Keratin 17 as biomarker for bladder cancer

Abstract

To solve the problem in which there is an unmet need to identify a novel diagnostic biomarker being used in conjunction with existing treatment methods enabling determining whether a subject has a bladder cancer or the bladder cancer has progressed so that patients in need of treatment can be identified at an early stage, and thus, such methodology is non-invasive, in particular, may be advantageous if such methodology can be deployed in a state of diagnostic test systems of the present art field capable of processing multiple tests and / or a plurality of samples at a time.SOLUTION: The present disclosure provides methods for detecting and analyzing a K17 expression in a bladder sample obtained from a subject. The present disclosure also pertains to methods and kits for identifying a mammalian subject with a bladder cancer by detecting the expression of K17 in a sample. The present methods include both cell-based and cell-free methods for determining a level of the keratin 17 in a sample obtained from the bladder of a subject.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 371,286, filed Aug. 5, 2016, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to a method for detecting the amount of keratin 17 in a sample obtained from a subject. The method can be used to diagnose bladder cancer in a subject. The method of the present disclosure can be performed using tissue biopsies and cell-based samples, as well as acellular samples such as urine samples. The present disclosure further provides a suitable method for determining the presence or absence of K17 in urine (which may or may not contain intact cells), and a method for estimating the expression level of cytokeratin 17 in bladder tissue by detecting K17 protein or mRNA in the acellular portion of a subject's urine to determine the presence of bladder cancer in the subject. [Background technology]

[0003] Bladder cancer is the fourth most common malignancy in men in the United States, accounting for approximately 5% of all newly diagnosed cancers. See American Cancer Society. Cancer Facts & Figures 2016, Atlanta, Ga: American Cancer Society (2016). Approximately half of all bladder cancers are initially identified in the lining of the bladder wall (i.e., the urothelium, also known as transitional epithelium) and are considered noninvasive or carcinoma in situ, or urothelial carcinoma (also known as transitional cell carcinoma). See Grossman, HB et al., JAMA. (2005) 293:810-816. Approximately one in three patients with bladder cancer will demonstrate invasion of the cancer into the underlying muscle layer of the bladder wall. Advances in treatment modalities have increased survival rates for subjects diagnosed early (i.e., stage 0 or stage I bladder cancer), with the average 5-year survival rate for subjects diagnosed early being between 85% and 98% [American Cancer Society. Cancer Facts & Figures 2016, Atlanta, Ga: American Cancer Society (2016)]. Conversely, later stages of diagnosis are associated with a more dire outcome, with the average 5-year survival rate for subjects diagnosed with stage II or III bladder cancer being between 45% and 65%. See Kaufman DS et al., Lancet. (2009) 374:239-249, American Cancer Society. Cancer Facts & Figures 2016, Atlanta, Ga: American Cancer Society (2016), and Soloway MS. Urology. (2006) 67: 3-10.

[0004] Current methods for detecting bladder cancer include urinalysis (i.e., detection of blood in urine), with a variety of commercially available tests in use: Immunocyt™, NMP22 BladderChek®, BTAstat® test, and UroVysion® Bladder Cancer Kit. Immunocyt™ tests a subject's urine for the presence of mucin and carcinoembryonic antigen (CEA). NMP22 BladderChek® screening determines whether a specific protein, NMP22, is present in a subject's urine. The BTAstat® test is designed to detect human complement factor H-related protein (hCFHrp) as a cancer biomarker. It is relatively sensitive for detecting high-grade urothelial carcinoma, but generally fails to detect low-grade lesions (i.e., stages 0-I bladder cancer), which offer the best chance of cure. The UroVysion® Bladder Cancer Kit tests for aneuploidies at chromosomes 3, 7, and 17, and deletions at the 9p21 locus, in urine specimens from subjects suspected of having bladder cancer by fluorescent in situ hybridization (FISH). Most low grade tumors are diploid and therefore will test negative with the UroVysion® Bladder Cancer Kit; therefore, like the BTAstat® test, UroVysion® cannot effectively detect low grade tumors of the bladder mucosa. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] American Cancer Society. Cancer Facts & Figures 2016, Atlanta, Ga: American Cancer Society(2016) [Non-Patent Document 2] Grossman, HB et al., JAMA. (2005) 293:810-816 [Non-Patent Document 3] Kaufman DS et al., Lancet. (2009) 374:239-249 [Non-Patent Document 4] Soloway MS. Urology. (2006)67:3~10 [Non-Patent Document 5] Escobar-Hoyos, LF et al., Modern pathology (2014) 27(4):621-630 [Non-Patent Document 6] Schmittgen and Livak, Nature protocols (2008) 3: 1101-1108 [Non-Patent Document 7] Edge SB and Compton CC. Annals of surgical oncology. (2010) 17:1471-4 [Non-Patent Document 8] L. Escobar-Hoyos et al. Cancer Res. 2015 Sep 1;75(17):3650~62 [Non-Patent Document 9] Schneider CA et al., Nat methods. (2012) 9:671-5 [Non-Patent Document 10] Ruifrok AC, Johnston DA, Anal Quant Cytol Histol. (2001) 23:291~9. [Non-Patent Document 11] Youden WJ. Cancer. (1950) 3:32~5 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is an unmet need to identify novel diagnostic biomarkers that can be used in conjunction with existing therapies to determine whether a subject has or has bladder cancer, so that patients who need treatment can be identified early.Such a methodology would be particularly advantageous if it could be deployed in state of the art diagnostic testing systems that are non-invasive and can process multiple tests and / or multiple samples at once.The method of the present disclosure addresses these unmet needs and allows for effective treatment, as well as better outcomes and longer survival times for patients than existing tests.

[0007] Keratin 17 (K17, KRT17, or cytokeratin 17), a member of the intermediate filament cytoskeleton family, has been identified as a prognostic and diagnostic biomarker for certain specific cancers, namely pancreatic and cervical cancer. See Escobar-Hoyos, LF, et al., Modern pathology (2014) 27(4):621-630. However, the present disclosure confirms that K17 is not overexpressed in all cancers. For example, FIG. 1 clearly shows that K17 is elevated (highly expressed) in certain cancers, such as cervical and pancreatic cancer, but not in many other cancers, such as liver, colon, kidney, brain, and lymph node cancer. K17 expression in cancer is generally organ and tumor type specific. In particular, the present disclosure is the first to detect elevated levels of K17 expression in bladder tissue from subjects with bladder cancer compared to K17 expression levels determined from bladder tissue and urine samples from healthy subjects, confirming that detection of K17 can enable a reliable diagnostic test for bladder cancer.

[0008] The present disclosure confirms and validates the utility of K17 as a diagnostic bladder cancer biomarker and provides a highly sensitive method for detecting bladder cancer in a subject.The method is also advantageous for use in patients not known to have bladder cancer, where a non-invasive and inexpensive test would provide an unexpected advance over existing diagnostics that require expensive and invasive procedures to confirm that the patient has bladder cancer. [Means for solving the problem]

[0009] This disclosure reveals that keratin 17 is a biomarker for diagnosing bladder cancer, including early-stage bladder cancer, which has the highest probability of favorable treatment outcomes. This disclosure also reveals that reliable, non-invasive bladder cancer diagnosis is possible using natural urine. For example, the data herein shows that improved diagnosis can be made based on urine testing alone, simply by reducing the amount of "false negative" readings that plague current non-invasive testing methods. The data presented herein also shows that K17 levels are elevated (when compared to control samples) in subjects with early-stage and high-grade bladder cancer, but are absent or detected at low levels in normal benign bladder mucosa (i.e., non-cancerous control tissue). In summary, this disclosure shows for the first time that increased K17 expression is a critical event in the development and progression of bladder cancer, and that K17 expression can be measured as a diagnostic indicator of bladder cancer in a subject.

[0010] Thus, in one aspect of the disclosure, a method is provided for determining the amount of K17 in a subject. In an embodiment, the subject has or may have bladder cancer. In an exemplary embodiment of the method, a sample of bladder cells is obtained from the subject, and the sample is then labeled to detect the expression of K17 in the sample. In an embodiment, the presence of K17 is detected through the binding of an antibody to K17 protein in the sample. In another embodiment, the presence of K17 is detected by measuring the level of K17 mRNA in the sample, such as by RT-PCR. In another embodiment, K17 expression is detected in the sample, and if the K17 level is increased compared to the K17 expression in a control sample (e.g., a reference sample or standard), the subject has bladder cancer. In an embodiment, the sample is a bladder tissue biopsy formalin-fixed paraffin-embedded tissue sample, or a urine sample. In some embodiments, the control sample is benign bladder mucosa, urothelial cells, or transitional epithelial cells from a healthy subject. In other embodiments, the control sample may be a standard, a reference sample containing a known amount of K17 protein or nucleic acid, or simply a readout or indication indicating a positive, negative, or no result based on a particular amount of K17 protein or nucleic acid detected in the sample. In other embodiments, the sample is urine, and the urine contains one or more bladder cells or bladder cancer cells. In some embodiments, the sample is found to contain no cells. In certain embodiments, the detected bladder cancer is urothelial cancer. In certain embodiments, the urothelial cancer is a papillary cancer, such as low-grade papillary urothelial neoplasm (PUNLMP), low-grade papillary urothelial carcinoma (LG), high-grade papillary urothelial carcinoma (HG), or transitional urothelial carcinoma. In other embodiments, the detected bladder cancer is a flat cancer, such as flat urothelial carcinoma in situ of the bladder (non-invasive flat urothelial carcinoma). In yet other embodiments, the detected bladder cancer is an invasive cancer, such as, for example, invasive urothelial cell carcinoma. In certain embodiments, the bladder cancer detected is squamous cell carcinoma, adenocarcinoma, small cell carcinoma, or sarcoma.In some embodiments of the method, the presence or level of K17 expression in the sample is determined by immunohistochemical staining or immunocytochemical analysis, or by mRNA detection.

[0011] In another aspect of the disclosure, a method is provided for detecting K17 expression in an acellular sample. In an embodiment, the subject has or may have bladder cancer. In an exemplary embodiment of the method, the sample is natural urine obtained from the subject. In other embodiments, the natural urine sample does not contain cells or has been processed to remove all cells from the sample. In a particular embodiment, an acellular urine sample is obtained from the subject. The sample is then contacted with an anti-K17 antibody to detect the amount of K17 in the sample. Here, the amount of K17 in the sample will reflect the amount of K17 in the subject's bladder. In an embodiment, the presence of K17 is detected through the binding of the antibody to K17 protein in the sample or through mass spectrometry. In an embodiment, the amount of K17 is determined by detecting K17 mRNA in the sample. In some embodiments, the method may be qualitative (i.e., any amount of K17 detected in the sample is indicative of bladder cancer) or quantitative (only amounts above a predetermined level are indicative of the subject having bladder cancer). In quantitative embodiments, K17 expression is determined based on the amount of K17 protein or K17 mRNA detected in the sample compared to the amount of K17 protein or K17 mRNA in a control sample, and if the K17 level is increased above that of the control sample, the subject has bladder cancer. In some embodiments, the control sample is urine from a healthy subject, or a solution containing a known amount of K17 protein or mRNA, or simply a standard set for a known or specific amount. In certain embodiments, the detected bladder cancer is urothelial cancer. In some embodiments, the detected urothelial cancer is a papillary cancer, such as low-grade papillary urothelial neoplasm (PUNLMP), low-grade papillary urothelial carcinoma (LG), or high-grade papillary urothelial carcinoma (HG). In other embodiments, the detected bladder cancer is a flat cancer, such as flat carcinoma in situ or non-invasive flat urothelial carcinoma of the bladder. In yet other embodiments, the detected bladder cancer is an invasive cancer, such as an invasive urothelial carcinoma or a transitional cell carcinoma, etc. In some embodiments, the detected bladder cancer is a squamous cell carcinoma, an adenocarcinoma, a small cell carcinoma, or a sarcoma.In some embodiments of the method, the level of K17 expression in the sample is determined by ELISA. In other embodiments of the method, the level of K17 expression is determined by detecting the amount of K17 mRNA in bladder cells or urine. [Brief description of the drawings]

[0012] [Figure 1] 1 is a histogram showing that keratin 17 expression varies among various cancer types. RNA expression data was evaluated by data mining the TCGA data portal from the National Cancer Institute. The histogram shows K17 expression in a logarithmic scale for certain cancer types with elevated K17 expression, such as cervical, bladder, and pancreatic cancers. The chart further shows that many cancer types, such as colon, liver, brain, and lymphatic cancers, express relatively low levels of K17. [Diagram 2] Image showing K17 immunocytochemical localization in a urine cytology sample. Keratin 17 expression can be demonstrated by detection of labeled cytoplasmic K17 (dark cells), whereas other non-cancerous cells show no K17 labeling (normal unlabeled cells). [Diagram 3] Figure 3A-D are images showing urinary keratin 17 immunocytochemistry. (a) K17 staining of rare benign cells, 4x magnification; (b) K17 staining of rare benign cells, 40x magnification; (c) increased (2+) K17 staining, 4x magnification; (d) increased (2+) K17 staining, 20x magnification. In rare cases, K17 was sparsely detected in benign cells from normal urinary cytology samples (a, b), but K17 staining was strongly positive in urothelial cells from subjects with urothelial carcinoma (c, d). [Figure 4] 4A-D are images showing immunocytochemistry of keratin 17. (a, b) Most normal squamous epithelial cells and urothelial cells (c, d) were negative for K17, i.e., they did not show keratin 17 staining. [Diagram 5]5A-D are images showing bladder histology (a, b, d). Only faint, focal K17 staining was detected in benign bladder mucosa (c). [Figure 6] Figure 6A-D are images showing that increased K17 was detected in all grades of urothelial tumors: (a) low-grade papillary urothelial neoplasms; (b) low-grade papillary urothelial carcinomas transitioning from adjacent control urothelial mucosa; (c-d) high-grade papillary urothelial carcinomas. Immunohistochemical analysis shows diffuse and intense keratin 17 expression throughout the abnormal urothelial cells (i.e., tumor tissue) (a, b), with more focal K17 expression in high-grade urothelial carcinomas (c, d). [Figure 7] Figures 7A-D are images showing increased keratin 17 immunohistochemistry in invasive urothelial carcinoma. (a) Invasive carcinoma found just beneath non-neoplastic urothelial mucosa; (b) Invasive carcinoma in perivascular soft bladder tissue; (c) K17 expression at the cancer invasion frontline; (d) Invasion of the lamina propria by tumor cells. All tissue samples examined show increased keratin 17 expression in tumor cells from subjects with invasive urothelial carcinoma compared to tissue from normal patients. [Figure 8] 8A-B show the detection of keratin 17 using a microfludic detection method. (a) HeLa and (b) C33 cell samples were analyzed. The data show that significantly more K17-labeled cells were detected in HeLa (90.4% K17 positive) compared to C33 (35.1% K17 positive) cells. These data show that detection of K17 can be efficiently obtained using the microfluidic device in combination with the K17 immunostaining method, and therefore, in addition to the K17 mRNA detection and quantification method, the microfluidic device is also a suitable means for carrying out the method.

[0013] Table 1: Keratin 17 staining in control urothelial mucosa and urothelial bladder cancer. Mean PathSQ scores for control samples (benign) and early stage urothelial bladder cancer (PUNLMP, LG, HG) show significant differences in K17 expression between control and urothelial cancer tissue samples (p<0.001).

[0014] Table 2: Immunocytochemical analysis of K17 expression in urine cytology samples. Data compare clinical diagnosis of urine samples from patients with bladder cancer (positive for malignancy) to clinical diagnosis of urine samples from patients without bladder cancer (negative for malignancy or suspicious for malignancy). These data show that samples from subjects with bladder cancer (T3-T7) all stained positive for K17. In contrast, no K17 labeling was observed in urine samples from non-cancerous patients (N1, N7, N8), even when detectable levels of bladder cells were present.

[0015] Table 3: Detection of K17 expression by immunocytochemical staining of cells isolated from urine. Table 3 shows the results from 4 independent studies for a total of 104 patient samples analyzed. 39 samples from subjects with bladder cancer and 65 samples from benign tissue were analyzed using the method. For each study, the sensitivity and specificity of the K17 detection method are shown. The results show that the K17-based diagnostic method has an average sensitivity of 93% and an average specificity of 91%.

[0016] Table 4: Detection limits of the ELISA used for the detection of keratin 17 protein in acellular urine. Table 4 shows the K17 test results obtained from K17 standards (recombinant K17 protein) tested by ELISA, with the mean, standard deviation and percent coefficient of variation (%CV). The cut-off value for the positive signal was determined from the negative control (zero standard / no K17 protein) such that a K17 positive call was 3 times the standard deviation of the mean of the negative controls (mean of the zero standard + [3 x standard deviation of the zero standard]).

[0017] Table 5: Detection of K17 protein in acellular urine samples from subjects with bladder cancer. Table 5 shows data from 21 urine samples tested (6 cancerous, 15 benign / control). Samples positive for K17 had a mean optical density (OD) of at least 0.081 and a %CV of less than 20%. The data shows that 4 of the 6 cancer cases were positive for K17 and none of the benign samples tested were positive for K17.

[0018] Table 6: Primer sets for detection of K17 mRNA by quantitative RT-PCR. Table 6 illustrates primer pairs for use in detection and / or quantification of K17 mRNA by RT-PCR. Suitable primers of the invention, such as those illustrated in Table 6, are designed to generate products that cover the 17K intron, such that PCR amplification of a product of the expected size occurs only if cDNA generated from K17 mRNA is present in the sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] To date, diagnostic markers (e.g., immunohistochemical markers) for bladder cancer, particularly early stage bladder cancer (e.g., urothelial carcinoma), have only marginally improved diagnostic accuracy compared to the gold standard of in situ and / or biopsy visualization, e.g., invasive procedures. In contrast, the present disclosure identifies, characterizes, and validates a novel biomarker, K17, and provides a novel methodology with improved diagnostic accuracy compared to current techniques, which is advantageous for use in detecting early stage bladder cancer using immunohistochemical staining of bladder tissue and immunocytochemical techniques for screening urine cytology samples, or nucleic acid-based tests for detecting K17 mRNA. The present disclosure also provides a cell-free based method for diagnosing a subject with bladder cancer through detection of K17 protein or mRNA levels in bladder tissue and urine samples, including cell-free urine samples.

[0020] Terminology The term "peptide" or "protein" as used in this disclosure refers to a linear chain of consecutive amino acid residues linked together by peptide bonds between the alpha amino and carboxy groups of adjacent amino acid residues. In one embodiment, the protein is keratin 17 (K17).

[0021] The term "nucleic acid" as used herein refers to one or more nucleotide bases of any kind, including single-stranded or double-stranded. In one embodiment of the present disclosure, the nucleic acid is DNA, and in another embodiment, the nucleic acid is, for example, mRNA. In carrying out the method of the present disclosure, the nucleic acid (e.g., K17 RNA) analyzed by the method is derived from one or more samples.

[0022] As used herein, the terms "cytokeratin 17", "keratin 17", "KRT17" and "K17" refer to the human keratin type II cytoskeletal keratin 4 gene or its product located on chromosome 17 and encoding type I intermediate filament chain keratin 17, as set forth in Accession No. NG_008625. The intended meaning of K17 includes the mRNA transcript of the keratin 17 cDNA sequence set forth in Accession No. NM_000422 and proteins translated therefrom, including, for example, the keratins set forth in Accession No. NP_000413, type 1 cytoskeletal protein 17 or homologs thereof.

[0023] As used herein, the phrases "subject," "test subject," or "patient" refer to any mammal. In one embodiment, the subject is a candidate for a bladder cancer diagnosis (e.g., urothelial carcinoma) or an individual with a precancerous lesion, such as a urothelial neoplasm. In certain embodiments, the subject has been diagnosed with bladder cancer, and the subject is a candidate for treatment thereof. The methods of the present disclosure can be performed on any mammalian subject at risk of developing cancer or who has been diagnosed with cancer. In particular, the methods described herein are most useful when performed on humans.

[0024] A "biological sample", "test sample" or "sample" as used in this disclosure can be obtained by any method known to one of skill in the art. A sample can be obtained from any part of a subject, including bladder tissue, urine, or a combination thereof. In certain embodiments, a sample is a tissue biopsy, fresh tissue, or live tissue removed from a subject's bladder. In certain embodiments, a sample is a collection of cells from the bladder wall, such as the transitional epithelium, connective tissue, muscle tissue, or adipose tissue of the bladder. In other embodiments, a sample is processed prior to use in the methods of the disclosure. For example, formalin fixation and paraffin embedding are beneficial for histological preservation and diagnosis of clinical tissue specimens, and formalin-fixed paraffin-embedded tissue is more readily available in large quantities than fresh or frozen tissue, so a formalin-fixed paraffin-embedded tissue sample isolated from a subject is useful in the methods of the disclosure. In some embodiments, cells are collected from urine by centrifugation or by filtration, and the collected cells are used for testing. Those skilled in the art will take care to ensure that the sample is collected, processed, and tested in a manner that prevents possible degradation of K17 protein or mRNA, which may vary depending on the test used. In certain embodiments, the sample is a volume of urine obtained from a subject, and thus the urine may or may not have a detectable amount of cells (i.e., the sample may or may not be acellular). In some embodiments, the sample is an acellular urine sample that contains K17 protein or mRNA.

[0025] As used herein, a "control sample", "non-cancerous sample" or "normal sample" is a sample that does not show elevated K17 and / or shows absence of K17 or reduced levels of K17 (where "reduced" is relative to a standard or other known value indicative of cancer). In certain embodiments, the control sample does not contain cancerous cells (e.g., benign tissue components including, but not limited to, normal bladder mucosa, benign bladder mucosal cells, and other non-cancerous cells derived from the urothelium or transitional epithelium of the bladder). In certain embodiments, the control or normal sample is a sample from benign tissue that does not show K17 staining or expression. Non-limiting examples of control samples used in this disclosure include non-cancerous tissue resections, surgical margins removed from a subject, isolated cells known to have normal or reduced levels of K17, or benign samples or urine from other healthy individuals. In one embodiment, the control sample is human urine from a healthy individual, or a compounded urine with no substitution or with a predetermined amount of K17 protein or nucleic acid substitution. In one embodiment, the control sample of the present disclosure is benign bladder tissue obtained from the subject. In certain other embodiments, the control sample is a urine cytology sample obtained from a healthy subject (i.e., a patient without cancer). In certain embodiments, the control sample is a volume of acellular urine obtained from a subject, such that the urine does not contain a detectable amount of K17 protein or nucleic acid. In other embodiments, the control sample is a volume of acellular urine obtained from a subject, such that the acellular urine sample contains a known amount of detectable K17 protein or nucleic acid.

[0026] The term "increase" or "higher" or "elevated" means at least a relative amount of an entity (such as K17 expression or amount) identified, measured, or analyzed in a control sample. Non-limiting examples include, but are not limited to, a 50% increase from the amount in a control sample, or an increase of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 1000% or more from the amount in a negative control sample. In certain embodiments, K17 expression indicative of a subject having bladder cancer includes an increase of at least 1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 13-fold, 15-fold, 20-fold, 25-fold, 30-fold or more compared to the amount of K17 expression exhibited by a control sample. As used in this disclosure, "increased level of K17 expression" refers to an increase in the amount of K17 protein or peptide fragments thereof or RNA present in a cell, organism or sample, in comparison to the level of control or normal K17 expression, or in comparison to the level of K17 expression measured in a reference sample or set of standards. In some embodiments, detection of any amount of K17 in a sample is an increase over the amount in a control. In certain embodiments, an increase in the level of keratin 17 expression corresponding to bladder cancer is demonstrated by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 13-fold, 15-fold, 20-fold, 25-fold, 30-fold, or more increase in K17 expression compared to the amount of K17 expression exhibited by a control sample or set of standards.

[0027] The term "reduced" or "reduction" means at least less than the relative amount of an entity identified, measured, or analyzed in a control sample or set of standards.

[0028] The phrase "bladder cancer" as used herein includes malignant tumors in cells or tissues of the bladder. Bladder cancer includes, for example, malignant tumors in the cell layer called urothelium or transitional epithelium, which lines the inside of the ureters, bladder, urethra, and parts of the kidney. In certain cases, bladder cancer includes the presence of cancerous cells in the connective tissue, muscle, or fat layer of the bladder wall. Bladder cancer of the present disclosure may be invasive or non-invasive papillary or flat cancer of the bladder. "Papillary cancer" is typified by elongated finger-like projections that grow from the inner surface of the bladder toward the hollow center of the bladder. Papillary tumors that grow toward the center of the bladder without growing into the deep tissue layers of the bladder wall are non-invasive papillary bladder cancers. In certain embodiments, the bladder cancer is a low-grade papillary urothelial neoplasm (PUNLMP). In other cases, the bladder cancer is low-grade (slow-growing) papillary urothelial carcinoma (LG) or high-grade papillary urothelial carcinoma (HG). In other embodiments, the bladder cancer is transitional urothelial carcinoma. "Flat carcinoma" is bladder cancer that does not grow toward the hollow of the bladder and mainly replaces the transitional epithelium of the bladder wall. In some embodiments, the bladder cancer is non-invasive flat carcinoma or flat carcinoma in situ (CIS). In other embodiments, the bladder cancer is squamous cell carcinoma, adenocarcinoma, small cell carcinoma, or sarcoma of the bladder wall. Bladder cancer detectable using the methods of the present disclosure can be classified as "grade", such as low or high grade cancer. "Low grade bladder cancer" is composed of urothelial cells that arise from normal or control bladder urothelial cells and generally has a good or favorable prognosis for response to treatment. "High-grade bladder cancer" is a less differentiated or undifferentiated subset of bladder cells that is distinguishable from normal or control tissue. High-grade bladder cancers are generally more invasive and have a poorer prognosis than low-grade cancers.

[0029] method This disclosure describes a method for using keratin 17 as a biomarker for bladder cancer, in which K17 RNA or protein expression is detected by immunohistochemistry, immunocytochemistry, solution or solid-phase protein assays (e.g., ELISA, microfluidics, flow cytometry, mass spectrometry), or K17 mRNA is detected or K17 mRNA levels are measured (e.g., by RT-PCR methodology such as Q-RTPCR) in microdissected tissue sections from formalin-fixed paraffin-embedded or urinary cytology samples for various diagnostic categories (i.e., non-cancerous bladder mucosa, low-grade papillary urothelial neoplasms (PUNLMP), low-grade papillary urothelial carcinoma (LG), and high-grade papillary urothelial carcinoma (HG), or transitional urothelial carcinoma). The data show that K17 showed approximately a 30-fold difference in expression between control (benign) and PUNLMP samples; a 15-fold difference in expression between control and LG bladder cancer samples; and a 13-fold difference in K17 expression between control and HG bladder cancer samples. Thus, the present disclosure shows that K17 expression can be measured and used as a diagnostic biomarker for the diagnosis of bladder cancer.

[0030] One aspect of the disclosure provides a method for diagnosing a subject with bladder cancer, comprising obtaining a test sample from the subject and detecting the level of K17 expression in the sample, thereby identifying the subject as having bladder cancer from an increase in the level of K17 expression in the sample compared to the level of K17 expression in a control sample. In another embodiment, the level of K17 expression is determined by detecting the presence of K17 mRNA in a qualitative or quantitative manner. In a particular embodiment, the level of K17 expression is determined by an immunoassay that detects the presence of K17 protein in a qualitative or quantitative manner.

[0031] In an embodiment, K17 expression is determined by detecting binding between K17 and anti-K17 antibodies in a sample. In one embodiment of the present disclosure, a biological sample (i.e., a test sample or a control sample) is obtained from the subject. Biological samples that can be used according to the present method may be collected by various means known to those skilled in the art. Non-limiting examples of sample collection techniques used in the present method include fine needle aspiration biopsy, surgical excision biopsy, endoscopic biopsy, and excision biopsy. In other embodiments, K17 levels can be detected in a urine sample obtained from a subject suspected of having cancer, and the urine sample is subjected to a test to determine the amount of K17 protein or K17 mRNA in the sample. In some embodiments, the urine sample may be analyzed for the presence of cells during the test, or may be processed to collect cells from the urine. However, in various other embodiments, the sample is processed in a manner that is not dependent on the presence or absence of cells. In certain embodiments, K17 expression can be detected in a cell-free sample from a bladder cancer patient, or even in a urine sample from the cell-free portion of natural urine. In other embodiments, the sample is a natural urine sample that contains a detectable amount (number) of cells. This method offers advantages over current diagnostics by enabling physicians to inexpensively detect bladder cancer in natural urine samples with high sensitivity (few false negatives) and accuracy (few false positives).

[0032] In some embodiments, the method includes a control sample that may be obtained from non-cancerous tissue or urine of a subject without cancer. The control sample may include natural urine containing a detectable amount of benign urothelial cells, as shown in Figures 3A-B and 4C-D. In other embodiments, the control sample is natural urine containing a detectable amount of normal squamous cells, as shown in Figures 4A-B. In yet other embodiments, the control sample (non-cancerous) may include bladder mucosal tissue (Figures 5A, B and D) or benign bladder mucosal tissue (Figure 5C) that does not show keratin 17 staining. In other embodiments, the control sample may be a positive control, such as a sample obtained from a subject known to have bladder cancer that shows an increased (strong) level of K17 staining. For example, the positive control sample is natural urine containing a detectable amount of urothelial cancer cells that show a strong (increased) amount of keratin 17 expression, as shown in Figures 3C-D. In yet other embodiments, the positive control sample (cancerous) comprises tissue obtained from a subject with low-grade papillary urothelial neoplasm (FIG. 6A), low-grade urothelial carcinoma (FIG. 6B), high-grade papillary urothelial carcinoma (FIGS. 6C-D), or invasive urothelial carcinoma (FIGS. 7A-D). Regardless of the type of cells or tissue utilized as the positive control sample, such cells must exhibit increased levels of K17 expression. Moreover, particularly in high throughput embodiments of the present disclosure, the method will be deployed with automated equipment and computer-controlled analytical procedures where there is no control, or where the control is a prepared liquid or material, or a pre-defined value set by the user or the device manufacturer.

[0033] While the presence of cancer cells in urine may be a result of bladder cancer, it is an unexpected aspect of the present disclosure that the amount of K17 in an acellular urine sample from a cancer patient is sufficient to distinguish such a sample from a normal (non-cancerous) sample. Furthermore, because the quality control and cell harvesting steps of cell-based assays are expensive, implementation of this embodiment of the present disclosure significantly reduces the costs associated with current invasive testing. Thus, in some embodiments, the sample is urine, a liquid that does not contain detectable amounts of cells, i.e., an acellular sample. For these particular embodiments, the control sample may be an acellular urine sample that contains a predetermined (known) amount of K17 protein or RNA, which may be none or below the detection limit. In some embodiments, the acellular urine sample contains a detectable amount of K17 protein or RNA (e.g., a sample from a bladder cancer patient, or a control sample). In other embodiments, the acellular urine sample does not contain a detectable amount of K17 protein or RNA (a negative control sample, or a sample from a subject without bladder cancer). In an embodiment in which the method employed analyzes keratin 17 expression in acellular portions of natural urine, the control sample may be a liquid sample (e.g., urine) containing a known amount of K17 protein (which may be zero for a negative control), or a liquid sample of normal bladder cells from a healthy patient. In other embodiments, the control sample may be a positive control, such as a liquid sample (e.g., urine) containing a known amount of K17 protein from a subject known to have bladder cancer, or containing an amount of K17 protein that is believed to be higher than would be expected if the subject did not have bladder cancer.

[0034] In some embodiments, the sample obtained from the subject is used directly without any prior treatment or processing, such as formalin fixation, deep freezing, or paraffin embedding. In certain embodiments, the biological sample may be obtained from the subject and processed by formalin processing and embedding the formalin-fixed sample in paraffin. In some embodiments, the sample may be stored before use. Thus, the diagnostic method can be applied using multiple preparation methods, including preparation for use with immunohistochemical staining devices (e.g., automatic staining devices), such as Autostainer Link (Dako), Discovery XT and Benchmark XT (Ventana Medical Systems), Leica® ST5010 Autostainer XL, Leica® Multistainer ST5020 (Leica Biosystems Nussloch), Autostainer Link48 (Agilant), and Lab Vision™ Autostainer 360-2D, Lab Vision™ Autostainer 480s (Thermo Fisher Scientific™).

[0035] In some embodiments, the sample used in the method is urine obtained from a subject.In some embodiments, the urine sample contains a detectable amount of cells that can be examined and analyzed for K17 expression.In some embodiments, the cells are from the bladder wall of the subject.In certain embodiments, the sample contains a detectable amount of cells that express K17.

[0036] After obtaining a suitable sample, the level of K17 expression in the sample can be determined using a variety of techniques known by those skilled in the art. In certain embodiments of the present disclosure, the level of K17 expression may be measured by immunohistochemistry (IHC), qRT-PCR, Northern blotting, Western blotting, enzyme-linked immunosorbent assay (ELISA), microarray analysis, or mass spectrometry. In certain embodiments, K17 is detected by a solution-phase protein assay (e.g., ELISA, microfluidics, flow cytometry). In certain embodiments, K17 expression is determined by immunohistochemical staining of a urine sample containing a detectable amount of bladder cells. In other embodiments, K17 expression is determined by detection of fluorescently labeled cells by flow cytometry or microfluidics-based detection methods. In certain embodiments, the fluorescently labeled cells express keratin 17. In some embodiments, the level of K17 expression is determined by detecting the presence of K17 mRNA or protein in the sample. In some embodiments, any amount of K17 mRNA or protein detected in the sample correlates with a diagnosis of bladder cancer. In other embodiments, the amount of K17 protein or mRNA in the sample should exceed the amount present in the control sample.

[0037] Any type of antibody may be used against the K17 antigen, including but not limited to anti-human K17 mouse monoclonal antibody [E3], polyclonal antibody against human K17, monoclonal or polyclonal antibody against mammalian K17 protein domain or epitope thereof. Also applicable are chimeric, single chain, Fc, Fab, Fab' and Fab2 fragments of immunoglobulin molecules, and Fab expression libraries against K17 protein. The antibodies include all classes, subclasses and types of immunoglobulins, as well as hapten molecules (e.g., nucleic acids, polymers) that bind to K17 protein.

[0038] In one embodiment, after incubation with the primary antibody, the samples are processed by an indirect avidin-biotin-based immunoperoxidase method using a biotinylated secondary antibody, developed, and counterstained with hematoxylin. Slides can then be analyzed for K17 expression.

[0039] In one embodiment, keratin expression is quantified by the PathSQ method, a manual semi-quantitative scoring system that quantifies the percentage of strongly stained cells blinded to the corresponding clinical data. In yet another embodiment, slides can be scored by the National Institutes of Health ImageJ 1.46, a Java-based image processing software that uses the DAB-hematoxylin (DAB-H) color deconvolution plug-in. See Schneider CA et al., Nat methods. (2012) 9:671-5, the entire contents of which are expressly incorporated herein by reference.

[0040] As shown in Table 1 of the present application, immunohistochemical analysis of K17 can be performed on formalin-fixed paraffin-embedded samples, in which normal bladder mucosa (control sample), PUNLMP, LG papillary urothelial carcinoma, and HG papillary urothelial carcinoma test samples are sectioned from hematoxylin and eosin stained tissue sections by laser capture microscopy to recover bladder parietal cells from each diagnostic category. The formalin-fixed paraffin-embedded tissue samples are then treated using an indirect immunoperoxidase method. Specifically, the samples are then incubated with a protease cocktail to promote the release of protein cross-links. After incubation, the tissue samples are deparaffinized and rehydrated at elevated temperature (>50°C). Antigen retrieval is then performed in a citrate buffer in an antigen retrieval device. Endogenous peroxidase is inhibited and the samples are labeled by incubation with a K17-specific antibody. After incubation with the primary antibody, the secondary antibody is added and the samples are developed and counterstained with hematoxylin. Negative controls from control tissue samples are processed using the same method.

[0041] In certain embodiments, immunohistochemical staining for K17 in negative control samples (benign) and bladder cancer samples (PUNLMP, LG, HG, transitional urothelial carcinoma) are scored by PathSQ, a manual semi-quantitative scoring system that quantifies the percentage of strongly stained [cancer] cells, blinded to the corresponding clinical data, and the fold increase from K17 expression levels in the control samples is calculated. See Figures 3C-D. For tissue biopsies, the PathSQ score should be at least 5%. In other embodiments, the PathSQ score is between 5% and 99%, 10% and 90%, 20% and 80%, 30% and 70%, or 40% and 60%. In other embodiments, one skilled in the art can determine the PathSQ score of a sample that corresponds to the presence of cancer.

[0042] In certain embodiments, K17 protein detection is performed by tissue microarray. For example, tissues containing normal bladder mucosa, PUNLMP, LG, HG or transitional urothelial bladder cancer cells can be obtained from paraffin blocks and arranged in tissue microarray blocks. In some embodiments, other sources of bladder cancer cells can be used as test samples. In particular, urine samples containing bladder parietal cells or urine samples that do not contain cells but contain proteins and / or mRNA from bladder parietal cells are ideal test samples. The control samples used to determine the fold increase of K17 in the sample from K17 in the control sample can be obtained from commercially available tissue microarray samples, such as tissue microarray samples obtained from HISTO-Array™. The tissue microarray slides used in the method can then be processed, i.e., deparaffinized with xylene and rehydrated using alcohol. In certain embodiments, the sample can be further processed by incubation with a citrate buffer, inhibiting endogenous peroxidases with hydrogen peroxide, or treating the sample with serum (e.g., bovine, human, donkey or horse serum) to inhibit non-specific binding. The sample is further labeled by incubation with a primary antibody against K17.

[0043] In some embodiments, and as shown in Figures 2, 3A-D through 5A-D, and Tables 2 and 3 of the present disclosure, K17 expression can be determined from a urine cytology sample by immunocytochemical analysis of detectable amounts of cells, where a natural urine sample is collected from the subject and bladder cells are isolated from the sample using techniques known to those skilled in the art, such as centrifugation or filtration. The isolated bladder cells are then fixed onto slides, labeled for K17 using standard immunocytochemical methods, counterstained for hematoxylin, and scored for K17 expression in both test and control urine samples using microscopy.

[0044] For example, Figure 2, as well as Figures 3A and B, show keratin 17 staining for control samples from cells isolated from natural urine obtained from healthy (non-cancerous) subjects. These cells show scattered ("control levels") of K17 staining. In comparison, Figures 3C-D show strong keratin 17 staining in urothelial cells from natural urine of a subject with bladder cancer (urothelial carcinoma). Figures 4A-D and Figures 5A-D are additional examples of negative control levels of keratin 17 expression, showing images of healthy squamous epithelial cells (Figures 4A-B) and healthy non-cancerous urothelial cells (Figures 4C-D), as well as non-cancerous bladder mucosal tissue (Figures 5A, B & D) and benign bladder mucosal tissue (Figure 5C).

[0045] In yet another embodiment, K17 mRNA expression can be determined using reverse transcriptase PCR (RT-PCR) in a qualitative format and by quantitative RT-PCR. More specifically, total RNA can be extracted from the sample using Trizol reagent. Reverse transcriptase-PCR can then be performed using methods known by those skilled in the art. For example, 1 μg of RNA can be used as a template for cDNA synthesis, and the cDNA template can then be mixed with gene-specific primers for K17 mRNA (i.e., forward 5'-3' primer sequence and reverse 3'-5' sequence). Examples of K17 primers are shown in Table 6. Probe sequences for detection can also be added (e.g., TaqMan™ from Applied Biosystems and SYBR® Green from Thermo Fisher Scientific systems are known in the art and useful for this purpose). Real-time quantitative PCR can then be performed for each sample, and the data obtained can be normalized to the control levels of K17 mRNA expression levels revealed in control or normal samples (i.e., benign bladder mucosa). See, e.g., Schmittgen and Livak, Nature protocols (2008) 3: 1101-1108. Other nucleic acid detection techniques, including, but not limited to, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), multiple displacement amplification (MDA), rolling circle amplification, ligase chain reaction (LCR), helicase-dependent amplification, ramification amplification, or branched DNA, can also be used to detect K17 mRNA or, in other circumstances, to quantify K17 mRNA expression.

[0046] In other embodiments where K17 protein is detected, the method of the present disclosure can be deployed using enzyme-linked immunosorbent assay (ELISA) to determine K17 expression in a sample. In addition, mass spectrometry and lateral flow protein detection assays can be used to detect and quantify K17 expression levels in a sample. For example, samples such as natural urine, as shown in Table 4 and Table 5, can be prepared by isolating bladder cells present in the sample by centrifugation. In certain embodiments, such as cell-free based diagnostic methods, a cell-free portion of the test sample can be isolated and tested for K17 protein expression using an ELISA system. For cell-free ELISA based detection methods, a microtiter plate is covered with a coating solution containing an anti-keratin 17 antibody, such as K17E3 monoclonal antibody, and incubated. The microtiter plate can then be blocked to prevent non-specific binding of K17 protein. A predetermined amount of the test sample is then added to each well of the microtiter plate and incubated. Then, a detection solution containing a K17 antibody (e.g., 2D10 monoclonal antibody labeled with a detectable marker such as peroxidase) is added to each well and incubated. In some embodiments, the wells may be washed before adding a detection substrate that can react with the detectable marker to emit a signal. After incubation, each plate is read by the emission of a detectable wavelength of light, such as 450 nm, to measure the amount of keratin 17 protein in the sample. In some embodiments, a control sample, such as a standard, is then run, and the amount of K17 protein in the test sample is then compared to the amount of K17 protein in the control, and the amount of K17 protein detected in conjunction with a cancer diagnosis that correlates to the amount of detected K17 protein exceeding the amount of the control or standard value can be inferred to determine whether the subject has bladder cancer.

[0047] In an embodiment, the method of the present disclosure can be deployed using flow cytometry. In one embodiment of the present disclosure, a microfluidic flow cytometry system is provided that can detect cells labeled with a detectable tag to detect and / or quantify K17 protein or mRNA in a sample, e.g., determine K17 expression or expression levels. As illustrated in Figures 8A-B, a cell-containing sample, such as a natural urine or bladder tissue sample, is obtained and prepared. In an embodiment, a urine sample is processed or prepared by fixing the cells with methanol and isolating the fixed cells by centrifugation. In some embodiments, the cells are suspended in phosphate buffered saline (PBS) and contacted with an anti-keratin 17 antibody (such as K17E3 monoclonal antibody (Abcam)) and incubated to bind to K17 present in the sample. The sample can then be washed to limit non-specific binding. The sample is then contacted with a secondary antibody containing a detectable marker, such as phycoerythrin, and incubated to specifically bind to the labeled K17 in the sample. The sample is then suspended in liquid and dispensed into a cartridge. The cartridge can then be read in a microfluidic detection system, such as Moxi Go system (Orflo Technologies), to determine the amount of K17 present in the sample.In one embodiment, a control sample, such as a reference sample, is run in the same manner, and the amount of K17 protein in the test sample is compared with the amount of K17 protein in the control to determine whether the subject has bladder cancer.In another embodiment, any amount of K17 detected in the test sample that exceeds a pre-defined standard amount is correlated with an increased level of K17 expression, and thus correlates with a diagnosis of bladder cancer.

[0048] In one embodiment of the current methodology of the present disclosure, the amount of K17 (protein or mRNA) in the sample is compared to a control amount of K17 present in either normal bladder cells or non-cancerous cells (or tissue or urine sample, or cell sample derived from either), or to the amount of K17 (protein or mRNA) in the control sample. The comparison can be made by any method known to those of skill in the art for detecting and quantifying protein or mRNA. In certain embodiments, the amount of K17 expression indicative of a subject having bladder cancer includes, but is not limited to, an increase of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 500%, 1000% or more over the amount of K17 expression in the control sample.

[0049] In other embodiments, K17 expression indicative of a subject having bladder cancer includes an increase of at least 1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 13-fold, 15-fold, 20-fold, 25-fold, 30-fold, or more compared to the amount of K17 expression exhibited by a control sample. In certain embodiments, K17 expression measured by PathSQ corresponding to an early stage of bladder cancer in a subject is demonstrated by K17 staining in the sample showing a 10-fold to 60-fold increase in K17 expression from the K17 expression in a control (benign) sample. In other embodiments, early stage bladder cancer in a subject is demonstrated by K17 staining in the test sample showing a 13-fold to 30-fold increase in K17 expression from the K17 expression in a control sample. In certain embodiments, early stage bladder cancer in a subject is demonstrated by K17 staining in the test sample showing an increase in K17 expression of about 13-fold, about 15-fold, or about 30-fold from the K17 expression in a control sample. In a preferred embodiment of the present disclosure, a test sample showing K17 expression with a PathSQ score of greater than 30 indicates that the subject has a lesion equivalent to or greater than low-grade papillary urothelial neoplasm (PUNLMP). In an embodiment where the test sample shows K17 expression with a PathSQ score between 30 and 35, the subject has low-grade papillary urothelial carcinoma (LG). In an embodiment where the test sample shows K17 expression with a PathSQ score of 29 or less, the subject has high-grade papillary urothelial carcinoma (HG). In a particular embodiment where the test sample shows K17 expression with a PathSQ score between 12 and 46, the subject has bladder cancer.

[0050] In yet other embodiments, K17 expression indicative of a subject having bladder cancer includes a test sample absorbance reading of at least 0.065, at least 0.070, at least 0.075, at least 0.080, at least 0.081, at least 0.085, at least 0.090, or higher. In some embodiments, K17 expression indicative of a subject having bladder cancer includes a test sample absorbance reading of between 0.065 and 0.2, 0.065 and 0.1, 0.075 and 0.2, 0.075 and 0.1, 0.08 and 0.2, 0.08 and 0.15, 0.08 and 0.1, 0.08 and 0.09. In certain embodiments, K17 expression indicative of a subject having bladder cancer is 0.065, 0.066, 0.067, 0.068, 0.069, 0.070, 0.071, 0.072, 0.073, 0.074, 0.075, 0.076, 0.077, 0.078, 0.079, 0.080, 0.081, 0.082, 0.083, 0.084, 0.085, 0.086, 0.087, 0.088, 0.089, 0. The absorbance reading of the test sample may include 0.090, 0.091, 0.092, 0.093, 0.094, 0.095, 0.096, 0.097, 0.098, 0.099, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, or higher.

[0051] The method can also be used to diagnose the presence of other K17-mediated diseases.Accordingly, the methods of the present disclosure are not intended to be limited by the foregoing description or the examples set forth below. EXAMPLES

[0052] Example 1 Materials and Methods. The study performed included the analysis of 84 formalin-fixed paraffin-embedded surgical tissue masses (Table 1). All surgical tissue masses were obtained from subjects (patients) suspected of having bladder cancer. The cases in these studies included the following diagnostic categories: benign urothelium (n=12), low-grade papillary urothelial neoplasm (PUNLMP) (n=9), low-grade papillary urothelial carcinoma (n=23), high-grade papillary urothelial carcinoma (n=14), and transitional / urothelial carcinoma (n=26). In some cases, tissue masses were selected by a pathologist who performed a histologic review (DCM) of hematoxylin-eosin stained sections from either transurethral resection (TURBT), bladder biopsy, or cystectomy specimens to confirm that the diagnostic tissue originally reported was adequately represented in the residual tissue mass. Cases with insufficient residual tissue or in which the diagnostic tissue could not be preserved for later clinical use were not included in the study. Bladder cancer was classified by (i) clinical stage and (ii) tumor grade (Table 1) as described in Edge SB and Compton CC. Annals of surgical oncology. (2010) 17:1471-4, the entire contents of which are incorporated herein by reference.

[0053] Immunohistochemistry. Immunohistochemical analysis was performed by indirect immunoperoxidase method as previously described in L. Escobar-Hoyos et al., Cancer Res. 2015 Sep 1;75(17):3650-62, specifically incorporated herein by reference in its entirety. Briefly, after incubation at 60°C, tissue sections were deparaffinized with xylene and rehydrated with alcohol. Antigen retrieval was performed in citrate buffer at 120°C for 10 min in an antigen retrieval apparatus. Endogenous peroxidase was inhibited with 3% hydrogen peroxide and sections were labeled with anti-human K17 mouse monoclonal antibody [E3] (Abcam®) or and Ki-67 for bladder cancer (clone MIB-1, 1:100 dilution, DAKO, Carpentaria, CA, USA) overnight at 4°C. Other antibodies used in this method that bind and label K17 include anti-cytokeratin 17 antibody [EP1623] cytoskeletal marker ab109725 (Abcam®), keratin 17 (D73C7) rabbit mAb (Cell Signaling Technology®), guinea pig polyclonal (IgG) against human K17 LS-C22650 (LifeSpan BioSciences), and rabbit polyclonal against keratin 17 orb22505 (Biorbyt). After application of the primary antibody, a biotinylated horse secondary antibody (RTUVectastain ABC kit; Vector Laboratories) was added to the samples. Revelation was performed with 3,3'-diaminobenzidine (DAB; Dako) and counterstained with hematoxylin. Negative controls were performed using the same concentrations of subclass-matched immunoglobulins for all assays. Immunohistochemical staining for K17 in bladder cancer was scored by PathSQ, a manual semiquantitative scoring system that quantifies the percentage of strongly stained tumor cells blinded to the corresponding clinical data. PathSQ is based on the proportion of tumor cells with strong (i.e., 2+) staining. Ki-67 staining was scored as the percentage of tumor cells with positive nuclear staining.

[0054] K17 detection by microfluidics. In addition to immunostaining of urine cytology samples, K17 positive cells in urine could be detected by applying flow cytometry or microfluidic systems that can detect cells labeled using fluorescent tags. One example of such a system is the Moxi Go system (Orflo Technologies), which utilizes a disposable microfluidic cartridge and a reader that can count the number of cells based on size (Coulter method) and fluorescent signal (phycoerythrin). Detection of K17 positive cells using the Moxi Go microfluidic device (Orflo Technologies) was examined using K17 positive (HeLa) and K17 negative (C33) cells as controls. Cells (approximately 1 × 10 5 The cells) were fixed with 70% methanol for 1 hour, pelleted by centrifugation at 1000g for 5 minutes, and then washed with 1 mL of 1x PBS. The cells were then pelleted and resuspended in 100 μL of phosphate buffer solution (PBS). 50 μL of cells were then mixed with 50 μL of keratin 17 antibody (E3, Abcam) and incubated for 2 hours at room temperature. The cells were then pelleted, washed once with 1 mL of PBS, and pelleted again. The pelleted cells were then resuspended in 50 μL of phycoerythrin (PE)-labeled anti-mouse antibody (1:100 dilution in PBS, Abcam) and incubated for 30 minutes at room temperature. The cells were washed twice with 1 mL of PBS and finally resuspended in 500 μL of PBS. The labeled cells were read on the Moxi Go microfluidic device by adding 75 μL of each sample onto the microfluidic cartridge and reading it on the device.

[0055] Detection of K17 using ELISA. Keratin 17 protein in the acellular component of urine was examined by ELISA on natural urine samples. Natural urine was centrifuged at 1000 g for 5 min to pellet cells. After centrifugation, the acellular liquid portion of urine was decanted and used for K17 ELISA examination. For ELISA, high-binding microtiter plates were covered with 100 μL per well of K17 coating solution using K17E3 monoclonal antibody (Nordic MUBio, Systeren, Netherlands) at a concentration of 2 μg / mL in 1×PBS and incubated overnight at 4° C. Each plate was then blocked with 200 μL per well of blocking buffer (Superblock PBS blocking solution, Thermo Fisher Scientific) and incubated at room temperature (RT) for 1 h.

[0056] Samples were tested in duplicate by adding 100 μL urine sample and 100 μL assay buffer (10% calf serum in 1×PBS) to each well and incubated for 1 h at RT. Samples were tested on the same plate along with 3-fold serial dilutions of K17 standard (keratin 17 recombinant protein, Abcam) starting at 1 μg / mL. Each plate was then washed 4 times with 400 μL wash buffer (0.05% Tween-20 in 1×PBS) and samples were incubated for 1 h at RT with 100 μL detection antibody solution containing peroxidase-labeled K17 2D10 monoclonal antibody (K17 2D10 monoclonal antibody-HRP, US Biologicals) at a final concentration of 1 μg / mL in assay buffer. Cells were then washed and 100 μL TMB substrate (Pierce) was added to each well and samples were incubated for 20 min at room temperature in the dark. The reaction was then stopped with 100 μL of stop solution (VWR) and the plates were read at 450 nm.

[0057] Scoring of keratin protein expression. Slides were scored by the National Institutes of Health ImageJ 1.46 (see Schneider CA et al., Nat methods. (2012) 9:671-5, the contents of which are incorporated herein by reference), a Java-based image processing software that uses the DAB-hematoxylin (DAB-H) color deconvolution plug-in (see Ruifrok AC, Johnston DA, Anal Quant Cytol Histol. (2001) 23:291-9, the entire contents of which are incorporated herein by reference), and by a manual semi-quantitative scoring system that quantifies the percentage of strongly positively stained cells (PathSQ) blinded to the corresponding clinical data. In one embodiment, the units of measurement for immunohistochemical analysis were the PathSQ core score and the average PathSQ score of all core scores. Differences in scores between diagnostic categories were determined by Kruskal-Wallis test or Wilcoxon rank sum test (not shown). Receiver operating characteristic curves and areas under the curve were calculated to evaluate the ability of biomarkers to discriminate between various diagnostic categories based on logistic regression analysis models. Optimal cut-off values ​​from the receiver operating characteristic curves were determined using Youden's index. See Youden WJ. Cancer. (1950) 3:32-5, the contents of which are incorporated herein by reference.

[0058] Example 2 Detection of keratin 17 in immunohistochemically stained cells as a diagnostic method for bladder cancer. To determine the diagnostic value of K17 in one or more diagnostic categories of early stage bladder cancer, immunohistochemical staining for K17 (Table 1) was performed on archived tissue samples from bladder cancer patients from four diagnostic categories: benign bladder mucosa (benign), low-grade papillary urothelial neoplasm (PUNLMP), low-grade papillary urothelial carcinoma (LG), high-grade papillary urothelial carcinoma (HG), and urothelial carcinoma.

[0059] K17 staining was only weakly detected in benign bladder mucosa (mean PathSQ score of 2.08), but was strongly present in all diagnostic categories of bladder cancer examined. For example, Table 1 shows that the mean PathSQ scores of HG and PUNLMP samples ranged from 12.3831954 to 45.1781993, respectively (Table 1), whereas the mean PathSQ score of control (non-cancerous) samples was about 2. Furthermore, when comparing the control samples shown in Figures 3A-D and Figures 4A-D and 5A-D with the stained cancerous samples in Figures 6A-D to 7A-D, it can be seen that strong (increased) keratin 17 staining is only present in samples obtained from subjects with bladder cancer. Taken together, these results indicate that K17 is a strong diagnostic marker for bladder cancer, such as urothelial carcinoma of the bladder (p<0.001).

[0060] Of the urine samples tested that contained bladder parietal cells, K17 staining was detected in 5 / 5 samples that were suspected or positive for bladder cancer. See Table 2. As shown in Table 2, no K17 expression was observed in cases that were negative for malignancy. These data clearly show that K17 was positively stained in all samples from subjects with bladder cancer (T3-T7). In contrast, urine samples from non-cancerous patients with detectable levels of bladder cells did not show K17 labeling (N1, N7, N8). These results provide experimental support that K17 detection is a diagnostic marker for bladder cancer.

[0061] The immunohistochemistry results obtained with bladder tissue samples supported the hypothesis that K17 detection could be a highly sensitive and specific marker that improves the accuracy of the diagnosis of bladder cancer in urine cytology specimens. As shown in Table 3 of the present disclosure, K17 detection shows sensitivity and specificity as a diagnostic marker for bladder cancer. For example, as the results of multiplex testing performed on natural urine samples containing bladder cells (Figure 2), K17 shows a sensitivity of 93% (number of true K17 positive samples / number of K17 positive samples detected) and a specificity of 91% (number of true K17 negative samples / number of K17 negative samples detected). For study 2, most false positive samples were performed by direct spotting the samples, whereas in study 3, they were performed using the ThinPrep sample preparation method. In summary, the data shown indicate that the presence of bladder cancer in subjects can be detected with high sensitivity and specificity by immunostaining bladder cells in urine samples to detect K17.

[0062] Example 3 Keratin 17 detection in cells by an automated instrument-based detection method. In addition to immunostaining of urine cytology samples, K17-positive cells in urine cytology samples can be detected by flow cytometry and a microfluidic device system that can detect fluorescently tagged proteins. Here, labeled cancer cells known to be positive for K17 (HeLa, Figure 8A) and control cells that are K17-negative (C33, Figure 8B) were isolated, fluorescently labeled with K17 antibody, and K17 expression was analyzed on a microfluidic detection device as described above. As can be seen in Figures 8A-8B, there are significantly more K17-labeled cells in HeLa (90.4% K17-positive) compared to control C33 cells (35.1% K17-positive). Taken together, these data indicate that detection of K17-positive cells can be efficiently achieved using a combination of K17 immunostaining and a microfluidic device.

[0063] Example 4 Cell-free K17-based diagnostic method. As shown in Table 4 and Table 5, test samples consisting of natural urine lacking bladder cells can be analyzed for K17 protein expression to determine whether a subject has bladder cancer. For example, Table 4 shows results obtained using an ELISA-based K17 detection method. Here, control samples (i.e., standards) with known amounts of K17 protein were analyzed to determine the sensitivity of the ELISA system test for cell-free samples. The cutoff value for increased K17 protein expression was determined from the negative control (zero standard) where the detectable signal was 3 times the standard deviation of the blank from the mean of the negative control. For example, the following formula was used: Mean of ELISA readings of negative control + [3 times the standard deviation of the negative control]. Based on the data, the limit of sensitivity of the ELISA is approximately 1 ng / mL of K17 protein.

[0064] Table 5 shows data from 21 cell-free urine samples (6 cancerous, 15 benign, positive samples in bold) obtained from subjects. Here, each of the 21 test samples was tested on an ELISA system to detect K17 expression in the cell-free portion of natural urine for each sample and compared to the control levels in Table 4. Test samples showing average optical density (OD) readings of 0.081 or greater with %CVs less than 20% correlated well with the presence of bladder cancer in the subject. For example, 67% of the cancer samples (4 out of 6 samples) showed OD readings of 0.081 or greater, while none of the non-cancerous control samples showed detectable levels of K17 greater than 0.078 OD. Since this was a model experiment and not an actual patient test, the lack of detectable K17 signal in two of the cancer samples may be due to uncontrollable assay factors such as time of sample collection, such that natural urine first thing in the morning accumulates K17 protein at higher levels than natural urine at other times of the day, or due to sample handling. Collectively, these data demonstrate that the present disclosure provides a method by which the measurement of detectable levels of K17 protein in acellular urine samples can be used to detect the presence of bladder cancer (e.g., diagnose bladder cancer) in a subject using ELISA or other protein-based detection platforms such as mass spectrometry or lateral flow instrumentation.

[0065] [Table 1]

[0066] [Table 2]

[0067] [Table 3]

[0068] [Table 4]

[0069]

Table 5

[0070]

Table 6

Claims

1. A method for detecting the expression of target keratin 17, A step of receiving a bladder biopsy tissue sample from a subject, wherein the sample contains multiple bladder cells, A step for detecting keratin 17 (K17) protein expression in the sample, comprising contacting the bladder cells with an anti-K17 antibody, and detecting the binding between the K17 protein and the anti-K17 antibody in the bladder cells by immunohistochemical analysis, A step of scoring the bladder cells for K17 expression by K17 staining, wherein if the K17 staining is 2+, the subject has bladder cancer cells. Methods that include... Methods used.

2. The method according to claim 1, wherein the plurality of bladder cells are transitional epithelial cells, connective tissue cells, muscle cells, adipocytes, or a combination thereof.

3. A method for detecting target bladder cancer, A step of receiving a bladder biopsy tissue sample from a subject, wherein the sample contains multiple bladder cells, A step of detecting the expression of K17 protein in the bladder cells of the sample, wherein the detection includes a step of contacting the bladder cells with an anti-K17 antibody, and a step of detecting the binding between the K17 protein and the anti-K17 antibody in the bladder cells, A step of receiving a control bladder tissue sample containing multiple bladder cells, A step of detecting K17 protein expression in the control bladder tissue sample, wherein the detection includes a step of contacting the control bladder tissue sample with an anti-K17 antibody, and a step of detecting the binding between K17 and the anti-K17 antibody in the control bladder tissue sample. If a 10- to 60-fold increase in K17 protein expression is detected in the bladder biopsy tissue sample compared to the amount of K17 expression in the control bladder tissue sample, the subject is detected to have bladder cancer. Methods that include...

4. The method according to claim 3, wherein the bladder cancer is urothelial carcinoma.

5. The method according to claim 4, wherein the urothelial carcinoma is a low-grade papillary neoplasm, a low-grade papillary urothelial carcinoma, a high-grade papillary urothelial carcinoma, or a transitional urothelial carcinoma.

6. The method according to claim 3, wherein the anti-K17 antibody is an anti-human K17 mouse monoclonal antibody [E3].

7. The method according to claim 3, wherein K17 protein expression in the sample is detected by a method selected from the group consisting of immunohistochemistry, tissue microarray, immunocytochemical analysis, Western blotting, flow cytometry, microfluidic detection, and ELISA.

8. The method according to claim 7, wherein K17 protein expression in the sample is detected by immunohistochemistry or immunocytochemical analysis.

9. The method according to claim 3, wherein the detection step includes the use of a handheld or mobile detection device.

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