Trop2 as a biomarker for detection of epithelial cancers and predicting and monitoring responses to therapies

Abstract

As disclosed herein, Trop2 is discovered to be a prognostic tissue biomarker for prostate and bladder cancers. We have discovered that elevated Trop2 expression in certain contexts is correlated with worse clinical features including Gleason score, age, and pre-operative PSA levels. More importantly, we demonstrate that elevated Trop2 expression at radical prostatectomy predicts worse overall survival in men undergoing radical prostatectomy. Additionally, we discovered that it is possible to detect shed Trop2 ECD in urine from men with clinically significant prostate cancer. Building upon these discoveries we have developed selected methods for detecting, for example, the presence Trop2 ECD levels in certain contexts in order to use it as a novel tissue prognostic biomarker and a non-invasive marker for cancers such as prostate cancer and bladder cancer.

Description

[0001]TROP2 AS A MINIMALLY INVASIVE AND NON-INVASIVE BIOMARKER FOR DETECTION OF EPITHELIAL CANCERS AND PREDICTING AND MONITORING RESPONSES TO THERAPIES CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. Section 119(e) of co- pending and commonly-assigned U.S. Provisional Patent Application No.63 / 613,576, filed December 21, 2023 entitled “TROP2 AS A MINIMALLY INVASIVE AND NON-INVASIVE BIOMARKER FOR DETECTION OF EPITHELIAL CANCERS AND PREDICTING AND MONITORING RESPONSES TO THERAPIES”,” the contents of which is incorporated by reference herein. TECHNICAL FIELD Embodiments of the disclosure concern at least the fields of molecular biology and medicine. BACKGROUND OF THE INVENTION Prostate cancer is the most common non-cutaneous cancer among men in the United States, accounting for about 29% of cancer diagnoses (1). It is projected that 34,700 men will die from prostate cancer in 2023, making it the second leading cause of cancer-related deaths among men in the United States (1). Current screening approaches for prostate cancer center on the measurement of serum prostate-specific antigen (PSA) in combination with adjuncts such as measurement of molecular forms of PSA, multiparametric MRI, and digital rectal examination (2-6). Clinically significant prostate cancer often fails local therapies, such as surgery or radiation therapy, and often leads to metastatic disease and mortality (7-9). Stratifying clinically significant prostate cancer from indolent prostate cancer using clinical features alone such as PSA level, Gleason Score, or T-stage is often challenging, particularly for intermediate-risk disease (3, 7, 9-11). The development of new molecular prognostic biomarkers can help with risk stratification and clinical decision-making by better identification of clinically significant prostate cancer (12, Trophoblastic cell surface antigen-2 (Trop2), an oncogenic transmembrane cell surface protein, is highly expressed in metastatic prostate cancer (14, 15). Trop2 defines a subpopulation of prostate basal cells that have self-renewal activity (16). Trop2 promotes tumorigenicity, metastasis, and neuroendocrine phenotype in prostate cancer (15-17). In our previous study, we demonstrated that high levels of tissue Trop2 predict a shorter time to recurrence in a prostate cancer cohort with 234 patients, providing evidence that Trop2 can serve as a prognostic tissue biomarker for early detection of clinically significant localized prostate cancer (15). Additionally, Trop2 is cleaved via a protease, disintegrin and metalloproteinase 17 (ADAM17), resulting in the release of the Trop2 extracellular domain into the extracellular environment (18), making it a potential liquid marker of prostate cancer. There is a need in the art for non-invasive methods of observing diagnostic and prognostic tissue biomarkers such as Trop2. SUMMARY OF THE INVENTION We have discovered that elevated levels of Trop2 can be used as a prognostic tissue biomarker and further demonstrated that clinically significant levels of Trop2 can be detected in the urine. Building upon these discoveries, we have developed methods for the detection of Trop2, for example in the prostate biopsy tissue or urine, alone or in combination with other markers, as a prognostic biomarker for clinically localized prostate cancer. As discussed below, we validated the prognostic power of Trop2 expression in prostate cancer tissue utilizing the Canary Prostate Cancer Tissue Microarray (CPCTA) which contains over 1100 patient samples, and assess urine shed Trop2 as a non-invasive biomarker for diagnosis and prediction of clinically significant prostate cancer. We demonstrate that elevated levels of Trop2 significantly correlate with shorter overall survival, higher Gleason Score, and higher pre-operative PSA levels. In addition, we discovered that clinically significant levels of shed Trop2 ECD can be detected in the urine from men with clinically significant prostate cancer. These findings allow Trop2 to be used as a novel prognostic tissue biomarker and diagnostic urine marker for prostate cancer, information that can be used to optimize treatment decision-making. As discussed below, the disclosure presented herein includes a number of embodiments of the invention including methods of detecting an epithelial cancer. Typically the methods comprise obtaining a sample of biological material from an individual (e.g., urine); and then characterizing the levels of shed extracellular domain (ECD) of tumor-associated calcium signal transducer 2 (Trop2) protein observed in the sample: and then assessing if levels of shed TROP2 ECD observed in the sample are characteristic of an epithelial cancer, for example a prostate cancer or a bladder cancer as shown in FIGS. 2 and 3. In some embodiments of the invention, urine is obtained from an individual considering a treatment for an epithelial cancer or undergoing a treatment for an epithelial cancer (e.g., a treatment comprising administering Sacituzumab Govitecan or TRODELVY to the individual). Illustrative embodiments of the invention include methods of detecting presence or levels of shed extracellular domain (ECD) of tumor-associated calcium signal transducer 2 (Trop2) protein in urine, the methods comprising obtaining a urine sample and performing and ELISA assay for proteins present in the urine, wherein at least one antibody used in the ELISA assay binds to the ECD of Trop2; and then correlating a signal obtained using the antibody in the ELISA assay with the presence or levels of Trop2 ECD in the urine sample. In certain embodiments, the urine is obtained from a person having or suspected of having an epithelial cancer (e.g., a prostate cancer or a bladder cancer). In some embodiments of these methods, the urine is obtained from a person undergoing a therapy for an epithelial cancer (e.g., a treatment comprising administering Sacituzumab Govitecan or TRODELVY to the individual). Embodiments of the invention also comprise ELISA assays that include a first antibody that binds extracellular domain (ECD) of tumor-associated calcium signal transducer 2 (Trop2) protein, wherein the first antibody is coupled to a solid matrix, as well as a second antibody that binds extracellular domain (ECD) of tumor-associated calcium signal transducer 2 (Trop2) protein, wherein the second antibody is coupled to a detectable marker. Certain embodiments of these ELISA assays comprise urine. In some embodiments, the urine is obtained from a person having or suspected of having an epithelial cancer, for example a prostate cancer or a bladder cancer. In certain embodiments, the urine is obtained from an individual considering a treatment for an epithelial cancer or undergoing a treatment for an epithelial cancer. Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Positive Trop2 expression is associated with worse overall survival time. A. Schematic representation of experimental design. The image was generated using BioRender (https: / / biorender.com). B. IHC staining of Trop2 in the Canary Prostate Cancer Tissue Microarray (CPCTA) cohort (n = 1153). Trop2 staining was subject to scoring from 0 to 3. 0 = no staining; 1 = not interpretable / core missing; 2 = faint staining; 3 = dense staining. The Canary TMAs included 4 cores / case (3 cancer cores and 1 benign core). Trop2 expression is summarized as (1) positive in cases with at least one core with a score of 3 and (2) low / negative in cases where all cores have scores of 2 or less. Cases with no cancer found in any cores are excluded from the analysis. Scale bar = 40 microns. C. Representative images of Trop2 staining in cancer cases from Canary TMAs. Scale bar = 100 microns (upper) and 40 microns (bottom). D. Kaplan-Meier survival curves of overall survival in Canary TMA cohort. Trop2 positive predicted worse overall survival of prostate cancer patients at radical prostatectomy. n = 1097. Patients without survival time were excluded from the analysis. p = 0.014, by log-rank test. Figure 2. Trop2 can be detected in urine from clinically significant prostate cancer patients. A. Schematic representation of experimental design (generated via BioRender (https: / / biorender.com). B. Trop2 levels were assessed by ELISA in urine from cancer-free patients (n=40) and prostate cancer patients (n=39). p = 0.0285, determined by Student-T test (Two-tailed). C. Trop2 levels in urine samples from patients with clinically significant prostate cancer were evaluated by ELISA (20ul / sample) or Western blot (30μl urine / sample). For Western Blot, 10 patients were run in the same gel with positive control, and two blots were developed at the same time with the same exposure time. D. The correlation of Trop2 levels from ELISA and WB analysis. P = 0.0065. E. Trop2 levels in patient-matched tissues were evaluated by IHC staining. Scale bar = 25 microns. Figure 3. Trop2 is a diagnostic and prognostic urine biomarker for epithelial cancers. A. Trop2 levels were assessed by ELISA in urine from cancer-free patients (n=85) and bladder cancer patients (n=77). p < 0.0001, determined by Student-T test (Two-tailed). B. shed Trop2 levels in urine samples from patients with / without progression p = 0.04, determined by Student-T test (Two-tailed). Higher shed Trop2 levels were associated with worse outcomes. C. shed Trop2 levels in urine samples from patients with different follow up status. p = 0.028. D. Kaplan-Meier survival curves of overall survival in bladder cancer patients with Trop2 high and Trop2 low levels. High urine Trop2 levels predicted worse overall survival of bladder cancer patients at time of surgery (n = 77). p = 0.0469, by log-rank test. DETAILED DESCRIPTION OF THE INVENTION In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized, and structural changes may be made without departing from the scope of the present invention. Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Early cancer detection of cancer in a minimally invasive way is still limited. Additionally, artisans are lacking cost-effective minimally invasive and non-invasive approaches to predicting and monitoring responses to cancer therapies. Early cancer detection of cancer in a minimally invasive way is still limited. Additionally, we are lacking cost-effective minimally invasive and non-invasive approaches to predicting and monitoring responses to cancer therapies. Trop2 is a transmembrane glycoprotein and is overexpressed in various epithelial cancers, including prostate, breast, ovarian, and others. We have recently demonstrated that high levels of Trop2 are predictive of localized prostate cancer recurrence post radical prostatectomy and are significantly elevated in late-stage metastatic prostate cancer and breast cancer. Additionally, we demonstrated a detailed mechanism of Trop2 action. In the prostate, Trop2 is activated through regulated intramembrane proteolysis (RIP) resulting in cleavage of the receptor at two distinct sites. These cleavages result in shedding of the TECD and translocation of the intracellular domain to the nucleus. Recently, the FDA approved the first Trop2 targeted therapy IMMU-132 (Sacituzumab Govitecan; TRODELVY) for metastatic triple-negative breast cancer (TNBC) patients who failed two prior therapies and urothelial cancer. Currently, TRODELVY is being evaluated in phase II trials in patients with metastatic castration-resistant PC (CRPC) (NCT03725761). However, currently there are no rapid, minimally invasive or noninvasive tools to predict and monitor responses to TRODELVY and other treatments in prostate cancer as well as other epithelial cancers. We recently demonstrated that TECD is shed from prostate cells by western blot. We also recently developed a plate ELISA by using two different anti-TECD capture and detection antibodies. Our results further demonstrate that shed TECD can be detected in prostate patient blood by immunoprecipitation followed by western blot and in urine from patients with high-risk prostate cancer by ELISA. These results show higher levels of blood TECD in patients with CRPC and recurrent prostate cancer when compared to cancer free patients and patients with non-recurrent prostate cancer. Additionally, our results conclusively demonstrate our ELISA represents a specific, sensitive, and inexpensive method to detect shed TECD levels in urine (p=0.0004). Our results prove that shed TECD can be detected in PC patient blood and urine. These important findings demonstrate that shed TECD may represent new minimally invasive and non-invasive biomarker for early detection of prostate and other epithelial cancers and prediction and monitoring of treatment responses to TRODELVY and other therapies. As Trop2 protein is overexpressed in nearly all epithelial cancers, the proposed test can be used for: 1) cancer screening; 2) detection of all epithelial cancers which account for 80 to 90 percent of all cancer cases; 3) predicting and monitoring responses to already FDA approved Trop2 targeting therapies, TRODELVY; and 4) monitoring responses to all therapies used for epithelial cancers. Aspects of the invention are described in Liu et al., “High expression of Trop2 is associated with aggressive localized prostate cancer and is a candidate urinary biomarker” Scientific Reports volume 14, Article number: 486 (2024), (hereinafter “Liu et al.”), the contents of which are incorporated by reference. The invention disclosed herein has a number of embodiments. As discussed below, the disclosure presented herein teaches methods of detecting an epithelial cancer, these methods comprising: obtaining a sample of a biological material from an individual, such as blood or urine; and then characterizing the levels of shed extracellular domain (ECD) of tumor-associated calcium signal transducer 2 (Trop2) protein observed in the sample: and then assessing if levels of shed TROP2 ECD observed in the sample are characteristic of an epithelial cancer, for example a prostate cancer or a bladder cancer as shown in FIGS.2 and 3. In typical embodiment of the invention, levels of shed TROP2 ECD are observed in an ELISA assay. In certain embodiments, levels of shed TROP2 ECD are observed in urine of the individual. In some embodiments of the invention, urine is obtained from an individual considering a treatment for an epithelial cancer or undergoing a treatment for an epithelial cancer (e.g., a treatment comprising administering Sacituzumab Govitecan or TRODELVY to the individual). Illustrative embodiments of the invention include methods of detecting presence or levels of shed extracellular domain (ECD) of tumor-associated calcium signal transducer 2 (Trop2) protein in urine, the methods comprising obtaining a urine sample and performing and ELISA assay for proteins present in the urine, wherein at least one antibody used in the ELISA assay binds to the ECD of Trop2; and then correlating a signal obtained using the antibody in the ELISA assay with the presence or levels of Trop2 ECD in the urine sample. In certain embodiments, the urine is obtained from a person having or suspected of having an epithelial cancer (e.g., a prostate cancer or a bladder cancer). In some embodiments of these methods, the urine is obtained from a person undergoing a therapy for an epithelial cancer (e.g., a treatment comprising administering Sacituzumab Govitecan or TRODELVY to the individual). Embodiments of the invention also comprise ELISA assays that include a first antibody that binds extracellular domain (ECD) of tumor-associated calcium signal transducer 2 (Trop2) protein, wherein the first antibody is coupled to a solid matrix, as well as a second antibody that binds extracellular domain (ECD) of tumor-associated calcium signal transducer 2 (Trop2) protein, wherein the second antibody is coupled to a detectable marker. Certain embodiments of these ELISA assays comprise urine. In some embodiments, the urine is obtained from a person having or suspected of having an epithelial cancer. In certain embodiments, the urine is obtained from an individual considering a treatment for an epithelial cancer or undergoing a treatment for an epithelial cancer. Further aspects of the methods of the invention are discussed below. In our studies, we validated the prognostic power of Trop2 expression in prostate cancer tissue utilizing the Canary Prostate Cancer Tissue Microarray (CPCTA) which contains over 1100 patient samples, and assess urine shed Trop2 as a potential non-invasive biomarker for diagnosis and prediction of clinically significant prostate cancer. We demonstrate that elevated levels of Trop2 significantly correlate with shorter overall survival, higher Gleason Score, and higher pre-operative PSA levels. In addition, shed Trop2 can be detected in the urine from men with clinically significant prostate cancer. These findings provide evidence that Trop2 holds promise to serve as a novel prognostic tissue biomarker and diagnostic urine marker for prostate cancer that can be used to optimize treatment decision-making. METHODS All methods were carried out in accordance with the relevant guidelines and regulations of Stanford University and University of California, Los Angeles. Immunohistochemistry (IHC) Trop2 staining was conducted on the Canary Prostate Cancer Tissue Microarray (CPCTA) (19). Details of the case selection, clinical data, statistical considerations, and TMA construction have been described previously (19). Briefly, microarrays were constructed using a standardized protocol and included 3 cores of cancer from the largest and highest-grade cancer lesion and a single core of normal peripheral zone prostate tissues from the same patient. Cases were selected at random from available radical prostatectomy specimens performed for localized prostate cancer. All patients had a minimum of 5-years of follow-up and complete clinical and pathological data was collected relevant to prostate cancer including patient age, pre- operative PSA level, clinical stage, pathological stage, Gleason Grade group, follow- up time, and patient status (no evidence of disease, biochemical recurrence, metastases, death from prostate cancer, and overall survival). The resource has been reviewed and approved by the Institution Review Board at Stanford University (#40197). The TMA slides were deparaffinized at 65°C for 1 hour and incubated with Clearify for 15 minutes, followed by rehydrated in 100%, 95%, and 70% of ethanol. 10 mM citrate buffer (pH = 6.0) was used for antigen unmasking for 20 minutes at 95°C. 3% of hydrogen peroxide was used to block endogenous peroxidase activity. 2.5% horse serum was used for blocking at room temperature for one hour. The primary antibody Goat-anti-Trop2-biotin antibody (R&D Systems; BAF650; 1:50) was incubated, and the slides were kept in a humidified chamber at 4°C overnight. The slides were washed for 5 minutes with phosphate-buffered saline (PBS) three times and incubated with a streptavidin horseradish peroxidase (HRP) (Vector Labs; SA-5004; 1:200) for one hour at room temperature. After washing the slides three times with PBS, slides were detected with a DAB kit (Dako). The slides were counterstained with hematoxylin followed by dehydration in ascending ethanol. Slides were mounted and scanned by a NanoZoomer (Hamamatsu) for scoring. To only score the cancer region with membrane Trop2 staining as well as grade cancer regions with accurate Gleason Scores, Trop2 staining intensity was scored by a genitourinary pathologist, who has expertise in prostate cancer, blinded to outcomes from 0 to 3 (0 is negative, 1 is weak, 2 is moderate, and 3 is strong as shown in Figure 1B). Statistical Analysis All Trop2 staining was scored by a pathologist with expertise in prostate cancer from the Department of Pathology at Stanford University. The Canary Prostate Cancer Tissue Microarray (CPCTA) cohort contains information on the overall patient survival from the date of radical prostatectomy. Overall survival is the time from radical prostatectomy to death with patients censored at last known alive date if the death date was unknown. The Canary TMA was built substantially around biochemical failure. Cases included samples from men with biochemically recurrent prostate cancer within 5 years of surgery and non-recurrent prostate cancer after 5 years of follow-up. Recurrence was defined as: a single PSA > 0.2 more than 8 weeks after prostatectomy; salvage or secondary therapy; clinical or radiologic evidence of metastasis. Fisher’s exact test or Wilcoxon test was used to assess the association between Trop2 staining and patient characteristics (seminal vesicle invasion, positive surgical margins, age, pre-operative PSA, Gleason Score, p-stage, extracapsular extension). The Kaplan-Meier (KM) method was used to estimate survival curves in Trop2 positive and Trop2 low / negative patients. Patient characteristics and Trop2 staining were evaluated as predictors of overall survival in both univariate and multivariate Cox proportional hazards models. All tests were two-sided and p-values of 0.05 or less were considered statistically significant. Statistical analysis was carried out using R [R Core Team (2019). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https: / / www.R- project.org / ]. Design and Collection of Urine Samples We used a case-control design to compare Trop2 urine in prostate cancer going to prostatectomy and non-cancer specimens with long term follow up known not to have ever received a prostate cancer diagnosis to represent the extremes of patient scenarios. After consent (IRB: HSC20050234H), we prospectively and serially enrolled men prior to prostatectomy for our cases. 39 samples from men with clinically significant prostate cancer were used in this study. After the prostatectomy, we requested an unstained representative pathologic slide for cancer and a non-cancer portion of the prostatectomy specimen. For the control group, we used a tissue bank of stored specimens from men who were followed over 10 years with PSA below 1.5 ng / ml and therefore they did not meet clinical criteria for biopsy or significant prostate cancer (n=40) (IRB HSC20000030H, Supplementary Table 1-2 in Liu et al.). All samples were collected at University of Texas Health Science Center at San Antonio under the approved Institutional Review Board (IRB) protocol (IRB: HSC20050234H). The experimental protocols were approved by University of Texas Health Science Center at San Antonio, Stanford University, and University of California, Los Angeles committee (IRB: HSC20050234H). All experiments were carried out in accordance with the relevant guidelines and regulations of Stanford University and University of California, Los Angeles ethical guidelines and regulations. Informed consent was obtained from all subjects. All methods were carried out in accordance with the relevant guidelines and regulations of Stanford University and University of California, Los Angeles. Enzyme-linked immunoassay (ELISA) Trop2 urine levels were determined by a Sandwich ELISA. Diluted anti-Trop2 capture antibody (SinoBiological, 10428-MM01, 1:100) was coated in a 96-well ELISA plate at 4°C overnight. The wells were washed three times with PBST (PBS supplied with 0.01% Tween-20).5% of BSA was used for blocking and incubated at 4 °C overnight. 20 ul of whole urine samples from cancer-free patients and clinically significant prostate cancer patients were incubated for 2 hours at room temperature. The plates were washed three times with PBST and then incubated with the Goat-anti- Trop2-biotin detection antibody (R&D Systems; BAF650; 1:250) for 2 hours at room temperature. The plates were washed three times with PBST and incubated with a streptavidin-HRP (Thermo Fisher Scientific, PI21134, 1:1000) at room temperature for 30 minutes. After washing with PBST three times followed by PBS two times, the signals were detected by ultra TMB-ELISA substrate (Thermo Scientific, #34028) for 20 minutes. After adding the stopping solution (Thermo Fisher Scientific, PIN600), plates were analyzed via plate reader (Promega) at 450 nm. The standard curve for Trop2 concentrations was determined using 0-500 pg / ml recombinant human Trop2 (SinoBiological, 10428-H08H-1). Western Blot (WB) 30ul of the whole urine samples from cancer-free patients and patients with clinically significant prostate cancer were denatured with sodium dodecyl sulfate (SDS) at 95°C for 5 minutes. Samples were separated by SDS-PAGE (Invitrogen™ XP08165BOX). The proteins were transferred onto a nitrocellulose membrane (GVS Life Sciences, 1212632). 5% non-fat milk was used for blocking, and the membrane was incubated with a primary antibody (R&D Systems; BAF650; 1:1000) at 4 °C overnight. The HRP-conjugated secondary antibody was incubated (Thermo Fisher Scientific, PI21134, 1:2000) for one hour at room temperature and then developed with ECL Substrate (Thermo Fisher Scientific, 32106). RESULTS Positive Trop2 Expression is Associated with Worse Overall Survival To validate Trop2 as a prognostic tissue biomarker for clinically significant prostate cancer, we assessed tissue levels of Trop2 in the Canary Prostate Cancer Tissue Microarray (CPCTA) that was constructed from prostate cancer tissues from 1328 patients from 7 institutions with a minimum of 5 years follow-up (Fig.1A) (19). Cores from 1153 patients (87%) had sufficient tissue to evaluate Trop2 levels by immunochemistry (IHC). The pathologists scored the intensity of the Trop2 IHC staining from 0 to 3 without knowledge of sample metadata (Fig. 1B). Samples with at least one core (1 out of 3 cancer cores) scored as 3 were categorized as positive Trop2 expression. Samples were categorized as low / negative Trop2 expression when all evaluable cores were scored as 2 or below. We found that 764 (66%) cases were Trop2 positive, and 389 (34%) cases were Trop2 low / negative (Table.1). We first analyzed the association between Trop2 expression and clinical- pathologic variables at radical prostatectomy. Trop2 positive expression correlated with unfavorable clinicopathological features including higher median age compared to Trop2 low / negative group (63 v.62 years; p=0.023, Wilcoxon Test), higher median log pre-operative PSA (1.92 v. 1.87; p=0.017, Wilcoxon Test), and a greater likelihood of Gleason Score >=7 in their radical prostatectomy specimen (57% v. 44%; p < 0.001, Pearson Chi-Squared Test) (Table 1, and Fig. 1C). There was no difference in the percentage of patients with positive surgical margins, extracapsular extension (ECE), seminal vesicle invasion (SVI), or P-stage T3 / T4 between Trop2 high and Trop2 low / negative expression (Table 1). Importantly, Trop2 positive expression is associated with a shorter overall survival time in Kaplan–Meier analysis (p = 0.014) (Fig. 1D). Trop2 positive expression has a trend with shorter disease specific survival but did not achieve significance due to few prostate cancer deaths in the cohort. In univariate Cox proportional hazards analysis, patients with Trop2 positive expression had lower overall survival compared to those with Trop2 low / negative expression (Hazard Ratio (HR) 2.07; 95% Confidence Interval (CI) 1.13, 3.29, p=0.018) (Table 2). Multivariate Cox proportional hazards analysis revealed that high Trop2 trended toward significant association with worse overall survival when adjusted for Gleason Score and age (Hazard Ratio (HR) 1.78; 95% Confidence Interval (CI) 0.96, 3.28, p=0.066) (Table 2). Only Gleason Score and age were significant predictors of overall survival when all clinical and pathological features were included in a multivariate model. Together, these data support the hypothesis that elevated Trop2 levels at the time of radical prostatectomy for localized disease can serve as a tissue marker for aggressive prostate cancer. Trop2 can be Detected in Urine from Clinically Significant Prostate Cancer Patients Trop2 is cleaved and released into the extracellular environment, making it a potential liquid biomarker for prostate cancer (18). Given our finding that elevated tissue levels of Trop2 were associated with poorer clinical features, we assessed whether shed Trop2 can be detected in the urine from men with clinically significant prostate cancer, thereby providing a non-invasive biomarker to identify clinically significant prostate cancer and monitor the disease. To test urine Trop2 levels, we developed a plate Sandwich enzyme-linked immunoassay (ELISA) by using two commercially available anti-Trop2 capture and detection antibodies (Fig. 2A). Urine samples from 40 patients with low serum PSA for 10 years were used as cancer-free controls (Supplementary Table 1 in Liu et al.). We obtained urine samples from 39 patients with clinically significant prostate cancer to test urine Trop2 levels. Approximately 69% of patients were defined as unfavorable intermediate-risk or above according to NCCN stratification (Supplementary Table 2 in Liu et al.). Compared to cancer-free patients, shed Trop2 was detectable and was significantly higher in the urine from clinically significant prostate cancer patients (p= 0.0191, Fig. 2B). Western Blot analysis of the urine samples showed detection of a single band at the expected size, confirming the validity of the ELISA (Fig. 2C, D, and Supplementary Fig. 1 in Liu et al.). Our Trop2 ELISA assay represents a specific, high-throughput, and inexpensive method to detect shed Trop2 levels in urine (p=0.0065) (Fig. 2C, D). We confirmed the expression of Trop2 protein in patient- matched tissues by IHC staining, and high levels of Trop2 were found in the cancer samples in all cases where shed Trop2 was found in the urine by ELISA (Fig. 2E). In summary, shed Trop2 can be detected in urine samples from clinically significant prostate cancer patients, providing evidence that it can be used as a non-invasive marker for early detection of clinically significant prostate cancer. DISCUSSION Numerous studies have shown that Trop2 is highly expressed in multiple epithelial cancers including breast, lung, urothelial, colon, and ovarian cancers (20- 25). Due to the high expression of Trop2 in epithelial cancers, Sacituzumab govitecan, an anti-Trop2-antibody-conjugated to SN-38, has been FDA-approved for triple-negative breast cancer, hormone-positive breast cancer, and urothelial cancer (26-28). Significant clinical benefits have been observed in clinical trials targeting Trop2 with Sacituzumab govitecan in Triple-negative breast cancer and hormone- positive breast cancer (26, 28, 29). In the context of prostate cancer, Trop2 is highly expressed in metastatic castration-resistant prostate cancer and Sacituzumab govitecan is currently tested in clinical trials for patients with metastatic prostate cancer (15, 17, 30, 31). As we demonstrated that Trop2 is a prognostic tissue biomarker for clinically significant prostate cancer and is associated with worse clinical features, providing evidence for the great potential of Trop2 targeted therapy as a therapeutic strategy for metastatic prostate cancer. Clinically significant prostate cancer, which accounts for approximately 15% of all prostate cancer diagnoses, frequently recurs after surgery or radiation therapy and shows high disease-specific mortality (7, 32). Establishing new prognostic biomarkers that can distinguish clinically significant prostate cancer will result in a decrease in overdiagnosis and overtreatment of patients with indolent prostate cancer (9, 33). Several risk stratification systems, such as D'Amico risk groups and the Cancer of the Prostate Risk Assessment (CAPRA) score commonly use clinical T stage, serum prostate-specific antigen (PSA), and biopsy Gleason Score as risk assessment criteria (4, 34-36). However, PSA is not cancer-specific, and the primary biopsy Gleason Score has a more than 20% mismatch compared to the pathologic Gleason Score and the mismatch rate increases to 50% in the second biopsy (37). Thus, the development of new companion prognostic molecular biomarkers in conjunction with the current risk stratification system will improve the early detection of clinically significant prostate cancer, and provide more accurate risk classification, ultimately guiding precise treatment for multimodal therapies versus active surveillance in prostate cancer patients. A molecular biomarker with prognostic value is a promising alternative and / or a marker to monitor prostate cancer progression and therapeutic response (38). The urine-based biomarker can be measured for disease diagnosis and prognosis in a non- invasive manner (39). In this context and to the best of our knowledge, this is the first study that evaluates shed Trop2 as a potential urine biomarker for clinically significant prostate cancer and a tissue-based predictor for the overall-survival time in prostate cancer. We demonstrate that tissue and urine Trop2 may supplement current clinical biomarkers for the early detection and prediction of clinically significant prostate cancer. We anticipate that patients with elevated urine Trop2, in cooperation with other high-risk criteria, can further facilitate risk stratification and outcome prediction of patients with prostate cancer. Our studies warrant further research in larger cohorts to assess the utility of the urine Trop2 assay as a clinical test and to establish shed Trop2 as a urine biomarker for the early identification of clinically significant prostate cancer and the monitoring of treatment response. REFERENCES: 1. Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA Cancer J Clin.2023;73(1):17-48. 2. Rebello RJ, Oing C, Knudsen KE, Loeb S, Johnson DC, Reiter RE, et al. Prostate cancer. Nat Rev Dis Primers.2021;7(1):9. 3. Mottet N, Bellmunt J, Bolla M, Briers E, Cumberbatch MG, De Santis M, et al. 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Expression and Therapeutic Targeting of TROP-2 in Treatment-Resistant Prostate Cancer. Clin Cancer Res.2023;29(12):2324-35. 31. Bardia A, Messersmith WA, Kio EA, Berlin JD, Vahdat L, Masters GA, et al. Sacituzumab govitecan, a Trop-2-directed antibody-drug conjugate, for patients with epithelial cancer: final safety and efficacy results from the phase I / II IMMU-132-01 basket trial. Ann Oncol.2021;32(6):746-56. 32. Cooperberg MR, Broering JM, Carroll PR. Time trends and local variation in primary treatment of localized prostate cancer. J Clin Oncol.2010;28(7):1117-23. 33. Rice. MA, Stoyanova. T. Biomarkers for Diagnosis and Prognosis of Prostate Cancer. In: Genadiev T, editor. Prostatectomy. InTechOpen.2018. 34. D'Amico AV, Whittington R, Malkowicz SB, Schultz D, Blank K, Broderick GA, et al. Biochemical outcome after radical prostatectomy, external beam radiation therapy, or interstitial radiation therapy for clinically localized prostate cancer. JAMA.1998;280(11):969-74. 35. Cooperberg MR, Hilton JF, Carroll PR. The CAPRA-S score: A straightforward tool for improved prediction of outcomes after radical prostatectomy. Cancer.2011;117(22):5039-46. 36. Becerra MF, Atluri VS, Bhattu AS, Punnen S. Serum and urine biomarkers for detecting clinically significant prostate cancer. Urol Oncol.2021;39(10):686-90. 37. Poulos CK, Daggy JK, Cheng L. Preoperative prediction of Gleason grade in radical prostatectomy specimens: the influence of different Gleason grades from multiple positive biopsy sites. Mod Pathol.2005;18(2):228-34. 38. Terada N, Akamatsu S, Kobayashi T, Inoue T, Ogawa O, Antonarakis ES. Prognostic and predictive biomarkers in prostate cancer: latest evidence and clinical implications. Ther Adv Med Oncol.2017;9(8):565-73. 39. Tomlins SA, Day JR, Lonigro RJ, Hovelson DH, Siddiqui J, Kunju LP, et al. Urine TMPRSS2:ERG Plus PCA3 for Individualized Prostate Cancer Risk Assessment. Eur Urol.2016;70(1):45-53. All publications mentioned herein (e.g. those listed numerically herein) are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further, the actual publication dates may be different from those shown and require independent verification. The following references include descriptions of methods and materials in this field of technology. CONCLUSION This concludes the description of the illustrative embodiments of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching.

Claims

CLAIMS:

1. A method of detecting an epithelial cancer comprising: obtaining a sample of a biological material from an individual; characterizing the levels of shed extracellular domain (ECD) of tumor-associated calcium signal transducer 2 (Trop2) protein observed in the sample: and assessing if levels of shed TROP2 ECD observed in the sample are characteristic of an epithelial cancer.

2. The method of claim 1, wherein levels of shed TROP2 ECD are observed in an ELISA assay.

3. The method of claim 2, wherein levels of shed TROP2 ECD are observed in urine or blood of the individual.

4. The method of claim 3, wherein the urine is obtained from an individual considering a treatment for an epithelial cancer or undergoing a treatment for an epithelial cancer.

5. The method of claim 4, wherein the treatment comprises administering Sacituzumab Govitecan or TRODELVY to the individual.

6. A method of detecting presence or levels of shed extracellular domain (ECD) of tumor-associated calcium signal transducer 2 (Trop2) protein in urine, the method comprising : obtaining a urine sample and performing and ELISA assay for proteins present in the urine, wherein at least one antibody used in the ELISA assay binds to the ECD of Trop2; and correlating a signal obtained using the antibody in the ELISA assay with the presence or levels of Trop2 ECD in the urine sample.

7. The method of claim 6, wherein the urine is obtained from a person having or suspected of having an epithelial cancer.

8. The method of claim 7, wherein the epithelial cancer is a prostate cancer or a bladder cancer.

9. The method of claim 6, wherein the urine is obtained from a person undergoing a therapy for an epithelial cancer.

10. The method of claim 9, wherein the treatment comprises administering Sacituzumab, Govitecan or TRODELVY to the individual.

11. An ELISA assay comprising a first antibody that binds extracellular domain (ECD) of tumor-associated calcium signal transducer 2 (Trop2) protein, wherein the first antibody is coupled to a solid matrix, and a second antibody that binds extracellular domain (ECD) of tumor-associated calcium signal transducer 2 (Trop2) protein, wherein the second antibody is coupled to a detectable marker.

12. The ELISA assay of claim 11, further comprising urine.

13. The ELISA assay of claim 12, wherein the urine is obtained from a person having or suspected of having an epithelial cancer.

14. The ELISA assay of claim 12, wherein the urine is obtained from an individual considering a treatment for an epithelial cancer or undergoing a treatment for an epithelial cancer.

15. The ELISA assay of claim 12, wherein the urine is obtained from an individual who has been administered Sacituzumab, Govitecan or TRODELVY.

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