NO: 15, and optionally at least one additional marker locus.

[0090] In some embodiments, the methods of the invention comprise amplifying a marker locus selected from SEQ ID NOs: 1-15 and a control locus set forth in SEQ ID NO:16.

[0091] In some embodiments, the methods of the invention involve amplifying multiple marker loci (ie, at least two marker loci) and a control locus.

[0092] In some embodiments, the plurality of marker loci comprises the locus set forth in SEQ ID NO: 1 and the locus set forth in SEQ ID NO: 5. In some embodiments, the plurality of marker loci further comprises at least one marker locus selected from the loci set forth in SEQ ID NO: 7 and SEQ ID NO: 11. In some embodiments, the plurality of marker loci comprise the loci set forth in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 11.

[0093] In some embodiments, the plurality of marker loci includes the loci set forth in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:11, and further includes at least one additional marker locus selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO: 15. Each option represents a separate embodiment of the present invention.

[0094] In some embodiments, the plurality of marker loci includes the locus set forth as SEQ ID NO: 1, and further includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 additional marker loci selected from the group consisting of SEQ ID NOs: 2-15. Each option represents a separate embodiment of the present invention.

[0095] In some embodiments, the plurality of marker loci includes the loci set forth as SEQ ID NOs: 1 and 5, and further includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 additional marker loci selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. Each option represents a separate embodiment of the present invention.

[0096] In some embodiments, the plurality of marker loci includes the loci set forth as SEQ ID NOs: 1, 5, and 7, and further includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 additional marker loci selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. Each option represents a separate embodiment of the present invention.

[0097] In some embodiments, the plurality of marker loci include the loci set forth as SEQ ID NOs: 1, 5, 7, and 11, and further include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 additional marker loci selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. Each option represents a separate embodiment of the present invention.

[0098] In some embodiments, the plurality of marker loci comprises the loci set forth in SEQ ID NOs: 1-15. In some embodiments, the plurality of marker loci consists of the loci set forth in SEQ ID NOs: 1-15. In some embodiments, the methods of the invention comprise amplifying a plurality of marker loci as set forth in SEQ ID NOs: 1-15.

[0099] As used herein, "amplification" refers to an increase in the copy number of one or more specific nucleic acid targets of interest. Amplification is typically carried out by polymerase chain reaction (PCR) in the presence of a PCR reaction mixture, which may include a DNA template, a polymerase (usually Taq polymerase), dNTPs, primers, and a suitable buffer supplemented with a probe (if necessary).

[0100] As used herein, the term "polynucleotide" includes a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. As used herein, the term "oligonucleotide" also includes a polymeric form of nucleotides, typically up to 100 bases in length.

[0101] "Amplification products" refers collectively to nucleic acid molecules of a specific target sequence that are produced and accumulated in an amplification reaction. This term generally refers to nucleic acid molecules produced by PCR using a given set of amplification primers.

[0102] As used herein, "primer" defines an oligonucleotide that can anneal to (hybridize with) a target sequence, thereby creating a double-stranded region that can serve as the initiation point for DNA synthesis under suitable conditions.The term "primer pair" refers to a pair of oligonucleotides that are selected to be used together in amplifying a selected nucleic acid sequence by one of several types of amplification processes, preferably PCR.As is generally known in the art, primers can be designed to bind to complementary sequences under selected conditions.

[0103] Primers can be of any suitable length, depending on the particular assay format and specific needs. In some embodiments, primers can be at least 15 nucleotides in length, preferably 15 to 25 nucleotides in length, or 19 to 25 nucleotides in length. Primers can be adapted to be particularly suitable for a selected nucleic acid amplification system. Oligonucleotide primers can be designed taking into account their melting temperature for hybridization with the target sequence (Sambrook et al., supra).

[0104] In some embodiments, marker and control loci can be amplified from the same DNA sample (digested sample) using pairs of reverse and forward primers designed to specifically amplify each locus. In some embodiments, primers can be designed to amplify a locus along with its 5' and 3' flanking sequences.

[0105] In some embodiments, the 5' flanking sequence can comprise 1 to 60 bases immediately upstream of the locus. In further embodiments, the 5' flanking sequence is between 10 and 50 bases immediately upstream of the locus. For example, the 5' flanking sequence can comprise 10, 15, 20, 25, 30, 35, 40, 45, or 50 bases immediately upstream of the locus. Each option represents a separate embodiment of the present invention.

[0106] In some embodiments, the 3' flanking sequence may comprise 1 to 90 bases immediately downstream of the locus, hi some embodiments, the 3' flanking sequence may comprise 5 to 80 bases immediately downstream of the locus. In some embodiments, the 3' flanking sequence may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 bases immediately downstream of the locus. Each option represents a separate embodiment of the present invention.

[0107] In some embodiments, primers may be designed to generate an amplification product between 75 and 225 bases in length when the locus is intact.

[0108] In some embodiments, the methods involve a process known as multiplex amplification or co-amplification, which is the simultaneous amplification of two or more target sequences (e.g., at least one marker locus and one control locus) in the same reaction mixture. This process requires the simultaneous use of multiple primer pairs. Primers can be designed to function at the same annealing temperature during amplification. In some embodiments, primers with similar melting temperatures (Tm) are used in the methods disclosed herein. A Tm variation of approximately 3-5°C is considered acceptable for primers used in pools.

[0109] In some embodiments, all marker loci and control loci can be amplified in a single reaction mixture. In other embodiments, due to technical limitations of a particular machine, for example, the digested DNA sample can be divided into several aliquots, each supplemented with primer pairs for amplifying one or more marker loci and control loci. Thus, even if the DNA sample is divided into several aliquots, the control loci are amplified in each aliquot, and the signal ratio calculation is performed for the control loci and marker loci amplified together, i.e., from the same aliquot.

[0110] In some embodiments, the method may use a control construct comprising a non-human DNA sequence in the digestion and amplification step(s). In some embodiments, the control construct comprises an artificial (synthetic) DNA sequence. The control construct can be used to control the digestion and amplification process, e.g., to monitor the effectiveness and quality of the digestion and amplification steps. In some embodiments, the control construct and the DNA sample are digested with at least one methylation-sensitive restriction enzyme simultaneously, and optionally in the same container (e.g., test tube, vial, etc.). In some embodiments, the method may include subjecting the control construct, together with the DNA sample, to digestion with at least one methylation-sensitive restriction enzyme. In some embodiments, the method may include adding the control construct to the DNA sample and subjecting both to digestion with at least one methylation-sensitive restriction enzyme.

[0111] In some embodiments, the methylation-sensitive restriction endonuclease may be used in the digestion step along with one or more of the following control constructs: a first control construct comprising a DNA sequence lacking a recognition sequence for the methylation-sensitive restriction endonuclease, and a second control construct comprising a DNA sequence containing a recognition sequence for the methylation-sensitive restriction endonuclease and being completely unmethylated, such that the first control construct remains intact while the second control construct is completely or at least partially digested. In a subsequent amplification step, primers (and optionally a probe) specific to the control construct may be added. Detection of adequate amplification for the first construct, concomitant with sufficiently low amplification for the second construct, indicates adequate DNA digestion.

[0112] In some embodiments, detecting adequate amplification of the first construct concurrently with sufficiently low amplification of the second construct can include detecting a difference of at least 5 cycles between the quantitation cycles (Cq) of the first control construct and the second control construct in real-time PCR. In some embodiments, the difference can be at least 6 cycles, at least 7 cycles, or at least 8 cycles. Each option represents a separate embodiment of the present invention.

[0113] In some embodiments, the first control construct and the second control construct may comprise SEQ ID NOs: 17 and 18, respectively. In some embodiments, primers and probes for amplifying and detecting the first control construct may comprise SEQ ID NO: 19 (forward), SEQ ID NO: 20 (reverse), and SEQ ID NO: 21 (probe). In some embodiments, primers and probes for amplifying and detecting the second control construct may comprise SEQ ID NO: 22 (forward), SEQ ID NO: 23 (reverse), and SEQ ID NO: 24 (probe).

[0114] In some embodiments, amplification of genomic loci may be performed using real-time PCR (RT-PCR), also known as quantitative PCR (qPCR), in which amplification and detection of amplification products are performed simultaneously.

[0115] In some embodiments, detection of amplification products in RT-PCR can be achieved using polynucleotide probes, generally fluorescently labeled polynucleotide probes.

[0116] As used herein, the terms "polynucleotide probe" and "oligonucleotide probe" are interchangeable and refer to a labeled polynucleotide complementary to a specific subsequence within the nucleic acid sequence of a locus of interest, e.g., the sequence of a marker locus or a control locus. In some embodiments, detection is achieved by using a TaqMan assay based on a combination of a reporter molecule and a quencher molecule (Roche Molecular Systems Inc.). In such assays, polynucleotide probes have a fluorescent moiety (fluorophore) attached to their 5' end and a quencher attached to their 3' end. During PCR amplification, polynucleotide probes selectively hybridize to target sequences on a template, and as the polymerase replicates the template, the 5'-nuclease activity of the polymerase also cleaves the polynucleotide probe. When the polynucleotide probe is intact, close proximity between the quencher and the fluorescent moiety typically results in low levels of background fluorescence. When the polynucleotide probe is cleaved, the quencher is separated from the fluorescent moiety, resulting in an increase in fluorescence intensity. The fluorescent signal correlates with the amount of amplification product, ie, the signal increases as amplification product accumulates.

[0117] As used herein, "selectively hybridize" (as well as "selective hybridization," "specifically hybridize," and "specific hybridization") refers to preferentially binding, duplexing, or hybridizing a nucleic acid molecule (such as a primer or probe) to a specific complementary nucleotide sequence under stringent conditions. The term "stringent conditions" refers to conditions under which a nucleic acid molecule will hybridize preferentially to its target sequence and will hybridize to a lesser extent, or not at all, to other non-target sequences. As is well known in the art, "stringent hybridization" in the context of nucleic acid hybridization is sequence-dependent and will vary under different conditions.

[0118] Polynucleotide probes may vary in length. In some embodiments, polynucleotide probes may comprise 15-30 bases. In additional embodiments, polynucleotide probes may comprise 25-30 bases. In some embodiments, polynucleotide probes may comprise 20-30 bases, e.g., 20 bases, 21 bases, 22 bases, 23 bases, 24 bases, 25 bases, 26 bases, 27 bases, 28 bases, 29 bases, 30 bases. Each option represents a separate embodiment of the present invention.

[0119] Polynucleotide probes can be designed to bind to either strand of the template. Additional considerations include the Tm of the polynucleotide probe, which should preferably be compatible with the Tm of the primer. Computer software can be used to design primers and probes.

[0120] As described above, the methods disclosed herein can include simultaneous amplification of two or more target sequences (at least one marker locus and one control locus) in the same reaction mixture. To distinguish between multiple target sequences amplified in parallel, polynucleotide probes labeled with different fluorescent colors can be used. In some embodiments, the polynucleotide probes form fluorophore / quencher pairs known in the art, including, for example, FAM-TAMRA, FAM-BHQ1, Yakima Yellow-BHQ1, ATTO550-BHQ2, and ROX-BHQ2. In some embodiments, the dye combinations can be compatible with the RT-PCR thermocycler of choice.

[0121] In some embodiments, fluorescence may be monitored during each PCR cycle, providing an amplification plot showing the change in fluorescent signal from the probe as a function of cycle number.

[0122] In the context of RT-PCR, the following terms are used: "Quantification cycle" ("Cq") refers to the cycle number at which fluorescence increases above a threshold, either automatically by the software or manually by the user. In some embodiments, the threshold may be constant for all loci or may be pre-set before performing amplification and detection. In other embodiments, the threshold may be defined separately for each locus after a run based on the maximum fluorescence level detected for that locus during an amplification cycle.

[0123] "Threshold" refers to the value of fluorescence used to determine Cq. In some embodiments, the threshold may be a value above baseline fluorescence and / or above background noise, and may be a value that is within the exponential growth phase of an amplification plot.

[0124] "Baseline" refers to the early cycles of PCR where there is little or no change in fluorescence.

[0125] Computer software can be used to analyze the amplification plots and determine the baseline, threshold, and Cq.

[0126] After digestion with at least one methylation-sensitive restriction enzyme, loci in which the CG dinucleotide(s) at the enzyme's recognition site(s) are methylated are amplified with high efficiency because the DNA molecules are protected from cleavage. Detectable amplification products are obtained after a relatively small number of amplification cycles, resulting in a relatively low Cq value. Conversely, loci in which the CG dinucleotide(s) at the enzyme's recognition site(s) are unmethylated are extensively cleaved during the digestion process, resulting in a high Cq value in the amplification and quantification process (i.e., detectable amplification products are obtained after a relatively large number of amplification cycles).

[0127] In other embodiments, amplification and detection of the amplified products can be performed by conventional PCR using fluorescently labeled primers, followed by capillary electrophoresis of the amplified products. In some embodiments, after amplification, the amplified products are separated by capillary electrophoresis, and the fluorescent signal is quantified. In some embodiments, an electropherogram can be generated that plots the change in fluorescent signal as a function of size (bp) or time since injection, with each peak in the electropherogram corresponding to the amplified product of a single locus. Peak height (e.g., provided using "relative fluorescent units" (rFU)) can represent the intensity of the signal from the amplified locus. Computer software can be used to detect the peaks and calculate the fluorescent intensities (peak heights) of the set of loci whose amplified products were run on the capillary electrophoresis device, followed by calculating the ratio between the signal intensities.

[0128] For DNA samples digested with a methylation-sensitive restriction enzyme, such as HhaI or HinP1I, loci in which the CG dinucleotide(s) at the enzyme's recognition site(s) are methylated will produce a relatively strong signal (high peak) on the electropherogram. Conversely, loci in which the CG dinucleotide(s) at the enzyme's recognition site(s) are unmethylated will produce a relatively weak signal (low peak) on the electropherogram.

[0129] In some embodiments, the fluorescent label of the primer comprises any one of fluorescein, FAM, Lissamine, phycoerythrin, rhodamine, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX, JOE, HEX, NED, VIC, and ROX.

[0130] C. Signal Ratio As used herein, the term "ratio" or "signal ratio" refers to the ratio between the intensities of signals obtained from the co-amplification of a pair of genomic loci, in particular the co-amplification of a marker locus and a control locus, in a single DNA sample (in the same reaction mixture). The signal ratio between a marker locus of the present invention and a control locus obtained after methylation-sensitive enzyme digestion and co-amplification reflects the methylation ratio between these marker loci and the control locus, and represents a "methylation value" according to the present invention.

[0131] As used herein, the term "signal intensity" refers to a measure reflecting the amount of locus-specific amplification product corresponding to the initial amount of intact copies of the locus. However, signal intensity may not indicate the actual amount of amplification product / intact locus, and may not involve calculation of the absolute amount of amplification product / intact locus. Therefore, when calculating the ratio of amplification product signals, a standard curve or reference DNA may not be required, since the actual DNA concentration or DNA methylation level itself does not need to be calculated.

[0132] In some exemplary embodiments, amplification and detection of the amplification products is performed by real-time PCR, and the signal intensity of a particular locus can be represented by the Cq calculated for that locus. The signal ratio in this case can be expressed by the following calculation: (対照遺伝子座のCq-マーカー遺伝子座のCq) .

[0133] In additional exemplary embodiments, detection of the amplification products is performed by capillary electrophoresis, where the signal intensity of a particular locus is the number of relative fluorescence units (rfus) of its corresponding peak. The signal ratio can be calculated by dividing the peak height of each marker locus by the peak height of the control locus.

[0134] In some embodiments, calculating the ratio between the signal intensities of the amplification products of the marker loci and the control loci in the DNA sample comprises (i) determining the signal intensities of the amplification products of the marker loci, (ii) determining the signal intensities of the amplification products of the control loci, and (iii) calculating the ratio between the two signal intensities.

[0135] In some embodiments, calculating the ratio between the signal intensities of the amplification products of the marker loci and the control loci in the DNA sample comprises determining the Cq of each locus and calculating the difference between the Cq of the control locus and the Cq of the marker locus. In some embodiments, calculating further comprises applying the formula: 2^(Cq of control locus - Cq of marker locus).

[0136] In some embodiments, calculating signal ratios can be calculating multiple signal ratios between each marker locus and a control locus.

[0137] In some embodiments, multiple loci among the loci set forth in SEQ ID NOs: 1-15 are amplified, and the methods of the invention include calculating multiple signal ratios, e.g., between each of the loci set forth in SEQ ID NOs: 1-15 and a control locus, e.g., between the locus set forth in SEQ ID NO: 1 and the control locus, between the locus set forth in SEQ ID NO: 2 and the control locus, etc.

[0138] In some embodiments, computer software can be used to calculate ratios between the signal intensities of the amplification products.

[0139] As described above, the signal ratio between the marker loci of the present invention and the control loci obtained after methylation-sensitive enzyme digestion and co-amplification reflects the methylation ratio between these marker loci and the control loci and represents a "methylation value" according to the present invention.

[0140] Methylation analysis using methylation-sensitive enzyme digestion and high-throughput sequencing "High-throughput sequencing" (also referred to as "next generation sequencing," abbreviated "NGS") involves sequencing using methods to determine large numbers (typically thousands to billions) of nucleic acid sequences in parallel. High-throughput sequencing generally involves three basic steps: library preparation, sequencing, and data analysis. Examples of high-throughput sequencing techniques include sequencing-by-synthesis and sequencing-by-ligation (e.g., employed by Illumina Inc., Life Technologies Inc., and Roche), nanopore sequencing, and electronic detection-based methods such as Ion Torrent™ technology (Life Technologies Inc.). High-throughput sequencing includes whole-genome high-throughput sequencing and target-specific high-throughput sequencing. Each option represents a separate embodiment of the present invention.

[0141] According to some embodiments of the invention, after extraction, the DNA is subjected to digestion with at least one methylation-sensitive restriction endonuclease, as described herein, followed by preparation of a sequencing library.

[0142] In some embodiments, preparing a sequencing library involves introducing adapter oligonucleotides, also referred to as "sequencing adapters," into DNA fragments and enriching DNA fragments corresponding to marker loci of interest and, optionally, one or more control loci. Enrichment of genomic regions of interest can be performed, for example, using locus-specific PCR or using capture agents in a liquid-phase or solid-phase hybridization-based process.

[0143] Sequencing adapters are oligonucleotides located at the 5' and 3' ends of each DNA fragment in a sequencing library. Sequencing adapters typically contain platform-specific sequences for fragment recognition by a specific sequencer, such as sequences that enable library fragments to bind to the flow cell of an Illumina platform. Each sequencing instrument typically uses a specific set of sequences for this purpose. Sequencing adapters may contain a sample index, which is a sequence that allows multiple samples to be sequenced together on the same instrument's flow cell or chip (i.e., multiplexing). Each sample index (typically 6-10 bases) is unique to a particular sample library and is used during data analysis to demultiplex and assign individual sequence reads to the correct sample. Sequencing adapters may contain single or dual sample indexes, depending on the number of libraries to be combined and the desired level of accuracy. Sequencing adapters can be introduced into the DNA fragments to be analyzed by ligation or via PCR. In some embodiments, a two-step PCR is used to enrich genomic regions of interest and introduce sequencing adapters into the enriched fragments. A first PCR is performed using a primer comprising a locus-specific sequence and an overhang sequence that introduces a first portion of a sequencing adaptor, and a second PCR is performed using a primer that introduces a second portion of the sequencing adaptor and optionally a sample index.

[0144] The sequencing library is subjected to sequencing to obtain multiple sequence reads.The sequence reads are analyzed using computer software to determine the read count (copy number) for each locus of interest.The read count of a marker locus reflects the number of methylated copies of this locus present in the DNA sample being tested (when the sample is digested with methylation-sensitive restriction endonuclease, the methylated copies remain intact).The relative copy number of a marker locus, for example, relative to a control locus, can be calculated as follows: Relative copy number = read counts at marker loci / read counts at control loci

[0145] The read count of a marker locus, or alternatively, the relative copy number of a marker locus, represents a "methylation value" according to the present invention.

[0146] In some embodiments, the analysis of methylation values ​​according to the present invention comprises: (a) subjecting a DNA sample to digestion with at least one methylation-sensitive restriction endonuclease, thereby obtaining restriction endonuclease-treated DNA; (b) generating a sequencing library from the restriction endonuclease-treated DNA, the sequencing library comprising DNA fragments corresponding to at least one marker locus disclosed herein (preferably, a plurality of marker loci disclosed herein) and at least one control locus; (c) subjecting the sequencing library to high-throughput sequencing to determine the copy number of each of the at least one marker locus and the control locus; (d) comparing the ratio between the copy number of each of the at least one marker locus and the control locus with at least one reference ratio to assess the presence of bladder cancer in the subject.

[0147] In some embodiments, generating a sequencing library comprises enriching DNA fragments corresponding to at least one marker locus and at least one control locus.

[0148] Standard ratio The terms "reference ratio" or "reference signal ratio" are used interchangeably and refer to a signal intensity ratio determined in DNA from a known source. The reference ratio for a given pair of marker and control loci can be expressed in several ways. In some embodiments, the reference ratio for a given pair of loci can be a single ratio. In some embodiments, the reference ratio for a given pair of loci can be a statistical value, for example, the average value of a large set of reference ratios obtained from a large set of DNA samples from a known source, such as an average value determined in a large group of cancer patients or an average value determined in a large group of healthy individuals.

[0149] In other embodiments, the reference ratio for a given pair of loci may be a plurality of ratios, such as a distribution of ratios determined for this pair of loci in a large series of DNA samples from a known source. In some embodiments, the reference ratio may be a reference scale.

[0150] In some embodiments, the reference scale for a given pair of loci can include signal ratios measured for this pair of loci in multiple DNA samples from the same reference source. For example, a reference scale for bladder cancer patients or a reference scale for healthy individuals. In other embodiments, the reference scale for a given pair of loci can include signal ratios from both healthy individuals and diseased individuals, i.e., a single scale combining reference ratios from both sources. Generally, when a single scale is used, the values ​​are distributed so that values ​​from healthy individuals are at one end of the scale, e.g., below a cutoff value, and values ​​from cancer patients are at the other end of the scale, e.g., above a cutoff value. In some embodiments, the signal ratio calculated for a test DNA sample from an unknown source can be compared to a reference scale of healthy and cancer reference ratios, and a probability / likelihood score for bladder cancer can be assigned to the calculated signal ratio based on its relative position within the scale. In some embodiments, the higher the calculated signal ratio, the higher the score assigned to it, and therefore the higher the probability for bladder cancer.

[0151] The terms "bladder cancer reference ratio" or "reference ratio in bladder cancer DNA" are interchangeable and refer to the signal intensity ratio measured between a given marker locus and a given control locus in DNA from a urine sample of a bladder cancer patient. The bladder cancer reference ratio represents the signal intensity ratio in bladder cancer DNA, i.e., DNA from a cancerous bladder. The bladder cancer reference ratio can be a single ratio, a statistical value, or multiple ratios (e.g., a distribution), as detailed above.

[0152] The terms "healthy reference ratio," "normal reference ratio," or "healthy DNA reference ratio" are interchangeable and refer to the signal intensity ratio measured between a given restriction locus and a given control locus in a urine sample from a normal individual. A "healthy individual" or "normal individual" is defined herein as an individual without detectable bladder disease or symptoms, bladder-related diseases, including bladder cancer, as determined by conventional diagnostic methods. A healthy reference ratio represents the signal intensity ratio in normal bladder DNA, i.e., DNA from normal, non-cancerous bladder tissue. A healthy reference ratio can be a single ratio, a statistical value, or multiple ratios (e.g., a distribution), as detailed above.

[0153] In some embodiments, the methods disclosed herein include pre-determining a reference ratio from cancerous bladder DNA. In some embodiments, the methods of the present invention include pre-determining a reference ratio from normal bladder DNA.

[0154] As mentioned above, the signal ratio can be determined by various methods, including, for example, measuring the peak after capillary electrophoresis or calculating Cq after real-time PCR. It should be understood that for determining bladder cancer, the reference ratio and the ratio measured for the test sample of unknown origin are obtained using the methods disclosed herein.

[0155] Determining the presence or absence of bladder cancer In some embodiments, the method disclosed herein includes calculating a score for the tested subject based on the methylation value and determining whether the score is above or below a predetermined cutoff, where detecting a score above the cutoff indicates a positive possibility of the presence of bladder cancer in the subject. Detecting a score below the cutoff indicates a negative possibility of the presence of bladder cancer in the subject (i.e., indicates a high possibility of the absence of bladder cancer in the subject). It should be understood that a negative result in the assay disclosed herein is still considered an assessment / determination of the presence of cancer according to the present invention. In some embodiments, the method disclosed herein is based on evaluating a signal ratio calculated for DNA from cells of a urine sample of unknown origin compared to a reference ratio to assess the presence or absence of bladder cancer.

[0156] In some embodiments, the calculated signal ratio indicates that the DNA is bladder cancer DNA.

[0157] Those skilled in the art will appreciate that the comparison of the calculated signal ratio for a test sample with the corresponding reference signal ratio can be performed in several ways, using a variety of statistical means.

[0158] In some embodiments, comparing the calculated test signal ratio for a given pair of loci to a reference signal ratio comprises comparing the test signal ratio to a single reference value, which may correspond to an average value obtained for the reference signal ratios from a large population of cancer patients or healthy individuals. In other embodiments, comparing the calculated test signal ratio for a given pair of loci to a reference signal ratio comprises comparing the test signal ratio to a distribution or scale of multiple reference signal ratios.

[0159] Known statistical tools can be used to determine whether a calculated signal ratio between a given marker locus and a control locus corresponds to a bladder cancer reference ratio or a normal reference ratio. In some embodiments, detecting a close approximation between the calculated ratio and a bladder cancer reference ratio identifies the subject as having bladder cancer. Conversely, in some embodiments, detecting a close approximation between the calculated ratio and a normal reference ratio identifies the subject as not having bladder cancer.

[0160] In some embodiments, the method includes comparing the calculated signal ratio with its corresponding bladder cancer reference ratio (i.e., a signal ratio determined for the same pair of loci for bladder cancer) to obtain a probability score reflecting the likelihood that the calculated signal ratio is a bladder cancer ratio. The closer the calculated signal ratio is to the reference ratio, the higher the probability score and the more likely the calculated signal ratio is a bladder cancer ratio. In some embodiments, the probability score is based on the relative position of the calculated signal ratio within the distribution of bladder cancer reference ratios.

[0161] In some embodiments, the methods comprise comparing a plurality of signal ratios calculated for a plurality of marker loci with respect to a control locus to their corresponding bladder cancer reference ratios.

[0162] In some embodiments, the pattern of signal ratios may be analyzed using statistical tools and computerized algorithms to determine whether it represents a bladder cancer pattern or a normal, healthy pattern. Exemplary algorithms include machine learning and pattern recognition algorithms.

[0163] In some exemplary embodiments, each calculated ratio (for each pair of marker and control loci) can be compared to a reference ratio scale generated for that pair from a large series of urine samples from both cancer patients and individuals without cancer. This scale can represent signal ratios calculated between pairs of marker loci and control loci in a large number of samples from cancer patients and normal individuals. The scale may indicate a threshold, hereinafter also referred to as a "cutoff" or "predefined threshold," above which the reference ratio corresponds to bladder cancer and below which the reference ratio corresponds to healthy individuals.

[0164] In some embodiments, lower ratios at the bottom of the scale and / or below the cutoff may be from samples from normal individuals (healthy, i.e., not suffering from bladder cancer), while higher ratios at the top of the scale and / or above a predetermined cutoff may be from cancer patients. Each ratio (between each marker locus and the control locus) can be assigned a score based on its relative position on the scale, and the individual scores for each locus can be combined to give a single score. In some embodiments, the individual scores can be summed to give a single score. In other embodiments, the individual scores can be averaged to give a single score. In some embodiments, the single score can be used to determine whether a subject has cancer, with a score above a predetermined threshold indicating bladder cancer.

[0165] In some embodiments, the score is a number between 0 and 100 that reflects the probability that the calculated signal ratio is a bladder cancer ratio, with 0 being the lowest probability and 100 being the highest probability. In some embodiments, a threshold score is determined, and a score equal to or greater than the threshold score is indicative of bladder cancer.

[0166] In additional exemplary embodiments, for each calculated ratio (between each marker locus and the control locus), the probability that it represents bladder cancer DNA may be determined based on a comparison with the corresponding bladder cancer reference ratio and normal reference ratio, and a score (probability / likelihood score) may be assigned. As a result, the individual probability scores calculated for each ratio (for each locus) are combined (e.g., summed or averaged) to provide a composite score. The composite score can be used to determine whether a subject has cancer, with a composite score above a predetermined threshold indicating bladder cancer.

[0167] Thus, in some embodiments, a threshold (or cutoff) score is determined, above which a subject is identified as having bladder cancer. The threshold score distinguishes between healthy and unhealthy populations.

[0168] In some embodiments, the methods of the present invention include providing a threshold score.

[0169] In some embodiments, determining the threshold score comprises measuring the signal ratio in a large population of subjects who are healthy or who have bladder cancer.

[0170] In some embodiments, the threshold is a statistically significant value. Often, statistical significance is determined by comparing two or more populations and determining a confidence interval (CI) and / or p-value. In some embodiments, a statistically significant value refers to a confidence interval (CI) of about 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, and 99.99%, with preferred p-values ​​being less than about 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001. Each option represents a separate embodiment of the present invention. According to some embodiments, the p-value of the threshold score is at most 0.05.

[0171] As used herein, the term "about," when referring to a measurable value, is meant to encompass a variation of + / - 10%, more preferably + / - 5%, even more preferably + / - 1%, and even more preferably + / - 0.1% from the stated value.

[0172] In some embodiments, the method further comprises comparing the calculated signal ratio between a given marker locus and a control locus with its corresponding normal bladder reference ratio to obtain a probability score, wherein detecting a low probability score of said ratio with respect to the corresponding healthy reference ratio indicates that the subject has bladder cancer.

[0173] In some embodiments, the sensitivity of the methods disclosed herein may be at least 65%. In some embodiments, the sensitivity of the methods may be at least 85%. In some embodiments, the methods disclosed herein for detecting or ruling out high-grade bladder cancer are characterized by a sensitivity of at least 85%. In additional embodiments, the methods disclosed herein for detecting or ruling out high-grade bladder cancer are characterized by a sensitivity of at least 87%.

[0174] In some embodiments, the "sensitivity" of a diagnostic assay as used herein refers to the percentage of diseased individuals who test positive (percent of "true positives"). Thus, diseased individuals not detected by the assay are "false negatives." Subjects who are not affected and test negative in the assay are referred to as "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method cannot provide a definitive diagnosis of a condition, it is sufficient if the method provides a positive indication that aids in diagnosis.

[0175] In some embodiments, the specificity of the methods disclosed herein is at least 90%. In additional embodiments, the specificity of the methods disclosed herein is at least 95%. In further embodiments, the specificity of the methods disclosed herein is at least 98%, e.g., 98-100%.

[0176] Management of subjects with hematuria and / or additional lower urinary tract symptoms suspected of malignancy Early detection and treatment may improve the prognosis of bladder cancer. However, the high prevalence of hematuria not associated with cancer (according to the 2020 published microscopic hematuria guidelines, the prevalence of bladder cancer was found to be 0.4% in low-risk groups, 1.0% in intermediate-risk groups, and 6.3% in high-risk groups) can lead to unnecessary and potentially harmful evaluations. The methods and compositions described herein can be used to increase this diagnostic yield for bladder cancer. According to embodiments of the present invention, hematuria is detected or confirmed in a urine sample from a subject, optionally with at least one additional LUTS. In some embodiments, the methods disclosed herein include detecting or confirming hematuria in a urine sample from a subject. The subject's urine sample is then tested using the methods and systems disclosed herein to determine whether a follow-up bladder cancer confirmation test, such as a cystoscopy, is required.

[0177] In addition to the methods described herein for assessing the methylation values ​​of marker loci, clinical evaluation of the patient can provide additional information. This clinical evaluation can be performed before assessing the methylation values ​​of marker loci, or can be triggered by the results of the methylation values. Patients with hematuria can have a variety of symptoms, which can include microscopic hematuria, gross hematuria evidenced by red or dark urine, or the presence of urinary clots. Associated lower urinary tract symptoms (LUTS) can include one or more of: urinary frequency, urgency, urge incontinence, nocturia, altered urinary flow, urinary hesitancy, straining and dribbling, and residual urine.

[0178] In some embodiments, a clinician may perform tests to distinguish between glomerular and non-glomerular causes of hematuria. For example, in some embodiments, a clinical evaluation of a subject according to the present invention may include a urinalysis. A urinalysis typically includes three components or tests: a physical examination, a chemical examination, and a microscopic examination. The physical examination includes examining one or more of the following: volume, color, clarity, odor, and specific gravity. The chemical examination includes measuring one or more of pH, red blood cells, white blood cells, protein, glucose, urobilinogen, bilirubin, ketone bodies, leukocyte esterase, and nitrite. The microscopic examination includes the detection of casts, cells, crystals, and / or microorganisms. The presence of three or more red blood cells per high-power field on the urine sediment defines microscopic hematuria. Urine appearance, pH, the presence of protein, white blood cells, nitrite, leukocyte esterase, crystals, and casts are also evaluated. A urine specimen with significant leukocytes and positive nitrite and leukocyte esterase suggests a urinary tract infection. The presence of excess protein with hematuria indicates glomerulonephritis.

[0179] Urine microscopy examines the urinary sediment for red blood cell (RBC) morphology. RBC casts can be examined to distinguish between glomerular and nonglomerular hemorrhage as a cause of hematuria. More than 25% dysmorphic RBCs per high-power field also indicate glomerulonephritis. Renal parameters such as serum creatinine and urine output can be examined to assess for the presence or absence of acute kidney injury.

[0180] Imaging studies can be in the form of ultrasound of the kidneys, ureters, and bladder. This can aid in the diagnosis of, for example, kidney stones or bladder or renal masses. It can also detect renal cysts. Abdominopelvic CT scans, with or without contrast, can be used to detect kidney stones and other morphological abnormalities of the kidneys. MRI of the abdomen and pelvis can also be used, for example, when a CT scan is contraindicated or not diagnostic.

[0181] In some embodiments, a cystoscopy is performed on subjects who have a positive result according to a methylation assay of the present invention to further evaluate the presence or absence of a tumor in the subject's bladder. In some embodiments, a urine cytology test can be performed to assess the presence or absence of malignant cells, but is typically not a substitute for a cystoscopy.

[0182] Computed tomography, with and without contrast, can be used to evaluate patients for bladder cancer, regardless of stage.

[0183] Cystoscopic examination under anesthesia (EUA) can be performed to determine whether a palpable mass is present and, if so, whether it is mobile. EUA during cystoscopy is useful for identifying locally advanced disease, which may present as gross extravesical extension, invasion of adjacent organs, or involvement of the pelvic sidewall. If a mass is felt, bimanual examination can be repeated after resection to determine whether the mass is still present and to distinguish clinical stage T2 from T3b disease. Low-grade noninvasive tumors are generally papillary with a thin stalk. High-grade invasive tumors frequently appear sessile, solid, or nodular. Carcinoma in situ (CIS) is a high-grade noninvasive tumor that may appear as a flat, velvety lesion or occur in a patchy pattern. CIS sometimes involves most of the urothelial lining.

[0184] Fluorescence cystoscopy can also be used, which uses intravesical photoactive protoporphyrins (e.g., 5-aminolevulinic acid [5-ALA] and hexylaminolevulinic acid [HAL]), which accumulate preferentially in neoplastic tissue over normal tissue. The photoactive agent enhances the visual difference between normal and neoplastic tissue after illumination with blue light of an appropriate wavelength. The photoactive agent is typically injected one hour before cystoscopy.

[0185] If a tumor is identified during cystoscopy, its size and location are recorded. Small papillary tumors can be removed using cold-cup biopsy forceps or by transurethral resection of the bladder tumor (TURBT). Narrow-band imaging can be used to aid in complete removal of the bladder tumor. Alternatively, fluorescent cystoscopy may be used.

[0186] Despite visually complete resection, there is a high recurrence rate in patients with non-muscle-invasive bladder cancer (NMIBC). Therefore, current American Urological Association (AUA) and European Association of Urology (EAU) guidelines, respectively, recommend the use of intravesical chemotherapy immediately after TURBT. Several chemotherapy agents, including intravesical docetaxel, mitomycin C (MMC), doxorubicin, and epirubicin, have demonstrated clinical efficacy in reducing recurrence rates in the post-TURBT setting. Intravesical MMC can also be used after nephroureterectomy. Intravesical bacillus Calmette-Guérin (BCG) immunotherapy is an established first-line treatment for the management of carcinoma in situ (CIS) and high-grade non-muscle-invasive urothelial carcinoma (UC). These agents can also be used in combination with each other, for example, by administering MMC before the scheduled BCG dose. Intravesical gemcitabine and valrubicin have shown modest activity, and valrubicin is an FDA-approved therapy for the treatment of BCG-refractory CIS. Systemic platinum-based chemotherapy is commonly used for the treatment of locally advanced metastatic bladder cancer.

[0187] Although response rates to traditional therapies are high, many bladder cancer patients suffer from recurrence within one to five years. For patients who do not respond to BCG, treatment options are limited, and radical cystectomy remains the standard of care. Immune checkpoint inhibitors have become an increasingly used treatment option for many solid tumors, including bladder cancer. PD-L1 expression in such tumors may be associated with resistance to intravesical BCG therapy. Therefore, patient evaluation may include determining the PD-L1 expression status in bladder tumor tissue, and anti-PD-L1 or anti-PD-1 antibodies may be administered alone or in combination with intravesical BCG or chemotherapy. Since 2016, four checkpoint inhibitor drugs have been approved for bladder cancer: atezolizumab (Tecentriq®), pembrolizumab (Keytruda®), nivolumab (Opdivo®), and avelumab (Bavencio®). Immune checkpoint inhibitors have also demonstrated greater efficacy in heavy CD8 immune cell infiltrated tumors and in tumors with a high tumor mutational burden (TMB), such as in the case of bladder cancer. Thus, patient evaluation may include determining the TMB of bladder tumor tissue. Administration of checkpoint inhibitors may be by traditional systemic routes, intravesical routes, or both.

[0188] Other biologic therapies used in bladder cancer include enfortumab vedotin, an antibody-drug conjugate containing monomethylauristatin E and targeting nectin 4, which is expressed in many bladder cancers. Nadofalagen filadenovec (also known as rAd-IFNa / Syn3) is a replication-deficient recombinant adenovirus that delivers human interferon alpha-2b cDNA to the bladder epithelium. These agents can be delivered systemically or intravesically, particularly in BCG-unresponsive non-muscle-invasive bladder cancer.

[0189] In some embodiments, the score calculated for a subject as disclosed herein based on the methylation value(s) of at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 is determined to be above a predetermined cutoff. In some embodiments, detecting a score above the predetermined cutoff identifies primary bladder cancer in the subject. In some embodiments, detecting a score above the predetermined cutoff determines a positive probability for the presence of bladder cancer in the subject, and cystoscopy is performed on subjects with a score above the predetermined cutoff (i.e., a positive probability for the presence of bladder cancer).

[0190] In some embodiments, provided herein is a method for analyzing a urine sample from a human subject having at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS), the method comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of the urine sample; using the methylation value to assess the likelihood that the subject has primary bladder cancer; and, if the likelihood is positive, performing a cystoscopy on the subject. If the likelihood is negative, cystoscopy is not necessary. In some embodiments, if the likelihood is negative, the subject is assigned for evaluation of the presence or absence of a non-malignant genitourinary pathology.

[0191] In some embodiments, the methods of the present invention include performing a cystoscopy on the subject. As an alternative to performing a cystoscopy, the methods of the present invention may include scheduling a cystoscopy procedure for the subject or allocating the subject for cystoscopy.

[0192] In other embodiments, a score calculated for a subject as disclosed herein based on the methylation value(s) of at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 is determined to be below a predetermined cutoff. In some embodiments, detecting a score below a predetermined cutoff rules out bladder cancer in the subject. In some embodiments, detecting a score below a predetermined cutoff rules out high-grade bladder cancer in the subject. Thus, in some embodiments, methods for ruling out bladder cancer are provided herein. In additional embodiments, methods for ruling out high-grade bladder cancer are provided herein. The latter can be performed with a sensitivity of at least 85% and a very high specificity of over 98% in subjects presenting with hematuria and / or other lower urinary tract symptoms suspicious of malignancy.

[0193] In some embodiments, detecting a score below a predetermined cutoff rules out bladder cancer (particularly high-grade bladder cancer) in the subject, and at least one of the following assays is performed on the subject to assess the presence of a non-malignant genitourinary pathology: serum creatinine measurement, glomerular filtration rate measurement, blood calcium measurement, blood uric acid measurement, urine pH measurement, urine calcium measurement, urine culture, uroflowmetry, post-void residual volume test, renal ultrasound, abdominal ultrasound, and prostate ultrasound.

[0194] Non-malignant genitourinary conditions may include one or more of a urinary tract infection, a kidney infection, kidney stones, bladder stones, benign prostatic hyperplasia (BPH), kidney disease or injury, congenital or acquired anatomical abnormalities, and gynecological conditions, with each option representing a separate embodiment of the present invention.

[0195] In some embodiments, the methods of the invention include detecting a score below a predetermined cutoff and administering repeat urine tests to the subject within a predetermined period of time, e.g., within 6 to 12 months (e.g., within 6, 7, 8, 9, 10, 11, 12 months, each option representing a separate embodiment of the invention).

[0196] The following are certain preferred embodiments of the present invention. Embodiment 1. A method of performing a cystoscopy on a human subject having at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS), comprising: (a) determining a methylation value for at least one marker locus, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 marker loci selected from the group consisting of SEQ ID NOs: 1-15, in DNA from cells present in a urine sample from a subject; (b) calculating a subject score based on the methylation value; (c) detecting that the calculated score is above a predetermined cutoff, thereby determining that primary high-grade bladder cancer is present in the subject; and (d) performing a cystoscopy to identify the location of malignant cells in the subject's bladder.

[0197] In some embodiments, step (d) comprises performing a cystoscopy to identify the location of malignant cells in the bladder and rule out the presence of upper tract urothelial carcinoma (UTUC) in the subject.

[0198] Embodiment 2. The method of embodiment 1, further comprising biopsying malignant cells in the subject's bladder during cystoscopy to obtain cancerous tissue.

[0199] Embodiment 3. The method of embodiment 1 or 2, further comprising resecting malignant cells in the subject's bladder during cystoscopy to obtain cancerous tissue.

[0200] Embodiment 4. The method of any one of embodiments 1-3, wherein the method further comprises treating the bladder lining with an agent that specifically stains malignant cells present in the bladder lining immediately prior to or during cystoscopy.

[0201] Embodiment 5. The method of any one of embodiments 1-4, wherein the method further comprises administering intravesical chemotherapy and / or intravesical immunotherapy during cystoscopy.

[0202] Embodiment 6 The method of any one of embodiments 2 or 3, further comprising distinguishing non-muscle-invasive bladder cancer from muscle-invasive bladder cancer from cancerous tissue.

[0203] Embodiment 7. The method of any one of embodiments 1 to 6, wherein a methylation value is calculated separately for each marker locus of SEQ ID NOs: 1-15, and a score is calculated using each of the calculated methylation values.

[0204] Embodiment 8. A method of treating bladder cancer in a human subject, comprising: (a) determining that the subject has at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS); (b) determining a methylation value for at least one marker locus, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 marker loci selected from the group consisting of SEQ ID NOs: 1-15, in DNA from cells present in a urine sample from the subject; (c) calculating a subject score based on the methylation value; (d) detecting that the calculated score is above a predetermined cutoff, thereby determining that primary high-grade bladder cancer is present in the subject; and (e) performing a cystoscopy to identify the location of malignant cells in the subject's bladder and rule out the presence of upper tract urothelial carcinoma (UTUC) in the subject; (f) administering intravesical chemotherapy and / or intravesical immunotherapy to the subject.

[0205] Embodiment 9 The method of embodiment 8, further comprising biopsying malignant cells in the subject's bladder during cystoscopy to obtain cancerous tissue.

[0206] Embodiment 10. The method of embodiment 8 or 9, further comprising resecting malignant cells in the subject's bladder during cystoscopy to obtain cancerous tissue.

[0207] Embodiment 11. The method of any one of embodiments 8-10, wherein the method further comprises treating the bladder lining with an agent that specifically stains malignant cells present in the bladder lining immediately prior to or during cystoscopy.

[0208] Embodiment 12 The method of any one of embodiments 9 or 10, further comprising distinguishing non-muscle-invasive bladder cancer from muscle-invasive bladder cancer from cancerous tissue.

[0209] Embodiment 13. The method of any one of embodiments 8-12, wherein the subject is administered bacillus Calmette-Guerin (BCG) as intravesical immunotherapy.

[0210] Embodiment 14. The method of any one of embodiments 8 to 12, wherein the subject is administered one or more of nadofalagen filadenovec (Adstiladrin), a checkpoint inhibitor, and enfortumab vedotin as intravesical immunotherapy.

[0211] Embodiment 15. The method of any one of embodiments 8 to 12, wherein the subject is administered intravesical chemotherapy with one or more of valrubicin, mitomycin, and gemcitabine.

[0212] Embodiment 16. A method of treating bladder cancer in an individual in need thereof, comprising: 1. A method comprising intravesically administering a therapeutically effective amount of one or more of BCG, valrubicin, mitomycin, and gemcitabine to an individual, wherein the subject has previously been diagnosed with bladder cancer, not including upper tract urothelial carcinoma (UTUC), and wherein the previous diagnosis of bladder cancer comprises a combination of (i) identifying cancerous tissue structures in bladder tissue obtained by biopsy from the individual during a cystoscopy procedure, and (ii) identifying aberrant methylation at at least one marker locus, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 marker loci selected from the group consisting of SEQ ID NOs: 1-15, present in DNA obtained from cells present in a urine sample obtained from the individual.

[0213] Systems and Kits In some embodiments, a kit is provided for assessing the presence or absence of bladder cancer in a subject having at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS) as disclosed herein. In additional embodiments, a system is provided for assessing the presence or absence of bladder cancer in a subject having at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS) as disclosed herein.

[0214] Systems according to the invention include a computer processor for performing the assays and / or processing the results, e.g., for performing calculations. In some embodiments, computer-implemented methods are provided herein.

[0215] In some embodiments, a system according to the present invention comprises: DNA extracted from cells of a urine sample from a human subject; Components for performing a methylation assay for at least one marker locus that is highly methylated in bladder cancer DNA compared to non-cancer DNA selected from the group consisting of SEQ ID NOs: 1-15; and computer software stored on a non-transitory computer-readable medium that instructs a computer processor to determine a methylation value for at least one marker locus based on a methylation assay and compare the methylation value of each of the at least one marker locus to at least one reference methylation value selected from a cancer methylation value and a non-cancer methylation value to assess the presence or absence of cancer in a subject according to the methods disclosed herein.

[0216] Components for performing methylation assays include biochemical components (e.g., enzymes, primers, probes, nucleotides), chemical components (e.g., buffers, reagents), and technical components (e.g., PCR systems such as real-time PCR systems, and equipment such as tubes, vials, plates, pipettes, etc.).

[0217] In some particular embodiments, the system according to the present invention comprises: DNA extracted from cells of a urine sample from a human subject; at least one methylation-sensitive restriction endonuclease that recognizes a sequence within at least one marker locus that is highly methylated in bladder cancer DNA compared to non-cancer DNA, selected from the group consisting of SEQ ID NOs: 1-15; and a plurality of primer pairs for simultaneous amplification of at least one marker locus and a control locus after digestion with at least one methylation-sensitive restriction endonuclease; components for detecting amplification products of at least one marker locus and a control locus; and computer software stored on a non-transitory computer-readable medium, the computer software instructing a computer processor to determine a signal intensity for each of the amplification products of the at least one marker locus and a control locus, and compare a ratio between the signal intensities of the amplification products of each of the at least one marker locus and the control locus to at least one reference ratio selected from a cancer reference ratio and a non-cancer reference ratio, to assess whether cancer is present or absent in the subject according to the methods disclosed herein.

[0218] The components for detecting the amplification products of at least one marker locus and control locus include, for example, a fluorescent label, for example, in the form of a fluorescent primer or fluorescent probe that can specifically hybridize to the amplification products.

[0219] In some embodiments, a system according to the present invention generates and communicates a report to the subject and / or the subject's healthcare provider based on the methylation value.

[0220] In some embodiments, provided herein is a system for assessing the presence or absence of bladder cancer in a subject according to the methods disclosed herein, the system including computer software stored on a non-transitory computer readable medium, which, when executed, performs the following steps: (i) receiving a methylation value determined in DNA extracted from cells of a urine sample of a subject for at least one marker locus that is highly methylated in bladder cancer DNA compared to non-cancer DNA selected from the group consisting of SEQ ID NOs: 1-15; (ii) comparing the methylation value of each of the at least one marker locus to at least one reference methylation value selected from a cancer methylation value and a non-cancer methylation value, and outputting an assessment of whether cancer is present or absent in the subject based on the comparison.

[0221] In some embodiments, the computer software further comprises instructions that, when executed, configure or direct the computer processor to determine a methylation value for each of the at least one marker locus based on data from a methylation assay.

[0222] In some embodiments, computer software according to the invention receives as input raw data from a real-time PCR run. In some embodiments, the computer software instructs a computer processor to analyze the real-time PCR data to determine a methylation value, e.g., a methylation ratio, as disclosed herein.

[0223] Computer software includes processor-executable instructions stored on a non-transitory computer-readable medium. Computer software may also include stored data. The computer-readable medium is a tangible computer-readable medium, such as a compact disc (CD), a magnetic storage device, an optical storage device, a random access memory (RAM), a read-only memory (ROM), or any other tangible medium of expression.

[0224] It will be understood that the computer-related methods, steps, and processes described herein are implemented using software stored in non-volatile or non-transitory computer-readable instructions that, when executed, configure or direct a computer processor or computer to perform the instructions.

[0225] Each of the systems, servers, computing devices, and computers described herein may be implemented in one or more computer systems and configured to communicate over a network. They may also all be implemented in a single computer system. In one embodiment, a computer system includes a bus or other communication mechanism for communicating information, and a hardware processor coupled with the bus for processing information.

[0226] The computer system also includes a main memory, such as a random access memory (RAM) or other dynamic storage device, coupled to the bus for storing information and instructions executed by the processor. The main memory may also be used to store temporary variables or other intermediate information during execution of instructions executed by the processor. Such instructions, when stored on a non-transitory storage medium accessible to the processor, render the computer system into a specialized machine customized to perform the operations specified in the instructions.

[0227] The computer system further includes a read only memory (ROM) or other static storage device coupled to the bus for storing static information and instructions for the processor. A storage device, such as a magnetic disk or optical disk, is provided and coupled to the bus for storing information and instructions.

[0228] The computer system may be coupled via the bus to a display for displaying information to a computer user.

[0229] Input devices, including alphanumeric and other keys, are coupled to the bus for communicating information and command selections to the processor. Another type of user input device is a cursor control, such as a mouse, trackball, or cursor direction keys, for communicating directional information and command selections to the processor and for controlling cursor movement on a display.

[0230] According to one embodiment, the techniques herein are performed by a computer system in response to a processor executing one or more sequences of one or more instructions contained in a main memory. Such instructions may be read into the main memory from another storage medium, such as a storage device. Execution of the sequences of instructions contained in the main memory causes the processor to perform the process steps described herein. In alternative embodiments, hardwired circuitry may be used in place of or in combination with software instructions.

[0231] The term storage medium as used herein refers to any non-transitory medium that stores data and / or instructions that cause a machine to operate in a specific manner. Common forms of storage medium include, for example, floppy disks, flexible disks, hard disks, solid state drives, magnetic tape, or any other magnetic data storage medium, CD-ROMs, any other optical data storage medium, any physical medium with a pattern of holes, RAM, PROMs, and EPROMs, FLASH-EPROMs, NVRAMs, or any other memory chips or cartridges.

[0232] Storage media are distinct from but can be used in combination with transmission media, which participate in transferring information to and from the storage medium. For example, transmission media include coaxial cables, copper wire and fiber optics, including the wires that comprise a bus.

[0233] In some embodiments, a kit according to the invention comprises components for performing a methylation assay for at least one marker locus described herein on DNA extracted from cells of a urine sample from a subject.

[0234] In some embodiments, the kit comprises at least one methylation-sensitive restriction enzyme, a pair of primers for amplifying at least one marker locus and at least one control locus, and components for detecting the amplification products of the at least one marker locus and at least one control locus. In some embodiments, the kit comprises instructions for performing an assessment of the presence or absence of cancer as disclosed herein.

[0235] In some embodiments, the instructions may include directions for carrying out the method steps described above.

[0236] In some embodiments, the kit or system comprises a single methylation-sensitive endonuclease, hi some embodiments, the methylation-sensitive endonuclease is HhaI.

[0237] In some embodiments, the kit or system comprises a single methylation-sensitive endonuclease, hi some embodiments, the methylation-sensitive endonuclease is HinP1I.

[0238] In some embodiments, the kit may further include a non-transitory computer-readable medium storing computer software comprising instructions that, when executed, configure or direct a computer processor to perform the method steps described herein. In some embodiments, the computer software may be computer software that calculates at least one of a signal intensity, a signal ratio, and a marker score.

[0239] In some embodiments, the kit or system comprises HhaI, a primer pair complementary to at least one marker locus and at least one control locus described herein, and a fluorescent polynucleotide probe complementary to at least one marker locus and at least one control locus.

[0240] In some embodiments, the kit or system comprises HinP1I, a primer pair complementary to at least one marker locus and at least one control locus described herein, and a fluorescent polynucleotide probe complementary to at least one marker locus and at least one control locus.

[0241] Exemplary primers for amplifying the marker loci set forth in SEQ ID NOs: 1 to 15 are set forth in SEQ ID NOs: 25 to 155 (for = forward, rev = reverse), as follows: Locus 1 for: SEQ ID NOs: 25-29; Locus 1 rev: SEQ ID NOs: 30-35 Locus 2 for: SEQ ID NOs: 36-38; Locus 2 rev: SEQ ID NOs: 39-42 Locus 3 for: SEQ ID NOs: 43-51; Locus 3 rev: SEQ ID NOs: 52-61 Locus 4 for: SEQ ID NOs: 62-64; Locus 4 rev: SEQ ID NOs: 65-67 Locus 5 for: SEQ ID NOs: 68-70; Locus 5 rev: SEQ ID NOs: 71-73 Locus 6 for: SEQ ID NOs: 74-76; Locus 6 rev: SEQ ID NOs: 77-79 Locus 7 for: SEQ ID NOs: 80-85; Locus 7 rev: SEQ ID NOs: 86-89 Locus 8 for: SEQ ID NOs: 90-92; Locus 8 rev: SEQ ID NOs: 93-95 Locus 9 for: SEQ ID NOs: 96-99; Locus 9 rev: SEQ ID NOs: 100-102 Locus 10 for: SEQ ID NOs: 103-106; Locus 10 rev: SEQ ID NOs: 107-112 Locus 11 for: SEQ ID NOs: 113-116; Locus 11 rev: SEQ ID NOs: 117-120 Locus 12 for: SEQ ID NOs: 121-125; Locus 12 rev: SEQ ID NOs: 126-130 Locus 13 for: SEQ ID NOs: 131 to 135; Locus 13 rev: SEQ ID NOs: 136 to 140 Locus 14 for: SEQ ID NOs: 141 to 145; Locus 14 rev: SEQ ID NOs: 146 to 151 Locus 15 for: SEQ ID NOs: 152-153; Locus 15 rev: SEQ ID NOs: 154-155.

[0242] Exemplary primers for amplifying the control locus set forth in SEQ ID NO: 16 are set forth in SEQ ID NOs: 156-167 as follows: Control for: SEQ ID NOs: 156-161; Control rev: SEQ ID NOs: 162-167.

[0243] In some embodiments, the kit or system includes at least one of a first control construct and a second control construct, each of which includes a non-human / artificial DNA sequence as described above. In some embodiments, the kit includes both the first control construct and the second control construct as described above.

[0244] In some embodiments, the kit or system includes one or more containers filled with at least one nucleotide primer pair. In some embodiments, each nucleotide primer pair included in the kit of the present invention may include a primer complementary to a subsequence within or a flanking sequence of a marker locus selected from the marker loci set forth in SEQ ID NOs: 1-15, and each nucleotide primer pair is designed to selectively amplify a fragment of the genome that includes the locus.

[0245] In some embodiments, the kit or system may include primer pairs for selectively amplifying combinations of the above loci.

[0246] In some embodiments, the kit or system may further comprise a nucleotide primer pair for selectively amplifying the first artificial control construct and the second artificial control construct.

[0247] In some embodiments, the kit or system may further include oligonucleotide probes for detecting amplification products of loci amplified using the primers in the kit. Each oligonucleotide probe may be complementary to and capable of hybridizing to a subsequence within the locus. In some embodiments, the oligonucleotide probes may be fluorescently labeled.

[0248] In some embodiments, the kit or system may further comprise at least one additional component required for DNA digestion, locus amplification, and detection of the amplification product, such as a DNA polymerase and a nucleotide mixture.

[0249] In some embodiments, the kit or system may further include appropriate reaction buffers for digestion and amplification, as well as a written protocol for performing bladder cancer identification. The written protocol may include instructions for performing any of the steps disclosed herein, including, but not limited to, DNA digestion parameters, PCR cycling parameters, signal ratio analysis, and comparison to a standard ratio.

[0250] The following examples are presented in order to more fully illustrate certain embodiments of the present invention. However, they should in no way be construed as limiting the broad scope of the invention. Those skilled in the art can readily devise numerous variations and modifications of the principles disclosed herein without departing from the scope of the invention. [Example]

[0251] Example 1 - Specificity of methylation markers SEQ ID NOs: 1-15 in a population of urological patients and healthy volunteers without bladder cancer A prospective multicenter study was conducted at a regional urology center to evaluate the specificity of the methylation markers SEQ ID NOS: 1-15 in a population of urological patients and healthy volunteers without a history or evidence of bladder cancer. The methylation markers SEQ ID NOS: 1-15 are detailed in Table 1 (previously disclosed in WO 2017 / 006317, assigned to the present applicant). A total of 171 patients were enrolled in the study. Of this cohort, 53 patients presented with either hematuria (microscopic or gross hematuria) and / or lower urinary tract symptoms (LUTS). Table 2 shows the demographics of the cohort.

[0252] [Table 1] * Descriptions refer to positions on the hg18 genome build

[0253] [Table 2] (a) Vaginal cancer, (b) Breast cancer (c) Other urinary conditions: benign prostate conditions such as high PSA, BPH, prostatitis, prostatic nodules; benign kidney and urinary tract conditions such as hydronephrosis, renal cysts, chronic kidney disease, urethral strictures; sexual and penile conditions such as erectile dysfunction, hematospermia, Peyronie's disease, balanitis, infertility, penile lumps and induration, retrograde ejaculation, low testosterone, herpes and fungal infections; testicular conditions such as epididymitis, testicular hypospermia, hydrocele, testicular pain; benign gynecological-urinary conditions such as utero-vaginal prolapse, vulvar and vaginal candidiasis; other conditions: hernia, back pain.

[0254] Urine samples were obtained from the subjects (at least 10 mL of urine from each subject) and methylation assays were performed as described in WO 2017 / 006317 as follows.

[0255] First, each urine sample was processed using centrifugation to separate cells (both normal and, if present, cancerous) from the urine fluid and create a cell pellet. Specifically, each urine sample container was thoroughly mixed to ensure sample homogeneity. Next, 10–50 mL of urine was transferred to a 50 mL centrifuge tube. For centrifugation, the volume was adjusted with PBS 0.01 M pH 7.4. Centrifugation was performed at 1000 g for 10 minutes at room temperature (15–25°C). After centrifugation, the supernatant (urine) was discarded, and the pellet (cells) was resuspended in the residual urine remaining in the tube. After resuspension, 40 mL of PBS 0.01 M pH 7.4 was added to the tube, and the pellet was mixed with the PBS by inverting the tube several times to ensure efficient washing of the cells. Next, a second centrifugation was performed at 1000 g for 10 minutes at room temperature (15–25°C). The PBS was discarded, and the pellet was resuspended in the remaining PBS in the tube. The entire pellet volume was then transferred to a 1.5 mL microcentrifuge tube. The volume required for the DNA extraction step is approximately 400 μL. If the volume was in the 400-500 μL range, the entire volume was used in the DNA extraction step. If the volume was 500 μL, the tube was centrifuged at 1000 g for 1 minute and the supernatant was carefully discarded until it reached approximately 400 μL.

[0256] DNA was then extracted from the cell pellet using a QIAamp blood mini kit (QIAGEN, Hilden, Germany) and subjected to digestion with the methylation-sensitive restriction endonucleases HhaI or HinP1I. The digestion reaction (total volume 120 microliters) contained total extracted DNA (not quantified) and HhaI or HinP1I in digestion buffer. Digestion was carried out at 37°C for 2 hours.

[0257] Quantitative real-time PCR was then performed on the digested DNA samples to amplify, for each DNA sample, the 15 marker loci detailed in Table 1 above, as well as the control locus shown in SEQ ID NO: 16. The control locus does not contain the recognition sequences for HhaI or HinP1I (it does not contain a nucleotide sequence recognized by either of these enzymes, whether methylated or not), and remains intact upon digestion with these enzymes, regardless of its methylation status.

[0258] More specifically, each digested DNA sample was divided into eight aliquots containing 10 microliters of digested DNA. Seven aliquots were supplemented with primer pairs for amplifying two of the 15 marker loci and a control locus (the control locus was amplified in all aliquots). The eighth aliquot was supplemented with primer pairs for amplifying one of the remaining marker loci and a control locus. Amplicons of 75 to 225 bases were amplified, each containing one of the 16 loci along with 5-80 bases of 5' and 3' flanking sequences. Each amplification reaction (total volume 25 microliters) also contained dNTPs, Taq DNA polymerase, and reaction buffer. Fluorescently labeled polynucleotide probes (one for each locus) were added to the reaction to allow detection of the amplification products during amplification. Real-time PCR reactions were performed in an ABI 7500 FastDx instrument using the following PCR program: initial activation of the enzyme at 95°C for 10 min, followed by 45 cycles of 95°C for 15 s, then 60°C for 1 min.

[0259] After amplification, quantitative PCR plots showing the change in fluorescent signal from the probe as a function of cycle number were analyzed to calculate the cycle of quantification (Cq) for each locus. The delta Cq between the Cq of the control locus and the Cq of each marker locus was then calculated and used in the following formula: 2 (対照遺伝子座のCq-マーカー遺伝子座のCq) .

[0260] It should be understood that calculations were performed for marker and control loci co-amplified in the same aliquot.

[0261] The numerical value obtained for each marker locus relative to the control locus represents the ratio between the signal intensities of the amplification products of the marker locus and the control locus, reflecting the methylation ratio between the marker locus and the control locus in the DNA sample. A marker score was then calculated for each marker locus. This score is the signal ratio normalized to the reference ratio, with the highest signal ratio being "100" and the lowest signal ratio being "0." Overall, 15 marker scores were calculated for each DNA sample. The 15 individual marker scores obtained for each DNA sample were combined into a single sample score called the "EpiScore," which ranges from 0 to 100 and reflects the overall relative methylation level of the DNA sample across the panel of 15 marker loci. A threshold score was established; above the threshold score, the DNA sample was classified as positive for bladder cancer. An EpiScore below the threshold classifies the DNA sample as negative for bladder cancer.

[0262] A total of 150 patients had valid methylation assay results, of which 3 were positive, resulting in a specificity of 98.0% (94.3%-99.6%). In the subset of patients with hematuria or LUTS, the specificity was similar at 97.8% (88.5%-99.9%). The presence of hematuria, urinary tract infection, or stones did not affect specificity (p>0.45 by Fisher's exact test). Table 3 shows the specificity by subgroup.

[0263] [Table 3]

[0264] Another prospective, single-center study was conducted in a urology outpatient clinic to evaluate the specificity of the methylation markers SEQ ID NOS: 1-15 in a population of urological patients presenting with hematuria and no history or evidence of bladder cancer. A total of 46 patients were enrolled in the study, of which 31 (67.4%) were men. Table 4 shows the clinical details of the cohort.

[0265] [Table 4] (a) One patient had both stones and BPH.

[0266] Urine samples were obtained from the subjects, and methylation assays were performed as described above. A total of 43 patients had valid methylation assay results, all of which were negative, resulting in a specificity of 100.0% (91.8-100.0%). Table 5 shows the specificity by subgroup.

[0267] [Table 5] (a) One patient had both stones and BPH.

[0268] Example 2 - Sensitivity of methylation markers SEQ ID NOS: 1-15 in patients with primary bladder cancer A prospective, single-center study was conducted to evaluate the sensitivity of methylation markers SEQ ID NOS: 1-15 in patients with primary bladder cancer. A total of 64 patients with newly diagnosed bladder cancer were enrolled in the study, and all had their diagnosis confirmed by pathology. A urine sample was collected from each subject before tumor resection, and a methylation assay was performed as described in Example 1. The median age was 74 years (range, 43-96 years), and 56 (87.5%) were male. A total of 62 patients had valid methylation assay results, of which 41 were positive, resulting in an overall sensitivity of 66.1% (53.0%-77.7%). Overall, 40 patients were diagnosed with high-grade tumors. The sensitivity for high-grade tumors was 87.5% (73.2%-95.8%), which was similar across all stages (p=0.5135 by Fisher's exact test). Table 6 shows the overall sensitivity as well as sensitivity by disease grade and stage.

[0269] [Table 6] (a) Five patients had no stage information: three low-grade and two high-grade. (b) Ten patients had no information on concomitant CIS.

[0270] To calculate the negative and positive predictive values ​​(NPV and PPV) of the methylation assay for bladder cancer detection, we used the incidence rates reported by Woldu et al. (Woldu et al., 2021, J Urol., 205(5):1387-1393) for the three risk categories defined by the American Urological Association (AUA) (Barocas et al., 2020, Microhematuria: AUA / SUFU Guideline. J Urol., 204(4):778-786). The calculations used the sensitivity and specificity of the methylation assay for high and low grade tumors, and assumed, in accordance with the literature, that 60% of newly diagnosed cancers are low-grade tumors and the remainder are high-grade. The results are shown in Table 7.

[0271] [Table 7]

[0272] The above table shows that even for the low and intermediate risk groups, the present invention is highly beneficial in reducing the number of unnecessary follow-up tests, especially cystoscopies.

[0273] A pre-test probability of 1% can be converted to a post-test probability of 10% or 21% for the low and intermediate groups, respectively, meaning that 1 in 10 or 1 in 5 cystoscopy tests would actually show the presence of bladder cancer, rather than only 1 in 100, substantially reducing the need for this invasive procedure. Such efficacy may, for the first time, allow for the selection of patients for urological evaluation based on urine tests, where the risk of missing high-grade disease is very small (0.02-0.32%).

[0274] The foregoing description of specific embodiments fully reveals the general nature of the invention, so that others, by applying their current knowledge, can easily modify and / or adapt such specific embodiments to various uses without undue experimentation and without departing from the general concept; therefore, such adaptations and modifications should be understood within the meaning and range of equivalents of the disclosed embodiments, and are so intended. It should be understood that the phraseology or terminology used herein is for purposes of description and not limitation. The means, materials, and steps for carrying out the various disclosed chemical structures and functions may take a variety of alternative forms without departing from the invention.

Claims

1. 1. A method for determining the presence or absence of primary bladder cancer in a human subject having at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS), comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from said subject; (b) calculating a score for the subject based on the methylation value; and (c) detecting that the calculated score is above a predetermined cutoff, thereby determining a positive probability for the presence of primary bladder cancer in the subject.

2. 1. A method for assessing a human subject for at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS), comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from said subject; (b) calculating a score for the subject based on the methylation value; and (c) detecting that the calculated score is above a predetermined cutoff, thereby determining a positive probability for the presence of bladder cancer in the subject; and (d) mandating a cystoscopy for said subject having said score above said predetermined cutoff.

3. 1. A method for ruling out bladder cancer in a human subject having at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS), comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from said subject; (b) calculating a score for the subject based on the methylation value; and (c) detecting that the calculated score is below a predetermined cutoff, thereby ruling out bladder cancer in the subject; and (d) avoiding cystoscopy in said subject.

4. 1. A method for assessing a human subject for at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS), comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from said subject; (b) calculating a score for the subject based on the methylation value; and (c) detecting that the calculated score is below a predetermined cutoff, thereby ruling out bladder cancer in the subject; and (d) performing on the subject at least one assay selected from the group consisting of serum creatinine measurement, glomerular filtration rate measurement, blood calcium measurement, blood uric acid measurement, urine pH measurement, urine calcium measurement, urine culture, uroflowmetry, post-void residual volume test, renal ultrasound, abdominal ultrasound, and prostate ultrasound; assessing the presence of a non-malignant genitourinary condition in said subject.

5. 5. The method of claim 4, wherein the non-malignant genitourinary condition is selected from the group consisting of a urinary tract infection, a kidney infection, kidney stones, bladder stones, benign prostatic hyperplasia (BPH), kidney disease or injury, and congenital or acquired anatomical abnormalities.

6. 1. A method for assessing a human subject with hematuria, comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from said subject; (b) calculating a score for the subject based on the methylation value; and (c) detecting that the calculated score is below a predetermined cutoff; and (d) administering a repeat urine test to the subject within 6 to 12 months.

7. 7. The method of claim 6, wherein the subject has one or more lower urinary tract symptoms (LUTS).

8. 1. A method for determining the presence or absence of high-grade primary bladder cancer in a human subject having at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS), comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from said subject; (b) calculating a score for the subject based on the methylation value; and (c) detecting that the calculated score is above a predetermined cutoff, thereby determining a positive probability for the presence of high-grade bladder cancer in the subject.

9. 1. A method for ruling out high-grade primary bladder cancer in a human subject having at least one symptom selected from hematuria and lower urinary tract symptoms (LUTS), comprising: (a) determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 in DNA from cells of a urine sample from said subject; (b) calculating a score for the subject based on the methylation value; and (c) detecting that the calculated score is below a predetermined cutoff, thereby ruling out high-grade bladder cancer in the subject; and (d) avoiding cystoscopy in said subject.

10. 10. The method of claim 9, further comprising assessing the subject for the presence or absence of a non-malignant genitourinary condition.

11. 11. The method of claim 10, wherein the assessment of the presence or absence of a non-malignant genitourinary pathology comprises at least one assay selected from the group consisting of serum creatinine measurement, glomerular filtration rate measurement, blood calcium measurement, blood uric acid measurement, urine pH measurement, urine calcium measurement, urine culture, uroflowmetry, post-void residual volume test, renal ultrasound, abdominal ultrasound, and prostate ultrasound.

12. 12. The method of any one of claims 1 to 11, wherein the lower urinary tract symptoms (LUTS) comprise at least one symptom selected from urination frequency, urgency, urge incontinence, nocturia, altered urinary flow, dysuria, straining and dribbling, and residual urine.

13. The method according to any one of claims 1 to 11, wherein the hematuria is microscopic hematuria or macroscopic hematuria.

14. 12. The method of any one of claims 1 to 11, wherein the at least one marker locus comprises a plurality of marker loci selected from the group consisting of SEQ ID NOs: 1 to 15.

15. 12. The method of any one of claims 1 to 11, wherein the at least one marker locus comprises the locus set forth in SEQ ID NO:

1.

16. 16. The method of claim 15, wherein the at least one marker locus further comprises the locus set forth in SEQ ID NO:

5.

17. 17. The method of claim 16, wherein the at least one marker locus further comprises the locus set forth in SEQ ID NO: 7 and the locus set forth in SEQ ID NO:

11.

18. 18. The method of claim 17, wherein the at least one marker locus further comprises at least one additional marker locus selected from the group of loci set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:

15.

19. 19. The method of claim 18, wherein the at least one additional restriction locus comprises the locus set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:

15.

20. The method of claim 19, wherein determining the methylation values ​​for a plurality of marker loci comprises determining a ΔCq for each marker locus and calculating a single integrated methylation value based on the ΔCq determined for each marker locus.

21. The method of claim 19, wherein the ratio between the signal intensities of the amplification products of each of the at least one marker locus and a control locus is calculated by determining the quantification cycle (Cq) of each locus and calculating 2 (Cq control locus - Cq limiting locus).

22. The method of any one of claims 1 to 11, wherein the methylation value is determined using one or more analytical methods selected from the group consisting of DNA sequencing, real-time PCR, and array hybridization.

23. 12. The method of claim 1, wherein determining the methylation value comprises, for a plurality of potential methylation sites in a plurality of marker loci, quantifying methylated read counts at the potential methylation sites, and calculating a methylation value based on the number of methylated read counts.

24. determining a methylation value for at least one marker locus selected from the group consisting of SEQ ID NOs: 1-15 and calculating a score; (i) subjecting the DNA from the cells of the urine sample of the subject to digestion with at least one methylation-sensitive restriction endonuclease that recognizes a sequence within the at least one marker locus that is highly methylated in cancer DNA compared to non-cancer DNA to obtain restriction endonuclease-treated DNA; (ii) extracting said at least one fragment from said restriction endonuclease-treated DNA co-amplifying the marker loci and the control loci, thereby generating an amplification product for each locus; (iii) determining the signal intensity of each amplification product generated; (iv) comparing the ratio between the signal intensities of the amplification products of each of the at least one marker locus and the control locus with at least one reference ratio selected from a cancer reference ratio and a non-cancer reference ratio to calculate a score.

25. 25. The method of claim 24, wherein step (i) is carried out using a single methylation-sensitive restriction endonuclease.

26. 26. The method of claim 25, wherein the methylation-sensitive restriction endonuclease is HhaI or HinP1I.

27. 25. The method of claim 24, wherein the control locus is a locus that does not contain a nucleotide sequence recognized by the methylation-sensitive restriction endonuclease.

28. 28. The method of claim 27, wherein the methylation-sensitive restriction endonuclease is selected from HhaI and HinP1I, and the control locus is the locus set forth in SEQ ID NO:

16.

29. 25. The method of claim 24, wherein step (ii) is carried out using real-time PCR.

30. 30. The method of claim 29, wherein step (ii) comprises adding fluorescent probes to assist in detection of the amplification products of the at least one marker locus and the control locus.

31. 31. The method of claim 30, wherein determining the methylation values ​​for a plurality of marker loci comprises determining a ΔCq for each marker locus and calculating a single integrated methylation value based on the ΔCq determined for each marker locus.

32. The ratio between the signal intensities of the amplification products of each of the at least one marker locus and the control locus determines the quantification cycle (Cq) of each locus; （Ｃｑ対照遺伝子座－Ｃｑ制限遺伝子座） 31. The method of claim 30, wherein the value is calculated by: