Systems and methods for evaluation and treatment of urothelial carcinoma

Abstract

Methods to determine risk of urothelial carcinoma and applications thereof are described. Generally, systems and methods analyze molecular markers indicative of urothelial carcinoma. Based on urothelial carcinoma risk, further diagnostics or treatments can be performed.

Description

SYSTEMS AND METHODS FOR EVALUATION AND TREATMENT OF UROTHELIAL CARCINOMACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The current application claims the benefit of and priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 689,535 filed August 30, 2024, the disclosure of which is incorporated herein by reference in its entirety for all purposes.TECHNICAL FIELD

[0002] The disclosure is generally directed to diagnostic systems and methods to evaluate urothelial carcinoma and treatments thereof.BACKGROUND

[0003] Urothelial carcinoma that arises due to proliferative growth of urothelial cells within the lining of the bladder, kidney, and ureter. Bladder cancer (BCa) and upper tract urothelial cancer (UTUC) are two types of urothelial carcinoma that are differentiated by the location the cancer develops and other factors, where BCa is more common and prevalent. Although BCa and UTUC have fairly similar symptoms and mostly affect older individuals, the cancers are molecularly different and may require different therapeutic interventions (see, e.g., J. P. Sfakianos, et al., Eur Urol Oncol. 2021 Apr;4(2): 170-179; and F. Lefort, et al., Cancers (Basel). 2023 Nov 23;15(23):5558; the disclosures of which are each hereby incorporated by reference).

[0004] Cystoscopy is the standard diagnostic assessment for BCa. To perform cystoscopy, a medical professional inserts a narrow tube with a camera (i.e., the cystoscope) through the urethra and into the bladder to examine the linings of these organs for signs of disease and / or retrieve a tissue biopsy. Although an outpatient procedure and generally performed with a local anesthetic to prevent pain, it is an uncomfortable procedure and undesirable, especially for routine screening procedures. These procedures may be required to be repeated often for patients with higher risk ofdeveloping urothelial cancer or patients that recently completed treatment and need to be screened for minimal residual disease and recurrence.

[0005] UTUC is rather difficult to diagnose, and endoscopic diagnosis is much more invasive. Generally, if a patient is experiencing symptoms such as side pain or blood in the urine, a CT scan can be performed, but ureteroscopy provides a much more reliable diagnosis. Similar to cystoscopy, a medical professional inserts a narrow tube with a camera (i.e., the ureteroscope) through the urethra, through the bladder and into ureter and kidney the to examine the linings of these organs for signs of disease and / or retrieve a tissue biopsy. Due to the invasiveness, this procedure is often considered a minor surgery. Despite being more uncomfortable and less desirable than cystoscopy, screening procedures are critical for patients with history of UTUC. In fact, because UTUC has a high rate of recurrence, patients often have multiple ureteroscopies in the first year after they are diagnosed and treated to screen for minimal residual disease and recurrence. Thus, it would be of great benefit to be able to screen and monitor patients for urothelial cancer in a less invasive manner.SUMMARY OF THE INVENTION

[0006] Several embodiments of the disclosure are directed to systems and methods for assessing an individual for risk related to presence of or degree of severity of urothelial carcinoma. In various embodiments, the assessment is for bladder cancer (BCa), upper tract urothelial cancer (UTUC), or BCa and UTUC collectively. In many embodiments, a molecular-based screening diagnostic is performed to assess whether urothelial carcinoma is present and / or degree of severity of BCa or UTUC. Due to the molecular differences in BCa and UTUC, it was unexpected that a set of molecular biomarkers useful in diagnosis of BCa is also useful for UTUC.

[0007] In many embodiments, RNA is collected from sediment of a patient’s urine sample. In many embodiments, the RNA is assessed for expression of one or more biomarkers. In some embodiments, biomarkers for assessment of urothelial carcinoma include one or more of receptor 1 (ROBO1 ), corticotropin releasing hormone (CRH), and insulin like growth factor 2 (IGF2). In some embodiments, ROBO1 , IGF2, and CRH areeach assessed to determine the presence of urothelial carcinoma. In some embodiments, a risk score for presence of urothelial carcinoma is computed utilizing ROBO1 , IGF2, and CRH RNA expression levels. In some embodiments, a risk score for a severity of a urothelial carcinoma is computed utilizing ROBO1 , IGF2, and CRH RNA expression levels. In many embodiments, a quality control biomarker is utilized to ensure the collected RNA used within the assessment has a requisite quality and / or quantity. In some embodiments, RNA expression levels of CDC42BPB is assessed to ensure the quality and / or quantity of the RNA used as input for assessment of RNA expression.

[0008] In some aspects, the techniques described herein relate to a method to determine an individual's risk of urothelial carcinoma.

[0009] In some aspects, the techniques described herein relate to a method to determine an individual's risk of urothelial carcinoma including: obtaining a sample including RNA, wherein the sample is derived from a urine sample of the individual.

[0010] In some aspects, the techniques described herein relate to a method to determine an individual's risk of urothelial carcinoma including: quantifying an amount of RNA transcripts for roundabout guidance receptor 1 (ROBO1), corticotropin releasing hormone (CRH), and insulin like growth factor 2 (IGF2) within the sample.

[0011] In some aspects, the techniques described herein relate to a method to determine an individual's risk of urothelial carcinoma including: computing a risk score using the amount of RNA transcripts for ROBO1 , CRH, and IGF2.

[0012] In some aspects, the techniques described herein relate to a method, further including: quantifying an amount of RNA transcripts for CDC42 binding protein kinase beta (CDC42BPB) within the sample; and determining the amount of RNA transcripts for CDC42BPB is greater than threshold.

[0013] In some aspects, the techniques described herein relate to a method further including: collecting the urine sample from the individual; sedimenting cells within the urine sample; and extracting RNA from the sedimented cells to yield the sample including RNA.

[0014] In some aspects, the techniques described herein relate to a method, wherein collecting the urine sample is performed at a location that is not a medical facility.

[0015] In some aspects, the techniques described herein relate to a method further including: storing the urine sample for at least 24 hours prior to RNA extraction, wherein the urine sample is stored with a preservative.

[0016] In some aspects, the techniques described herein relate to a method, wherein the urine sample of the individual was collected prior to a diagnosis of the individual having urothelial carcinoma.

[0017] In some aspects, the techniques described herein relate to a method, wherein the individual is experiencing a symptom of urothelial carcinoma.

[0018] In some aspects, the techniques described herein relate to a method, wherein the symptom of urothelial carcinoma is hematuria or pain during urination.

[0019] In some aspects, the techniques described herein relate to a method, wherein the urine sample of the individual was collected during a treatment regimen for urothelial carcinoma.

[0020] In some aspects, the techniques described herein relate to a method, wherein the urine sample of the individual was collected after a treatment regimen for urothelial carcinoma.

[0021] In some aspects, the techniques described herein relate to a method, wherein the treatment regimen includes surgical resection orfulguration and the risk score is used to assess disease burden.

[0022] In some aspects, the techniques described herein relate to a method, wherein the urothelial carcinoma is bladder cancer.

[0023] In some aspects, the techniques described herein relate to a method, wherein the urothelial carcinoma is upper tract urothelial cancer.

[0024] In some aspects, the techniques described herein relate to a method, wherein the risk score indicates a risk of urothelial carcinoma greater than a threshold, the method further including: performing cystoscopy, ureteroscopy or biopsy within the urethra, bladder, ureter or kidney.

[0025] In some aspects, the techniques described herein relate to a method, wherein the risk score indicates a risk of urothelial carcinoma less than a threshold, the methodfurther including: repeating steps (a), (b), and (c) at a later timepoint in lieu of performing cystoscopy, ureteroscopy or biopsy.

[0026] In some aspects, the techniques described herein relate to a method, wherein the risk score indicates a risk of urothelial carcinoma greater than a threshold, the method further including: administering a treatment, wherein the treatment includes at least one of: surgical resection, radiation therapy, chemotherapy, immunotherapy, or targeted therapy.

[0027] In some aspects, the techniques described herein relate to a method further including: repeating steps (a), (b), and (c) after administration of the treatment to assess for minimal residual disease.

[0028] In some aspects, the techniques described herein relate to a method, wherein repeating steps (a), (b), and (c) is performed at multiple timepoints over a period of time.

[0029] In some aspects, the techniques described herein relate to a method, the method further including: administering a treatment regimen over a period of time, wherein the treatment regimen includes at least one of: surgical resection, radiation therapy, chemotherapy, immunotherapy, or targeted therapy; and repeating steps (a), (b), and (c) at multiple timepoints during the period of time to assess response to treatment.

[0030] In some aspects, the techniques described herein relate to a method, wherein when the risk score indicates an updated risk of urothelial carcinoma, the method further includes: altering a treatment regimen in accordance with the updated risk.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The description and claims will be more fully understood with reference to the following figures and data graphs, which are presented as examples of the disclosure and should not be construed as a complete recitation of the scope of the disclosure.

[0032] Figure 1 provides a flowchart of an exemplary method to assess an individual for risk of urothelial carcinoma.

[0033] Figure 2 provides Table 1 summarizing the bladder cancer cohort demographics used within the study to generate and assess a molecular diagnostic assay for urothelial cancer.

[0034] Figures 3A and 3B each provide a flowchart of the urine samples collected and analyzed for validation of a molecular diagnostic assay for urothelial cancer.

[0035] Figure 4 provides Table 2 summarizing the bladder cancer sample characteristics used within the study to generate and assess a molecular diagnostic assay for urothelial cancer.

[0036] Figures 5A-5D provide data summarizing the Urine RNA Profile (uRNAp) score results for detection and surveillance of bladder cancer. Figs. 5A and 5C provide waterfall plots of uRNAp scores by tumor grade from patient samples without urothelial carcinoma (No UC), and with low grade (LG) or high grade (HG) UC. A uRNAp score of 0.23 (horizontal dashed line) was the cutoff for UC diagnosis. Detection scores (Fig. 5A) were generated from patients without a prior history of UC (detection cohort, n = 458 samples). Surveillance scores (Fig. 5C) were generated from patients with a history of bladder cancer (surveillance cohort, n = 1253 samples). Figs. 5B and 5D provide box and whisker plots summarizing uRNA score and tumor size. In the detection cohort (Fig. 5B), 10 tumors were defined as small (< 1 cm; n=7 LG; n=3 HG), 21 were medium (between 1 and 3 cm; n=9 LG; n=12 HG), and 71 large (> 3 cm; n=9 LG; n=62 HG). Tumor size information was not available in 2 cases. In the surveillance cohort (Fig. 5D), 70 tumors were defined as small (< 1 cm; n=41 LG; n=29 HG), 77 were medium (between 1 and 3 cm; n=29 LG; n=48 HG), and 71 large (> 3cm; n=14 LG; n=57 HG). Tumor size information was not available in 27 cases.

[0037] Figure 6 provides Table 3 summarizing the performance of uRNAp as compared to cytology for detection and surveillance of bladder cancer.

[0038] Figures 7A and 7B provide receiver operating characteristic curves for uRNAp for detection cohort (Fig. 7A) and surveillance cohort (Fig. 7B). The detection cohort consisted of 458 samples at unique timepoints from 448 patients and the surveillance cohort consisted of 1253 samples at unique timepoint from 392 patients. AUC = area under the receiver operating characteristic curve.

[0039] Figure 8 provides cystoscopy images of bladder cancers with false-negative uRNAp scores. All uRNAp false negatives were biopsy-confirmed bladder cancer and images taken during surgery were available for 13 of the 16 cases. Of the uRNAp falsenegatives were 14 were low grade tumors; 9 small (<1 cm aggregate tumor, panels 1 , 2, 3, 4, 6, 7, 9, 11 ; no image available n=2) and 5 medium (1-3 cm aggregate tumor size, panels 10, 12, 13; no image available n=1 ). There were 2 high-grade uRNAp false negatives: one inside a diverticulum (panel 5) and the other a 5-10 mm patch of CIS (panel 8). All cases of uRNAp false negative results without available images were from surveillance patients the 2 small low-grade tumors had uRNAp scores of 0.1015 and 0.2191 and 1 medium low-grade had a uRNAp score of 0.21578.

[0040] Figure 9 provides Table 4 summarizing the IITUC cohort demographics used within the study to generate and assess a molecular diagnostic assay for urothelial cancer.

[0041] Figure 10 provides Table 5 summarizing the UTUC sample characteristics used within the study to generate and assess a molecular diagnostic assay for urothelial cancer.

[0042] Figure 11 provide data summarizing the Urine RNA Profile (uRNAp) score results for detection of UTUC. Fig. 11 provides a waterfall plot of uRNAp scores by tumor grade from patient samples without urothelial carcinoma (No UC), and with low grade (LG) or high grade (HG) UC. A uRNAp score of 0.23 (horizontal dashed line) was the cutoff for UC diagnosis.

[0043] Figure 12 provides Table 6 summarizing the performance of uRNAp as compared to cytology for detection of UTUC.DETAILED DESCRIPTION

[0044] The description discloses various systems and methods to assess an individual’s risks related to urothelial carcinoma. Some embodiments of the systems and methods are employed to assess risk of whether a urothelial carcinoma is present. Some embodiments of the systems and methods are employed to assess risk of associated with the degree of severity of a urothelial carcinoma. The description further discloses systems and methods to be utilized based on the individual’s predicted risk, which can include (but are not limited to) confirmatory diagnostics and / or treatments.

[0045] Urothelial carcinoma includes Bladder Cancer (BCa) and upper tract urothelial cancer (UTUC). Various embodiments of the disclosed systems and methods can be utilized for assessment of BCa and / or UTUC. In various embodiments, systems and methods are utilized to assess BCa and UTUC collectively. In various embodiments, systems and methods are utilized to assess BCa and UTUC individually. Whether BCa and UTUC are to be assessed individually or collectively can be based on a condition of the patient. For example, systems and methods are utilized to screen a patient without any history of BCa or UTUC, which can assess the presence of and / or severity of both BCa and UTUC. In another example, systems and methods are utilized to screen a patient that has been diagnosed with and treated for BCa, which can assess the presence of and / or degree of severity of minimal residual disease and / or recurrence. In yet another example, systems and methods are utilized to screen a patient that has been diagnosed with at treated for UTUC, which can assess the presence and / or degree of severity of minimal residual disease and / or recurrence.

[0046] In several embodiments, systems and methods for assessment of BCa and / or UTUC is based on RNA expression of one or more biomarkers. In many embodiments, RNA from a patient sample is assessed for expression of one or more biomarkers. In many embodiments, RNA expression level of one or more biomarkers is utilized to compute a risk for presence of and / or degree of severity of a urothelial carcinoma. In some embodiments, a screening result that signals a patient is likely to have urothelial cancer present can be used as an indicator to perform further clinical actions. In some embodiments, a screening result that signals the degree of severity of a patient’s urothelial cancer can be used as an indicator to perform further clinical actions. Examples of further clinical actions include (but are not limited to) administration of a confirmatory diagnostic and administration of a treatment.

[0047] The systems of methods disclosed herein have been developed to enhance identification and stratification of risk of urothelial cancer, which is essential for determining the appropriate monitoring activity and therapeutic regimen. These systems and methods greatly improve on the current clinical procedures because the course of urothelial cancer can vary greatly based on grade, stage, size of tumor, multifocality,presence of carcinoma in situ (CIS), and interval of recurrence (see., S. Cambier, et al., Eur Urol. 2016 Jan;69(1 ):60-9 and R. J. Sylvester, et al., Eur Urol. 2006 Mar;49(3):466-5 (discussion 475-7); the disclosure of which is hereby incorporated by reference). Currently, cytology is the only urine-based assay recommended for regular use by the American Urologic Association (AUA) for BCa and UTUC surveillance. Unfortunately, cytology has high specificity but low sensitivity, and thus multipole cystoscopy or ureteroscopy procedures are needed to performed routinely to check for cancer presence. Because the systems and methods of disclosure provide a more sensitive and less invasive procedure to screen for urothelial carcinoma, they can significantly improve patient experience for routine checkups, ongoing treatment assessments, and. And with improved experience that merely requires sample collection, medical professionals are more likely to administer more screening procedures and at earlier timepoints, resulting in better outcomes for patients. Furthermore, the utilization of the improved systems and methods of the current disclosure will increase urothelial carcinoma patient follow-through during post-treatment monitoring of minimal residual disease.Biomarkers Indicative of Urothelial Carcinoma Risk

[0048] Several embodiments are directed towards the assessment of an individual’s risk for urothelial carcinoma, which can be utilized to screen, diagnose, and / or monitor the individual. In some embodiments, a risk assessment comprises a prediction of the presence of urothelial carcinoma in the individual. In some embodiments, a risk assessment comprises a prediction of the severity of a urothelial carcinoma in the individual. For purposes of this disclosure, severity of a urothelial carcinoma is a correlative of an amount of urothelial cancer present in an individual. In many embodiments, a risk assessment comprises assessing expression of a set of one or more biomarkers Generally, RNA transcripts levels of biomarkers are assessed, which correlate with a risk of urothelial carcinoma.

[0049] A prediction of a risk of urothelial cancer can signal whether a clinical action is to be performed, such as administration of confirmatory diagnostics and / or administration of treatments. In some instances, a risk prediction of urothelial carcinoma is performed toscreen individuals to determine if they should undergo confirmatory diagnostic assessment (e.g., cystoscopy or ureteroscopy). In some instances, a risk prediction of urothelial carcinoma is performed to determine whether an individual is to be treated for urothelial carcinoma. In some instances, a risk prediction of urothelial carcinoma is performed when an individual has one or more symptoms of urothelial carcinoma. Common symptoms of urothelial carcinoma include hematuria or pain when urinating. In some instances, a risk prediction of urothelial carcinoma is performed to determine which treatment regimen is to be administered to the individual. In some instances, a risk prediction of urothelial carcinoma is performed to determine treatment efficaciousness, which can be performed during and / or after administration of a treatment regimen. In some instances, a risk prediction of urothelial carcinoma is performed to detect minimal residual disease or recurrence.

[0050] An example of a method for assessing an individual’s risk for urothelial carcinoma in accordance with various embodiments of the disclosure is provided in Fig. 1. Method 100 can begin by measuring (101 ) RNA transcript levels of a patient sample. In many embodiments, RNA transcript levels of one or more biomarkers indicative of urothelial carcinoma are measured. In some embodiments, the one or more biomarkers comprise at least one biomarker selected from roundabout guidance receptor 1 (ROBO1 ), corticotropin releasing hormone (CRH), and insulin like growth factor 2 (IGF2). In some embodiments, the one or more biomarkers comprise at least two biomarkers selected from ROBO1 , CRH, and IGF2. In some embodiments, the one or more biomarkers comprise at least at least three biomarkers selected from ROBO1 , CRH, and IGF2. It has been found that the assessment of RNA expression level of the combination of ROBO1 , CRH, and IGF2 yields a prediction of urothelial carcinoma risk with high sensitivity and specificity (see section on Examples and Experimental Data).

[0051] In some embodiments, RNA transcript levels of one or more quality control biomarkers are measured. RNA levels of one or more quality control biomarkers can be utilized to ensure that the biomarker assay yields an accurate assessment of urothelial carcinoma risk. One example of a quality control is an assessment of the quantity or the quality of RNA used for input. A determination of RNA level of a control biomarker can beused to determine whether the input RNA had the requisite amount of input material to ascertain a risk. Ensuring the requisite amount of input material increases the confidence that a negative result is not a false-negative result. It has been found that an assessment of RNA transcript quantity of the biomarker CDC42 binding protein kinase beta (CDC42BPB) can be utilized to determine whether enough RNA from urothelial cells was present within the input to yield a quality assessment of the risk of urothelial carcinoma.

[0052] Biomarker transcripts can be sourced from biological any sample comprising bladder, ureter, or kidney cell RNA, especially urothelial cell RNA. Sources include (but are not limited to) a urine sample or a biopsy of the bladder, urethra, ureter, or kidney. In some embodiments that utilize a urine sample, at least a certain volume of urine is utilized to collect RNA for assessment. In various embodiments that utilize a urine sample, at least 1 mL, at least 2 mL, at least 5 mL, at least 10 mL, at least 15 mL, or at least 20 mL of urine is utilized to collect RNA for assessment.

[0053] In some implementations, prior to isolation of RNA, cells of a urine sample are concentrated (or otherwise enriched), which can be done by (for example) sedimentation or centrifugation. Cells can be concentrated by a number of methodologies. Examples of methodologies to concentrate cells of urine samples include (but are not limited to) centrifugation, filtration, dialysis, or to passively allow cells to sediment via gravity. In some implementations, cell-free RNA molecules derived from urine is used as input to determine RNA transcript levels. Cell-free RNA molecules derived from urine can comprise high concentrations of RNA derived from urothelial cells, especially within urine samples collected from individuals having urothelial carcinoma.

[0054] In some embodiments, a preservative is utilized to prevent RNA transcript degradation in a sample after collection. For example, EDTA can be utilized to preserve a patient sample (e.g., a urine sample) to prevent RNA degradation. In some embodiments, the preservative allows for stable storage of urine for at least twenty-four hours at room temperature (e.g., 25°C) and at least 96 hours refrigerated (e.g., 4 °C). In various embodiments, a urine sample is collected at a medical facility, a diagnostic center, a phlebotomy center, or at a remote location that is not a medical facility. A patient within a remote location (e.g., within a patient’s home) can send the patient sample to a site toperform processing and / or assessment. In some embodiments, a kit is utilized is utilized to collect a patient sample. For example, a urine collection kit can comprise a collection container and one or more preservatives.

[0055] RNA transcripts can be extracted from cells or a urine sample prior to quantification. RNA transcripts (or cell-free RNA molecules) can also be concentrated or otherwise enriched prior to quantification. Common techniques to concentrate or enrich RNA include chemical reagent extraction (e.g., acid-guanidinium-phenol based reagent) and column purification (e.g., silica membrane columns).

[0056] RNA transcripts can be measured utilizing any technique that provides relative RNA quantification. One common technique is quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). Dye intercalation, dye probe release, or other RT- qPCR techniques can be utilized. PCR primers and probes can be designed to cross an exon-exon boundary to help reduce false-positive signal derived from contaminant DNA remaining within the input.

[0057] In one particular example, RNA transcripts are measured as follows. Urine of a patient is collected and preserved using EDTA and other preservatives (e.g., Norgen urine preservative of Norgen Biotek Corp., Ontario, Canada). At least 10 mL of the urine sample is centrifuged to sediment cells. RNA is extracted and collected from the sediment by lysing the cells and using column purification. The extracted RNA is used as input in an RT-qPCR reaction to quantify transcript level of one or more biomarkers.

[0058] In one example, the biomarkers assessed are CDC42BPB, ROBO1 , CRH, and IGF2. If the cycle threshold (Ct) for CDC42BPB is greater than a threshold, then the amount of assayable RNA transcripts derived from urothelial cells was insufficient. Any appropriate threshold can be utilized as appropriate to the reaction conditions and starting material. In various embodiments, the Ct threshold for determining an insufficient amount of assayable RNA transcripts is greater than 34, the Ct threshold for determining an insufficient amount of assayable RNA transcripts is greater than 35, the Ct threshold for determining an insufficient amount of assayable RNA transcripts is greater than 36, the Ct threshold for determining an insufficient amount of assayable RNA transcripts is greater than 37, the Ct threshold for determining an insufficient amount of assayable RNAtranscripts is greater than 38, the Ct threshold for determining an insufficient amount of assayable RNA transcripts is greater than 39, or the Ct threshold for determining an insufficient amount of assayable RNA transcripts is greater than 40. In practice and utilizing 10 mL of patient urine, a Ct threshold of 36 for CDC42BPB was found to be a useful threshold to determine whether the amount of assayable RNA transcripts derived from urothelial cells was insufficient. In experimentation assessments performed, it was found that the use of a Ct threshold of 36 resulted in about 4% of samples were insufficient for risk analysis. If the amount of assayable RNA transcripts derived from bladder cells was sufficient (e.g., CDC42BPB less than or equal to 36), then ROBO1 , CRH, and IGF2 Ct values were utilized for further assessment.

[0059] Method 100 can further determine (103) urothelial carcinoma risk based on RNA transcript levels of one or more biomarkers. In several embodiments, a risk score is computed using an equation that combines the determined quantity of the one or more biomarker RNA transcripts. In some embodiments, each biomarker is weighted to compute a risk score. In some embodiments, one or more thresholds are utilized to classify the individual’s risk for urothelial carcinoma. The computation of risk for BCa and UTUC may be the same or different. It has been found that the sensitivity to detect BCa and UTUC are both good, but are not the same. Prior to experimentation, it was unexpected that the assay would be sensitive enough to detect UTUC due to the location of the carcinoma origin.

[0060] In one example, Ct values of ROBO1 , CRH, and IGF2 are utilized within an equation to determine whether an individual is at risk. Any equation to assess the values of ROBO1 , CRH, and IGF2 as related to urothelial carcinoma risk assessment can be utilized. One method of generating an equation is determine a concordance (e.g., the best concordance) between the Ct values of the biomarkers and the risk of urothelial carcinoma. Several computational models exist that can be used to learn concordance correlation coefficients between two sets of variables, which can be utilized to derive a urothelial carcinoma risk equation. Accordingly, in some implementations, a urothelial carcinoma risk equation comprises concordance correlation coefficients between Ctvalues one or more of ROBO1 , CRH, and IGF2 and the risk of having urothelial carcinoma.

[0061] In some embodiments, the expression levels of the one or more biomarkers (e.g., ROBO1 , CRH, IGF2, or a combination thereof) is used to compute a risk score. The computed risk score is an indication that urothelial carcinoma is present within the patient sample. In some embodiments, the likelihood that urothelial carcinoma is present correlates with the computed risk score. A risk score can be positively or inversely correlated with the likelihood that urothelial carcinoma is present. In some embodiments, the risk score is utilized to indicate a clinical action is to be performed. In some embodiments, when the risk score is greater (or less) than a threshold or within a certain range, a clinical action is performed. Examples of clinical actions that can be performed include (but are not limited to) repeat the risk assessment, administer a confirmatory diagnostic, initiate administration of a treatment regimen, alter a treatment regimen, initiate patient monitoring, and alter patient monitoring.

[0062] The computed risk score is alternatively or additionally an indication of the degree of severity of urothelial carcinoma that is present within the patient sample. The degree of severity can be a correlative of an amount of urothelial carcinoma present within the patient. In some embodiments, the degree of severity of urothelial carcinoma correlates with the computed risk score. A risk score can be positively or inversely correlated with the degree of severity of urothelial carcinoma. In some embodiments, the risk score is utilized to indicate a clinical action is to be performed. In some embodiments, when the risk score is greater (or less) than a threshold or within a certain range, a clinical action is performed. Examples of clinical actions that can be performed include (but are not limited to) repeat the risk assessment, administer a confirmatory diagnostic, initiate administration of a treatment regimen, alter a treatment regimen, initiate patient monitoring, and alter patient monitoring. In various embodiments, the value of a risk score can stratify patients, which can be used to indicate how a patient is treated. For example, a higher risk score (or lower risk score if inversely correlated) can indicate that a more aggressive treatment regimen is to be administered. A risk score can further indicatetypes of treatment to be included within a treatment regimen. In various embodiments, one or more thresholds and / or one or more ranges are utilized to stratify patients

[0063] Method 100 also optionally repeats (105) risk assessment. An assessment can be repeated for various reasons, such as (for example) failure to meet sample sufficiency, failure to meet a requisite quality, or as part of repeated monitoring or surveillance. Risk monitoring or risk surveillance is to be understood as intentional repeated risk assessments over a period of time, which may be performed for a variety of reasons. For example, urothelial carcinoma risk assessment can be repeated periodically for individuals over a certain age, for an individual with risk factors (e.g., family history of urothelial carcinoma), or for individuals having genetic indication for risk of urothelial carcinoma.

[0064] In some instances, when an individual is assessed for urothelial carcinoma and the individual’s sample yields a risk score that indicates a certain level of risk but not treatment, the individual is further assessed at later timepoint (e.g., resulting in monitoring of the patient). In some instances when a risk score that indicates a certain level of risk but not treatment, risk assessment is repeated until the risk score until the risk score indicates that treatment is to be performed.

[0065] In some instances, urothelial carcinoma risk assessment can be repeated periodically as individual receives treatment to monitor the treatment. In some instances, treatment monitoring comprises one or more assessments before treatment is initiated (e.g., to determine a baseline risk score). In some instances, treatment monitoring comprises one or more assessments during treatment (e.g., to monitor progress via the risk score). In some instances, treatment monitoring comprises one or more assessments after the completion of treatment (e.g., to confirm carcinoma is not present). In some implementations, one or more risk assessments are performed to assess disease burden after surgical resection or fulguration.

[0066] In some instances, urothelial carcinoma risk assessment can be repeated periodically after an individual completes a treatment regimen to detect minimal residual disease and / or recurrence. Monitoring for detection of minimal residual disease and / or recurrence can be performed over a period of time. Various timelines that may be usefulfor monitoring can be (for example) for 6 months, for 12 months, for 18 months, for 24 months, for 36 months, for 48 months, for 60 months, for 120 months, for 180 months, for 240 months, etc. Urothelial carcinoma risk assessments can be repeated with any periodicity during monitoring (e.g., every 3 months), which can be altered based on risk score results. For example, when several risk scores repeatedly and consistently indicate no risk of carcinoma, the period between risk assessments can be increased, or the monitoring can be completed. In another example, when a risk score indicates minimal residual disease may be present, the period between risk assessments can be decreased, or the risk assessment can be immediately repeated.

[0067] Method 100 also optionally performs (107) confirmatory diagnostics. One advantage of the biomarker assay described herein is that it is noninvasive and easily repeatable, providing a facile screening diagnostic for urothelial carcinoma. In some implementations, an individual’s risk for urothelial carcinoma indicates a confirmatory diagnostic is to be performed. In some implementations, when an individual’s risk score is greater (or less) than a threshold or within a range, a confirmatory diagnostic is performed. In many instances, a confirmatory diagnostic is a more invasive diagnostic than the biomarker assay. More invasive diagnostics include cystoscopy, ureteroscopy, and biopsy, in some implementations, an individual’s risk score indicates cystoscopy is to be performed within the urethra and / or bladder. In some implementations, an individual’s risk score indicates ureteroscopy is to be performed within the ureter and / or kidney. In some implementations, an individual’s risk score indicates a biopsy is to be performed in the urethra, bladder, ureter, and / or kidney. Cystoscopy, ureteroscopy, and biopsy can be performed concurrently in a single procedure, individually within independent procedures, or a combination thereof. Further, BCa and UTUC can have different thresholds and / or ranges. In some implementations, a set of thresholds or ranges are utilized to indicate which confirmatory diagnostic(s) is / are to be performed.

[0068] Method 100 also optionally performs (109) a treatment on the individual for urothelial carcinoma. Treatments appropriate for urothelial carcinoma include (but are not limited to) surgical resection, radiation therapy, chemotherapy, immunotherapy, and targeted therapy, which can be combined. In some implementations, an individual’s riskfor urothelial carcinoma indicates that administration of a treatment regimen is to be initiated. In some implementations, when an individual’s risk score is greater (or less) than a threshold or within a range, administration of a treatment regimen is to be initiated. In some implementations, an individual’s risk for urothelial carcinoma indicates that a treatment regimen is to be altered, such as when an updated risk is computed. In some implementations, when an individual’s risk score is greater (or less) than a threshold or within a range, a treatment regimen is to be altered. In some implementations, an individual’s risk for urothelial carcinoma indicates which treatments are to be included within the patient’s treatment regimen. In some implementations, when an individual’s risk score is greater (or less) than a threshold or within a range, certain treatments are to be included within the patient’s treatment regimen. Each treatment option can have different thresholds and / or ranges. In some implementations, a set of thresholds or ranges are utilized to indicate which treatment(s) is / are to be included within the patient’s treatment regimen.

[0069] Common surgical techniques for BCa include (but are not limited to) transurethral resection (TUR), TUR with fulguration, partial cystectomy, and radical cystectomy with urinary diversion. Surgery can be combined with neoadjuvant and / or adjuvant chemotherapy. Chemotherapy can be administered systemically and / or or via intravesical administration. Common chemotherapeutics that can be utilized for BCa include (but are not limited to) carboplatin, cisplatin, doxorubicin, fluorouracil, gemcitabine, methotrexate, mitomycin, paclitaxel, and vinblastine. Common immunotherapeutics that can be utilized for BCa include (but are not limited to) immune checkpoint inhibitors (e.g., avelumab, nivolumab, atezolizumab, and pembrolizumab). Common targeted therapeutics that can be utilized for BCa include (but are not limited to) FGFR inhibitors (e.g., erdafitinib and rogaratinib) enfortumab vedotin, VEGF inhibitors (e.g., ramucirumab), and sacituzumab govitecan-hziy.

[0070] Common surgical techniques for UTUC include (but are not limited to) nephroureterectomy, segmental resection of ureter, endoscopic tumor ablation. Surgery can be combined with neoadjuvant and / or adjuvant chemotherapy. Chemotherapy can be administered systemically and / or via intracavitary administration. Commonchemotherapeutics that can be utilized for IITUC include (but are not limited to) mitomycin. Common immunotherapeutics that can be utilized for UTUC include (but are not limited to) immune checkpoint inhibitors (e.g., avelumab, nivolumab, atezolizumab, and pembrolizumab). Common targeted therapeutics that can be utilized for UTUC include (but are not limited to) FGFR3 inhibitors (e.g., erdafitinib, infigratinib, rogaratinib, AZD4547).

[0071] While specific examples of assessing an individual’s risk for urothelial carcinoma are described above, one of ordinary skill in the art can appreciate that various steps of the process can be performed in different orders and that certain steps may be optional according to some embodiments of the disclosure. As such, it should be clear that the various steps of the method could be used as appropriate to the requirements of specific applications. Furthermore, any of a variety of processes for determining an individual’s risk for urothelial carcinoma appropriate to the requirements of a given application can be utilized in accordance with various embodiments of the invention.EXAMPLES AND EXPERIMENTAL DATA

[0072] Biological data support the methods and systems of assessing urothelial carcinoma risk and applications thereof. In the following, examples and experimental data results of urothelial carcinoma risk assessment utilizing transcript levels of biomarkers and data thereof are provided.Prospective Multicenter Validation of Urine RNA Profile (uRNAp) for Urothelial Carcinoma Detection and Surveillance in over 1700 Cases

[0073] Urothelial carcinoma (UC) is the most common malignancy of the urinary tract accounting for over 90% of the cancers of the bladder and upper urinary tract. Bladder cancer is the 7thmost common cancer in the United States with more than 80,000 new diagnoses and 16,000 deaths in 2024. The incidence of bladder cancer is about 4 times higher in men compared with women. Most bladder cancer patients initially present withhematuria or lower urinary tract symptoms that indicate the need for radiographic imaging and direct visualization of the bladder and urethra with cystoscopy.

[0074] Non-muscle invasive bladder cancer accounts for nearly 75% of cases at presentation and is primarily managed with transurethral resection of bladder tumor (TURBT) followed by intravesical therapies based on risk stratification. For muscle- invasive bladder cancer, radical cystectomy with or without neoadjuvant chemotherapy is the first line treatment. The risk of recurrent non-muscle invasive bladder cancer varies greatly and can be stratified based on grade, stage, tumor size, multifocality , presence of carcinoma in situ (CIS), and the interval since the last recurrence. With recurrence rates for bladder cancer of 15-61% at 1 year and 31-78% by 5 years, frequent surveillance after initial treatment is mandatory.

[0075] Initial detection and long-term surveillance of bladder cancer rely on cystoscopy in the clinic setting. If bladder tumors are found, TURBT is performed in the operating room for pathological diagnosis and local staging. Cystoscopy has a diagnostic accuracy of 70-80% and may miss small papillary tumors and flat-appearing CIS. The need for frequent cystoscopic surveillance negatively impact patient’s quality of life and is burdensome in low-resource clinical settings. Cystoscopies require in-person clinic visits as often as every 3 months contributing to the high healthcare cost associated with bladder cancer. Additionally, most patients experience some level of anxiety and discomfort / pain associated with the procedure and the majority of patients report at least one adverse event associated with cystoscopy including hematuria, dysuria, and urinary tract infection. Surveys of patients undergoing cystoscopy indicate they would be receptive to urine biomarker assays with about 75% of surveillance patients indicating that they would be willing to accept a urine test with least 90% sensitivity and 60% of detection patients indicating a preference for a urine test instead of cystoscopy.

[0076] Biomarkers to identify patients at high risk of bladder cancer would be beneficial for patients. While hematuria and lower urinary tract symptoms are common signs of bladder cancer, majority of patients with these symptoms do not have cancer. Current guidelines acknowledge that urine biomarker assays with high negative predictive values have the potential to further stratify risk of bladder cancer and direct prioritization ofbladder cancer work up in patients most likely to have disease while reducing invasive procedures in those unlikely to have bladder cancer.

[0077] There is also great interest in urine-based assays to direct cystoscopy scheduling for surveillance of bladder cancer. While guidelines acknowledge the high sensitivity and negative predictive values of newer commercially available urine-based assays, cytology is currently the only urine-based assay recommended by guidelines for regular use for bladder cancer surveillance. With high specificity (>90%) but low sensitivity (16-84%), a negative cytology does not negate the need for cystoscopy, limiting its utility in reducing the frequency of invasive surveillance procedures. As such, improved assays and additional validation are needed before clinical recommendation of the use of biomarkers to replace or delay detection or surveillance cystoscopy.

[0078] To address the need for a noninvasive test with the potential to assess the need for cystoscopy, our group developed uRNAp, a noninvasive urine gene expression test based on RT-qPCR of ROBO1 , CRH, and IGF2 in urine sediments, for detection and surveillance of non-muscle invasive bladder cancer. Here, we present the results of a prospective, multi-center validation study of uRNAp for initial detection of bladder cancer and for surveillance in patients undergoing cystoscopy for a history of bladder cancer.Materials and MethodsPatient Population

[0079] The study protocol was approved by Stanford University Institutional Review Board and Veterans Affairs Palo Alto Health Care System (VAPAHCS) Research and Development Committee. Informed consent was obtained from participants. Adult patients undergoing diagnostic cystoscopy for signs and / or symptoms of bladder cancer or surveillance of bladder cancer at VAPAHCS and Stanford Health Care (SHC) between May 2019 to March 2025 were study eligible. Exclusion criteria included a history of muscle-invasive bladder cancer or muscle invasive upper-tract urothelial carcinoma. Provision of follow-up samples was not required for study participation, therefore there was no attrition. Risk stratification factors including age, sex, smoking history, and the degree of hematuria were collected for all participants. Given that bladder cancer affectingmales 4:1 compared to females and the recruitment took place at a VA medical center, the majority of the patients were male (Fig. 2). The detection cohort included patients without a prior diagnosis of bladder cancer undergoing cystoscopy for gross hematuria, microhematuria, lower urinary tract symptoms, or abnormal findings on radiologic imaging. The surveillance cohort included patients with a history of non-muscle invasive bladder cancer undergoing surveillance cystoscopy. Tumor size was defined based on a combination of radiographic imaging studies (if available) and as estimated by the experienced surgeon and reflected in operative reports. Small tumors are defined as <1 cm, medium 1 -3 cm, and large > 3cm. Images of tumors were taken at the discretion of the surgeon and obtained from patient medical records. Risk categorization was based on AUA guidelines.

[0080] A minimum sample of 10 mL urine was collected from participants at scheduled clinic appointments throughout patients’ standard clinical care. For patients undergoing cystoscopy, voided urine was collected prior to the procedure. If patients were unable to void, urine collection was performed via the cystoscope upon insertion. For patients providing multiple samples included in analysis, samples were collected at unique appointments at least 3 months apart. Visual assessment by cystoscopy was required for inclusion in statistical analysis. Patients with visualized lesions suspicious for malignancy or indeterminant regions of interest on cystoscopy underwent TURBT and patients with concern for possible upper urinary tract tumor underwent ureteroscopy. The presence of cancer was based on histopathology of biopsy samples. For patients who provided samples prior to both clinic cystoscopy and subsequent biopsy, the urine sample from the cystoscopy was used in the analysis, as it more closely mirrors the potential clinical application for uRNAp. Treating clinicians were blinded to uRNAp assay data prior to procedures.Sample Preparation and Analysis

[0081] Ten milliliters of urine was separated by centrifugation to collect the sediment for RNA isolation and uRNAp assay. RNA was isolated from the sediment using an RNA isolation kit via chloroform extraction or column purification. No significant difference wasfound with different methods of RNA preparation. RT-qPCR was performed to evaluate expression of R0B01 , CRH, IGF2, and CDC42BPB, a housekeeping gene to ensure adequate sample cellularity. Cycle threshold (Ct) values for gene expression were determined, with a Ct of 41 assigned for undetermined Ct values. R0B01 , CRH and IGF2 were used to calculate the uRNAp score. CDC42BPB served as a sample adequacy control to ensure the sample input contained enough cells for reliable assay performance. Samples with CDC42BPB Ct values >36 were excluded due to low cellularity.Statistical Analysis

[0082] Statistical analyses were performed in accordance with established guidelines.26Of the samples collected, 458 samples in the detection cohort and 1253 in the surveillance cohort were suitable for analysis (Figs. 3A and 3B). With 104 incidences of bladder cancer in the detection cohort and 245 incidences in the surveillance cohort, the study exceeded the criterion of 30 events per variable for prediction model development using 3 genomic predicitors. All samples included in the study are described at both patient and sample level (Figs. 2 and 4) using mean with interquartile range (IQR) for continuous characters, and percentage for categorical characters. A uRNAp score of each sample was calculated. Based on RT-qPCR methodology, an adjustment was made using retrospective data to the coefficients and intercept in the regression model. From the modified model, a cutoff of 0.23 was established as the threshold for test positivity as described previously. The primary outcome was the presence of pathologically confirmed urothelial carcinoma of the bladder. Patients with adenocarcinoma or squamous cell carcinoma of the bladder were excluded.

[0083] The performance of uRNAp to detect the presence of pathologically confirmed bladder cancer on cystoscopy was assessed using receiver operating characteristic (ROC) curves using the area under the curve (AUC), sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). Potential clinical benefit was assessed by decision curve analysis (DCA). All analyses were performed on detection and surveillance cohorts separately.

[0084] R studio (version 2024.04.2, https: / / www.r-project.org) was used to perform all the statistical analyses including key packages pROC, dcurves and ggplot2. Analysis of Variance (ANOVA) test were used for the comparisons on uRNAp scores across tumor sizes with significance level of p-value < 0.05 for a two-sided test. Study data and presentation was assessed for rigor using STROBE criteria.Results

[0085] A total of 1736 urine samples were prospectively collected from 856 patients across a tertiary academic medical center and a VA medical center. Seventy-four samples were excluded from analysis: 71 samples had insufficient cellularity (4%, CDC42BPB Ct > 36) and 3 samples were from patients with non-urothelial carcinoma of the bladder (Figs. 3A and 3B). There were 458 samples from 448 patients included in the analysis for the detection cohort and 1253 samples from 392 patients included in the analysis for the surveillance cohort. Patients in the detection cohort gave a median of one sample (range 1-2), while those in the surveillance cohort gave a median of 3 samples (range 1 -15) at unique time points.

[0086] In the detection cohort cystoscopy was performed due to gross hematuria (n=260), microhematuria (n=103), lower urinary tract symptoms (n=63), or incidental imaging findings (n=32). In this cohort, there were 104 histologically confirmed cases of bladder cancer (25 low-grade, 79 high-grade). Of these 104 cases of bladder cancer using a uRNAp score cutoff of 0.23, 101 had true positive (TP) uRNAp tests. Three- hundred fifty-four samples correlated with negative cystoscopy or benign biopsy, of which 180 had true negative (TN) uRNAp tests. The distribution of uRNAp scores for patients in the detection cohort is shown in Figure 5A. For these patients without a prior history of bladder cancer, uRNAp demonstrated 97% sensitivity, 51 % specificity, 98% negative predictive value (NPV), and 37% positive predictive value (PPV). Clinical cytology was available for 66% (n=305) of the patients in the detection cohort. As expected, cytology had high specificity (99%) with lower sensitivity of 32% overall and 48% for detection of high-grade bladder cancer (Fig. 6). ROC analysis of uRNAp in the detection cohort demonstrated an AUC of 0.91 (Fig. 7A). While the detection cohort included all patientsundergoing cystoscopy for signs and symptoms of bladder cancer, the majority of patients were being evaluated due to hematuria (n=369). uRNAp assay performance in the subset of patients being evaluated for hematuria was similar to the full cohort, with 98% sensitivity and 52% specificity.

[0087] In patients with a prior history of bladder cancer (surveillance cohort) there were 245 histologically confirmed recurrences (84 low-grade, 161 high-grade). This cohort included 1 ,008 samples from negative cystoscopy or benign biopsy with 414 true negative uRNAp tests and 245 incidences of bladder cancer with 232 true positive uRNAp results using a uRNAp cutoff of 0.23. Overall, uRNAp demonstrated 95% sensitivity, 41 % specificity, 97% NPV and 28% PPV (Fig. 6) in bladder cancer surveillance patients. For high-grade recurrence uRNAp sensitivity was 99% and for low-grade recurrences the sensitivity was 86%. Distribution of uRNAp scores for the surveillance cohort is shown in Figure 5C. For the surveillance population 82% of the patients had corresponding clinical cytology that had an overall sensitivity of 33% and specificity of 99% for identification of recurrent disease and 49% sensitivity for high-grade recurrence. ROC analysis of uRNAp in the surveillance population demonstrated an AUC of 0.82 (Fig. 7B). There was no difference in model performance between medical centers for either the detection or surveillance populations.

[0088] Thirteen patients undergoing surveillance for a history of bladder cancer were found to have pathologically confirmed upper tract urothelial carcinoma (UTUC) with no evidence of disease in the bladder. Investigation of the upper tract was initiated for 6 of these patients due to positive cytology with negative cystoscopy. Three patients had new onset of gross hematuria with negative cystoscopy, and subsequent imaging indicated upper tract disease. Two patients underwent imaging for other symptoms that revealed potential upper tract tumors. In one patient, a tumor emanating from the ureter was seen on cystoscopy. Four of the UTUC were low-grade and nine were high-grade. In all cases the uRNAp score predicted the presence of UTUC.

[0089] In both the detection and surveillance cohorts we found that high-grade tumors had significantly higher uRNAp scores for comparably sized low-grade tumors. Tumor size was reported in the clinical records for 98% of the patients with bladder cancer in thedetection cohort (n=102) and 89% of the patients in the surveillance cohort (n=218). The results within Figures 5B and 5D show that uRNAp score significantly increases with both tumor size and grade (p<0.02). Large (>3cm), high-grade (HG) tumors had the highest median uRNAp score of 0.8364 in the detection cohort and 0.8175 in the surveillance cohort, while small (<1 cm) low-grade (LG) tumors had the lowest scores (median 0.2745 detection and 0.3087 surveillance). Consistent with this finding, the majority of false negative uRNAp scores observed were from patients with small low-grade tumors (10 of 16).

[0090] Variant histology was reported in the pathology report for 12 patients in the detection cohort and 11 patients in the surveillance cohort. Variants included squamous differentiation, micropapillary, plasmacytoid, neuroendocrine, small cell carcinoma, and poorly differentiated subtypes. Consistent with the performance for high-grade disease, uRNAp predicted bladder cancer in all cases with variant histology.

[0091] Potentially confounding factors such as a history of prostate cancer had no discernable effect on uRNAp assay performance. One-hundred six men with a history of prostate cancer provided 218 urine samples (detection n=72, surveillance n=146). All new incidences of bladder cancer (n=15, detection cohort) in men with a history of prostate cancer were positively detected by uRNAp with specificity of 52%. In the surveillance cohort 27 of the 28 recurrences in men with a history of prostate cancer were detected, yielding a sensitivity of 96% and specificity of 38%.

[0092] To better understand the assay performance, we further examined the false negative uRNAp assays (Fig. 8). Based on clinical descriptions, available intraoperative photographs, and pathology, we found that 10 of the 16 uRNAp false negative tumors were small (<1 cm) low-grade Ta (8 surveillance, 2 detection). Four of the uRNAp false negatives were low-grade Ta recurrences that fell in the medium size category (1 -3 cm) and two high-grade tumors were missed. The one high-grade false negative from the surveillance cohort was a small (~1 cm) patch of CIS. Notably, the high-grade false negative in the detection cohort was a 2 cm T1 tumor in a bladder diverticulum with a narrow opening. Twelve of the false negative uRNAp cases had corresponding cytology, however cytology was also negative in all cases.

[0093] The potential clinical benefit of uRNAp as assessed by decision curve analysis (DCA) demonstrated standardized net benefit at threshold probabilities greater than 3% in the detection population, and 3.5% in the surveillance population compared to standard of care. For detection patients, 51 % of negative cystoscopies could be deferred, missing diagnoses of two small LG Ta tumors and one HG tumor. For surveillance patients, 41 % of cystoscopies could have been deferred with uRNA, missing 12 LG and one HG recurrences.

[0094] Assessments were also performed on patients with UTUC. Cohort demographics are provided within Fig. 9. Sample characteristics are described within Fig. 10. The uRNAp assay was performed on patient samples (Fig. 11 ). Accordingly, samples were assessed by RT-qPCR to evaluate expression of ROBO1 , CRH, IGF2, and CDC42BPB, a housekeeping gene to ensure adequate sample cel lularity . The sensitivity of the uRNAp assay was very good on UTUC samples (Fig. 12). This data provides the rationale of using the uRNAp assay for detection or surveillance.

Claims

WHAT IS CLAIMED IS:1 . A method to determine an individual’s risk of urothelial carcinoma, comprising:(a) obtaining a sample comprising RNA, wherein the sample is derived from a urine sample of the individual;(b) quantifying an amount of RNA transcripts for roundabout guidance receptor 1 (ROBO1 ), corticotropin releasing hormone (CRH), and insulin like growth factor 2 (IGF2) within the sample; and(c) computing a risk score using the amount of RNA transcripts for ROBO1 , CRH, and IGF2.

2. The method of claim 1 , further comprising: quantifying an amount of RNA transcripts for CDC42 binding protein kinase beta (CDC42BPB) within the sample; and determining the amount of RNA transcripts for CDC42BPB is greater than threshold.

3. The method of claim 1 further comprising: collecting the urine sample from the individual; sedimenting cells within the urine sample; and extracting RNA from the sedimented cells to yield the sample comprising RNA.

4. The method of claim 3, wherein collecting the urine sample is performed at a location that is not a medical facility.

5. The method of claim 4 further comprising: storing the urine sample for at least 24 hours prior to RNA extraction, wherein the urine sample is stored with a preservative.

6. The method of any one of claims 3-5, wherein the urine sample of the individual was collected prior to a diagnosis of the individual having urothelial carcinoma.

7. The method of claim 6, wherein the individual is experiencing a symptom of urothelial carcinoma.

8. The method of claim 7, wherein the symptom of urothelial carcinoma is hematuria or pain during urination.

9. The method of any one of claims 3-5, wherein the urine sample of the individual was collected during a treatment regimen for urothelial carcinoma.

10. The method of any one of claims 3-5, wherein the urine sample of the individual was collected after a treatment regimen for urothelial carcinoma.11 . The method of claim 10, wherein the treatment regimen includes surgical resection or figuration and the risk score is used to assess disease burden.

12. The method of any one of claims 1 -11 , wherein the urothelial carcinoma is bladder cancer.

13. The method of any one of claim 1 -11 , wherein the urothelial carcinoma is upper tract urothelial cancer.

14. The method of any one of claims 1-13, wherein the risk score indicates a risk of urothelial carcinoma greater than a threshold, the method further comprising: performing cystoscopy, ureteroscopy or biopsy within the urethra, bladder, ureter or kidney.

15. The method of any one of claims 1-13, wherein the risk score indicates a risk of urothelial carcinoma less than a threshold, the method further comprising: repeating steps (a), (b), and (c) at a later timepoint in lieu of performing cystoscopy, ureteroscopy or biopsy.

16. The method of any one of claims 1-13, wherein the risk score indicates a risk of urothelial carcinoma greater than a threshold, the method further comprising: administering a treatment, wherein the treatment comprises at least one of: surgical resection, radiation therapy, chemotherapy, immunotherapy, or targeted therapy.

17. The method of claim 16 further comprising: repeating steps (a), (b), and (c) after administration of the treatment to assess for minimal residual disease.

18. The method of claim 17, wherein repeating steps (a), (b), and (c) is performed at multiple timepoints over a period of time.

19. The method of any one of claims 1 -13, the method further comprising: administering a treatment regimen over a period of time, wherein the treatment regimen comprises at least one of: surgical resection, radiation therapy, chemotherapy, immunotherapy, or targeted therapy; and repeating steps (a), (b), and (c) at multiple timepoints during the period of time to assess response to treatment.

20. The method of claim 18 or 19, wherein when the risk score indicates an updated risk of urothelial carcinoma, the method further comprises: altering a treatment regimen in accordance with the updated risk.

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