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Urine Cytology and Urinary Biomarkers
Chapter 6
Urine Cytology and Urinary Biomarkers Ok-Jun Lee, Ho-Won Kang and Seok Joong Yun Chungbuk National University, Cheongju, Republic of Korea
Chapter Outline Introduction Urine Cytology Normal Cytology Neoplasms of the Urinary Bladder Urinary Biomarkers
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Soluble Urinary Biomarkers Cell-Based Markers Conclusions References
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INTRODUCTION Diagnosis of bladder cancer currently depends on urine cytology and cystoscopy. Cystoscopy is the gold-standard method for diagnosis, but ideally most of patients would be preferable to avoid cystoscopy because of its invasive nature. The contact between bladder tumors and urine suggests that urine should be a valuable source of biomarkers for diagnosis of bladder cancer. However, although urinary cytology is an established diagnostic tool, only a limited number of biomarkers have yet been used in clinical practice, and it remains a challenge to urologists to develop convenient and accurate assays for measurement of biomarkers in urine. Urinary biomarkers could have a role in detection of bladder cancer, which is generally diagnosed following presentation with hematuria or voiding symptoms. Cystoscopy remains the standard tool for diagnosis of bladder cancer, but identification of urinary biomarkers with high negative-predictive value would be helpful to reduce the need for cystoscopy. Urinary biomarkers with high positive-predictive values could also be used for follow-up of patients with bladder cancer for accurate and early diagnosis of cancer recurrence or progression. Many reports have been published on experimental assessment of tumor DNA, RNA, microRNA, methylation, and proteins as urinary biomarkers for Bladder Cancer. DOI: http://dx.doi.org/10.1016/B978-0-12-809939-1.00006-0 © 2018 Elsevier Inc. All rights reserved.
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bladder cancer. However, only a few markers have progressed to clinical trials. In general, these experimental urinary markers are not close to clinical application. We therefore describe here existing urinary markers and tests, including urinary cytology, nuclear matrix protein-22 (NMP22), bladder tumor antigen (BTA), and BCLA-4.
URINE CYTOLOGY Urine cytology is an important noninvasive approach for the detection of urothelial carcinoma. The combined use of urine cytology with cystoscopic biopsy has a major role in the diagnosis of urothelial carcinoma. Despite many attempts to develop tests with better diagnostic accuracy, urine cytology remains one of the best ways to diagnose a variety of cancers of the urinary tract. Conventional urine smear has been popular as a diagnostic procedure since the 1940s [1]. An improved technique, liquid-based cytology (LBC), was developed in the 1990s as an alternative to conventional cytology for processing gynecological specimens [2]. LBC proceeds via cell enrichment to reduce levels of background inflammatory cells and blood cells, after which a uniform layer of cells is deposited on a microscope slide by an automated process, facilitating the identification of malignant cells. LBC has now become the preferred method for urine diagnosis, but differences with conventional cytology have been reported [3 5]. Although differences occur according to the LBC method that is adopted, results suggest that LBC and conventional cytology are comparable for the morphological assessment of tumor cells, and no significant improvement in sensitivity is observed with LBC [6,7]. However, the automation and excellent slide quality associated with LBC techniques are notable advantages of this approach. Regardless of the difference between conventional smear and LBC preparations, it should be noted that urine cytological diagnosis is very effective in highgrade urothelial carcinomas, but less effective in low-grade urothelial tumors. The mean sensitivity of urine cytology is B50% for detecting urothelial carcinoma (B80% in high-grade carcinomas and B25% in lowgrade lesions) [8]. To make any cytological diagnosis, it is necessary to differentiate between urothelial cancer cells and detached, normal urothelial cells, which requires an understanding of the cytological differences between these cell types.
Normal Cytology The two types of urine specimen are voided urine and instrumented urine [9]. Instrumented samples are generated from cystoscopy, bladder wash-outs, catheterization, and direct brushing (Figs. 6.1 and 6.2). Voided and instrumented samples, as well as samples that are not suitable for diagnostic purposes, have the following cytological characteristics:
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FIGURE 6.1 Normal (noncancerous) voided-urine liquid-based cytology. Most voided-urine samples reveal a mixture of urothelial cells and squamous cells. Most of the urothelial cells are basal or intermediate.
FIGURE 6.2 Liquid-based cytology of a sample prepared by bladder washing. (A) Superficial umbrella cells are large and have abundant cytoplasm and prominent nucleoli. Binucleation and multinucleation are common. (B) Basal urothelial cells are tightly clustered. These cells show round nuclei with evenly distributed granular chromatin.
1. Voided-urine samples G Samples have variable cellularity and are often sparsely cellular G Usually consist of isolated individual cells; tight clusters of urothelial cells are uncommon G Contain intermediate and superficial (umbrella) cells G Contain bland urothelial cells with homogeneous, granular, or finely vacuolated cytoplasm, round nuclei, and small nucleoli G Contain squamous cells G Contain epithelial cells of the prostate or seminal vesicles in men (rare)
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2. Instrumented samples G Samples have high cellularity G Contain clusters of urothelial cells, often quite large G Contain basal, intermediate, and superficial cells 3. Samples that are not suitable for diagnostic purposes G Samples with low cellularity G Samples with obscuring inflammation G Samples consisting of blood only G Samples with marked degenerative changes
Neoplasms of the Urinary Bladder Multiple grading schemes have been used to define urothelial neoplasms, but the 2004 classification system of the World Health Organization (WHO) and the International Society of Urological Pathology (ISUP) is now widely accepted [10,11]. Cytological diagnoses based on the WHO/ISUP 2004 classification can be divided into two groups: low-grade urothelial lesions and high-grade urothelial carcinomas. Cytological diagnosis according to the WHO/ISUP 2004 classification system defines the following types of urothelial neoplasm: 1. Low-grade urothelial lesions G Papillary urothelial neoplasm of low malignant potential (PUNLMP) G Papillary carcinoma, low grade 2. High-grade urothelial lesions G Papillary carcinoma, high grade G Carcinoma in situ (CIS)
Low-Grade Urothelial Lesions In the WHO/ISUP 2004 classification system, a PUNLMP consists of delicate papillae with little or no fusion, and its covering urothelium is usually thickening but shows minimal. Nuclei are normal in size or slightly enlarged, without notable nuclear atypia. Low-grade papillary urothelial carcinoma shows papillae that are mostly delicate and separate, with some fusion. The nuclei tend to be uniformly enlarged, with mild atypia (Fig. 6.3). The cytological features of these lesions are similar, but PUNLMPs are more difficult to recognize cytologically than low-grade carcinomas because PUNLMPs have less atypia. Diagnosis of low-grade urothelial lesions is made by the following cytological criteria: 1. Papillary fragments with fibrovascular cores rarely identified 2. Cytological features that suggest underlying tumors G Increased homogeneity of urothelial cytoplasm G Mild increase in nuclear-to-cytoplasmic ratio G Irregular nuclear membranes
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FIGURE 6.3 Features of a low-grade urothelial lesion. (A) Voided-urine liquid-based cytology shows an increased nuclear-to-cytoplasmic ratio compared with noncancerous samples and irregular nuclear outlines, but these changes are subtle. (B) Subsequent histology of a low-grade papillary urothelial carcinoma from the same patient.
High-Grade Urothelial Lesions Histologically, high-grade urothelial carcinoma is defined as a tumor with moderate-to-marked cytological atypia and disordered growth. Urothelial CIS is most often seen in association with high-grade papillary carcinoma or invasive urothelial carcinoma. De novo CIS accounts for only 1% 3% of newly diagnosed cases of bladder cancer [12,13]. Urothelial CIS is a flat and noninvasive lesion that is confined to the epithelium and shows marked cytological atypia. The cytological distinction between high-grade urothelial carcinoma and CIS is very difficult to determine, and they are considered together in the following description of cytological diagnostic features (Figs. 6.4 and 6.5): 1. 2. 3. 4. 5. 6. 7.
Increased cellularity Individual cells and cohesive groups Enlarged nuclei with marked hyperchromasia High nuclear-to-cytoplasmic ratio Irregular nuclear outlines Coarsely granular chromatin Necrosis and/or red blood cells in the background
Other Malignant Lesions Other primary cancers of the urinary tract include squamous-cell carcinoma, adenocarcinoma, urachal carcinoma, and small-cell carcinoma. The limited diagnostic accuracy of urinary cytology, and the infrequent occurrence of these tumors, means that accurate diagnosis is challenging, and they are likely to be interpreted as urothelial carcinomas. Despite these limitations, urinary cytology is valuable for diagnosis of rare tumors when combined with histological confirmation.
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FIGURE 6.4 Features of high-grade urothelial carcinoma. (A) Voided-urine liquid-based cytology shows loosely cohesive clusters and individually dispersed cells with enlarged nuclei with marked hyperchromasia. Irregular nuclear outlines are evident. (B) Histology of a high-grade papillary urothelial carcinoma from the same patient.
FIGURE 6.5 Features of urothelial carcinoma in situ. (A) Voided-urine, liquid-based cytology shows loosely cohesive urothelial cells with enlarged nuclei with coarsely granular chromatin, high nuclear-to-cytoplasmic ratios, and irregular nuclear outlines. Based on cytological features, high-grade urothelial carcinoma and carcinoma in situ are indistinguishable. (B) Histological features of carcinoma in situ from the same patient.
URINARY BIOMARKERS Urinary biomarkers have been investigated for roles as potential alternatives or adjuncts to standard tests for the initial diagnosis of bladder cancer or the identification of recurrent disease. The FDA has approved qualitative BTA Stat (Polymedco, Cortland, NY, USA), quantitative BTA TRAK (Polymedco), quantitative NMP22 (Alere, Waltham, MA, USA), qualitative NMP22 BladderChek Test (Alere), and UroVysion (Abbot Laboratoris, Abbott Park, IL, USA) for diagnosis and follow-up, whereas ImmunoCyt/UCyt (DiagnoCure, Que´bec, Canada) is approved for follow-up [14,15]. The qualitative NMP22 and BTA tests can be performed as point-of-care tests, whereas the others are performed in a laboratory. These biomarker tests are summarized in Table 6.1.
TABLE 6.1 Currently Available Urinary Biomarker Assays Urinary Biomarker
Assay Type
Sensitivity Mean (Range)
Specificity Mean (Range)
Advantages
Disadvantages
NMP22
Immunochromatic assay or sandwich ELISA
67.5% (31.0% 91.7%)
74.4% (5.1% 94.3%)
Unaffected by BCG, detects lowgrade tumors
High false-positive rate, no clearly defined cutoff value
BTA stat (complement factor H-related protein)
Dipstick immunoassay
68.7% (52.8% 89.0%)
73.7% (54.0% 93.0%)
Sensitivity and specificity
Influenced by benign genitourinary conditions
BTA TRAK
Sandwich ELISA
62.0% (17.0% 77.5%)
73.6% (50.5% 95.0%)
BLCA-4
ELISA
93% (90% 95%)
97% (95% 98%)
Sensitivity and specificity
Needs further study
UroVysion (chromosomal aneuploidy)
Multicolor, multiprobe FISH
77% (73% 81%)
98% (96% 100%)
Unaffected by BCG
Labor-intensive and expensive
ImmunoCyt
Immunocytochemistry
58.2% (38.5% 86.1%)
78.8% (73.0% 83.9%)
Survivin
Bio-dot test, ELISA
64% (35% 83%)
93% (88% 93%)
UBC (cytokeratins)
Sandwich ELISA or point-ofcare test
60.7% (48.7% 70.0%)
83.8% (72.0% 95.0%)
CYFRA 21-1 (cytokeratin fragment)
Immunoradiometric assay or electrochemiluminescent immunoassay
74.2% (69.0% 79.3%)
91.3% (88.6% 94.0%)
High interobserver variability Sensitivity and specificity
Needs further study Influenced by benign genitourinary conditions and vesical instillations
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Soluble Urinary Biomarkers Nuclear Matrix Protein-22 Nuclear matrix proteins (NMPs) form part of the internal structural framework of the cell nucleus. This nonchromatin structure supports the nuclear shape, organizes DNA, and has important roles in DNA replication, transcription, and gene expression. NMP22 is a nuclear mitotic protein that is involved in the proper distribution of chromatin to daughter cells during cellular replication [14]. Healthy individuals have low levels of urinary NMP22, whereas patients with bladder cancer may have levels that are 25-fold higher. Urinary elevation of the ubiquitous cellular protein NMP22 is associated with increased cell death [16,17]. The standard test for NMP22 is a quantitative microtiter sandwich ELISA that uses two antibodies, each of which recognizes a different epitope of the protein. This assay should be carried out in a laboratory by trained personnel, so it is not a point-of-care test. Because the NMP22 test is quantitative, it is important to note the cutoff value that is used in any particular study. Although the manufacturer’s recommended cutoff value is 10 U/mL, variable limits ranging from 3.6 U/mL to 27 U/mL have been applied, depending on the optimum sensitivity and specificity determined by the receiver operating curve. A qualitative point-of-care NMP22 assay has now become available, which involves addition of four drops of urine to a proprietary immunochromatographic-assay device; the results can be read 30 50 minutes later [18 20]. Results from various studies have demonstrated that the sensitivity of the NMP22 ELISA ranges from 47% to 100%, and is typically 60% 70%. Notably, these studies mostly involved patients without a history of bladder cancer. The sensitivity of the NMP22 assay increases with tumor size, grade, and stage. NMP22 measurement has a lower sensitivity to detect recurrent tumors than primary tumors because recurrent tumors often are smaller. The reported specificity of the ELISA assay is between 60% and 90%, depending on the cutoff value used. The NMP22 test has higher false-positive rates (33% 50%) in patients with urolithiasis, inflammation, benign prostatic hyperplasia, or urinary tract infections. Because of the high false-positive rate, the assay cannot be recommended without cystoscopy [15,21 23]. Both tests for NMP22 have been approved by the FDA for surveillance of bladder cancer, and BladderChek test is also approved for diagnosis in high-risk patients. Bladder Tumor Antigen BTA is also known as human complement factor H-related protein [24,25]. The BTA TRAK and BTA stat assays both measure levels of the protein in urine. BTA stat is a qualitative immunoassay that can be performed at the point-of-care within several minutes. BTA TRAK is a quantitative test that is
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performed in a laboratory [20]. The sensitivity of both the BTA stat and BTA TRAK tests varies with tumor grade and stage and the cutoff limit used on the test. The specificity of BTA stat and BTA TRAK tests in healthy individuals is $90%. However, these tests have lower specificity in patients with urinary tract infections, urinary calculi, nephritis, renal stones, cystitis, benign prostatic hyperplasia, hematuria, or proteinuria. Complement factor H is present in human serum at high concentrations (0.5 mg/mL), so the BTA stat test might give false-positive results in many benign conditions that cause hematuria. The manufacturer recommends that the BTA stat and BTA TRAK tests should only be used when information is available for the clinical evaluation of the patient and alongside other diagnostic procedures, and that the tests should not be used for screening [14,18,22,26,27]. The FDA has approved both BTA stat and BTA TRAK tests for use in the management of bladder cancer in combination with cystoscopy.
BLCA-4 Six NMPs that are specifically expressed in bladder cancer have been identified, including BLCA-4. Overexpression of BLCA-4 seems to increase the cellular growth rate and also causes cells to express a more tumorigenic phenotype [28]. Urinary levels of BLCA-4 are analyzed by ELISA, which has a reported sensitivity of 89% 96.4% and specificity of 95% 100%. These levels of sensitivity and specificity for detecting bladder cancer are promising but should be confirmed in a larger trial. In addition to BCLA-4, BLCA-1 is a potentially useful marker for bladder cancer that is currently under investigation [27,29,30]. Cytokeratins Cytokeratins are proteins of cytoskeletal intermediate filaments, and their main function is to enable cells to withstand mechanical stress. In humans, 20 different cytokeratin isotypes have been identified. Cytokeratins 8, 18, 19, and 20 have been associated with bladder [31]. Cytokeratin fragment 21-1 (CYFRA 21-1) is a soluble fragment of cytokeratin 19 that can currently be measured either by a solid-phase sandwich immunoradiometric assay or an electrochemiluminescent immunoassay. On the basis of a limited number of studies, these CYFRA 21-1 assays have been estimated to have sensitivities of 75% 97% and specificities of 67% 71% for the detection of bladder cancer [18]. Although CYFRA 21-1 seems to be the best cytokeratin-associated antigen for use as a urinary biomarker for bladder cancer, it does not perform well as a marker for low-stage bladder cancer, and urinary CYFRA21-1 levels are strongly influenced by benign urological diseases and therapeutic intravesical instillations [32]. The urinary bladder cancer tests UBC Rapid (Concile, Freiburg, Germany) and UBC-ELISA detects fragments of cytokeratins 8 and 18 in urine. UBC
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Rapid is a point-of-care test, whereas UBC-ELISA is a 2 hour sandwich ELISA test. The results of several retrospective cohort and case control studies have demonstrated that the sensitivities of UBC tests for detection of bladder cancer (both primary and recurrent) are between 35% and 79%. In addition to low overall sensitivity, these tests have especially low sensitivity for detection of both low-grade and low-stage tumors [33,34].
Survivin Survivin is a member of a family of proteins that regulate cell death, known as the inhibitor of apoptosis family [35]. Overexpression of survivin inhibits extrinsic and intrinsic pathways of apoptosis. In bladder cancer, survivin is expressed in urine, and its expression is associated with disease recurrence, stage, progression, and mortality [32,35]. Immunohistochemical studies have shown that survivin expression is elevated in bladder cancer tissues, and that nuclear localization of survivin may be related to disease-free survival. The survivin levels in urine have been measured by immunoblotting with the BioDot microfiltration detection system, in which urine samples are blotted as dots on nitrocellulose membranes, and survivin present in the samples is detected using a rabbit polyclonal antisurvivin antibody and standard dotblot detection reagents. In studies with limited numbers of patients with bladder cancer, the survivin dot-blot assay had 100% sensitivity. Its specificities among healthy individuals and patients with benign genitourinary conditions were 100% and 87%, respectively. Results from a limited number of studies have shown that survivin may be a useful marker for the detection of bladder cancer. However, more cohort studies are needed to evaluate this marker [18,35,36]. HA HAase Test The HA HAase test is a combination of two tests that measure urinary levels of hyaluronic acid (HA) and hyaluronidase (HAase) [37]. HA is a glycosaminoglycan that promotes tumor metastasis, and its concentration is elevated in several tumors. Small, angiogenic fragments of HA are generated when HAase degrades HA. HYAL1-type HAase is the major tumor-derived HAase secreted by tumor cells, and a molecular determinant of bladder tumor growth and invasion. The HA test is an ELISA-like assay based on the competitive-binding principle that measures urinary HA levels (ng/mL), which are then normalized to total urinary protein (mg/mL). Urinary HA levels $500 ng/mg total protein (cutoff limit) constitute a positive HA test result. The HAase test is also an ELISA-like assay that measures urinary HAase activity (mU/mL), which is normalized to total urinary protein. Urinary HAase levels $10 mU/mg total protein constitute a positive HAase test result. The HA test detects bladder cancer, regardless of the tumor grade, whereas the HAase test preferentially detects bladder tumors of grades G2
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and G3. The HA HAase test is a combination of the HA and HAase tests. For a positive HA HAase test result, either or both individual tests must be positive [18,37]. In case control and cohort studies, the sensitivity of the HA HAase test varies between 83% and 94% for detection of both primary and recurrent bladder tumors. The sensitivities of the test for detection of G1, G2, and G3 tumors are 75% 90%, 84% 100%, and 92% 100%, respectively. The overall specificity of the HA HAase test in healthy individuals, patients with benign genitourinary conditions, and patients with a history of bladder cancer varies between 77% and 84%. This test may also be useful in screening a high-risk population for bladder cancer. However, the test is not commercially available [14,18,23,29].
Cell-Based Markers uCyt Immunocytology is based on the visualization of tumor-associated antigens in urothelial carcinoma cells via binding of labeled monoclonal antibodies [38]. uCyt, formerly known as ImmunoCyt, is a commercially available immunocytological assay that detects bladder cancer cells through the use of fluorescein-labeled monoclonal antibodies M344 and LDQ10 that are directed against sulfated-mucin glycoproteins, and a Texas red-linked monoclonal antibody 19A211 against glycosylated forms of high-molecular-weight carcinoembryonic antigens [34]. The sensitivity of uCyt varies from 38% to 100%, and the specificity ranges from 75% to 90%. The uCyt assay is sensitive and reasonably specific, and is suitable for use in combination with voided-urine cytology. However, issues associated with a steep learning curve, observerdependent inference, and quality control should be addressed to improve the general acceptability of this test [18,28]. The assay has been approved by the FDA for surveillance of patients with a history of bladder cancer. DD23 DD23 is a monoclonal antibody that detects a protein dimer expressed on bladder cancer cells [39]. This immunoglobulin G1 murine monoclonal antibody resulted from the immunization of a BALB/c mouse with fresh human bladder cancer. The DD23 antibody is used in an alkaline phosphataseconjugated immunohistochemical assay and to visualize tumor cells in urine specimens. In a case control study, the sensitivity for detection of bladder cancer was 85%, with a specificity of 95% [14,18,40]. UroVysion UroVysion is a multitarget, multicolor fluorescence in situ hybridization (FISH) assay that involves staining of exfoliated cells in urine with four denatured,
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fluorescent, centromeric chromosome-enumeration probes [41]. These probes detect chromosome 3, chromosome 7, chromosome 17, and the chromosome 9p21 locus. After staining, cells are observed under a fluorescence microscope. The criteria for detection of bladder cancer by the UroVysion test are observation of five or more cells with a gain of two or more chromosomes, $10 cells with a gain of one chromosome, or $ 20% of cells with a loss of the 9p21 locus. Currently, however, no universally accepted criteria exist to determine the positivity of a FISH test. In case control studies, the sensitivity of the UroVysion test ranges from 69% to 87%. The test has a high sensitivity to detect high-grade and high-stage tumors (83% 97%) and also to detect CIS (B100%). The specificity of the UroVysion test, as reported in various studies, is high (89% 96%) in patients with a variety of benign genitourinary conditions. A potential advantage of a FISH-based method is the ability to detect occult disease not visible on urethrocystoscopy. A false-positive FISH test result can predict future recurrence within 3 12 months in 41% 89% of patients. Another advantage of FISH is that it is unaffected by Bacillus Calmette-Gue´rin (BCG) therapy and can be used for surveillance in patients who have been treated with intravesical BCG instillation. A disadvantage is that this test is laborintensive and has a learning curve for reliable operation [18,29,35,42]. The UroVysion test has been approved by the FDA both for monitoring patients with a history of bladder cancer and for detection of bladder cancer in patients with hematuria.
CONCLUSIONS Urinary cytology is undoubtedly effective for the detection of urothelial carcinomas, especially in high-grade cancer. Although technological improvements have been introduced to the process, urinary cytology has so far been limited to a mean sensitivity of B50% for the detection of urothelial carcinoma. Urinary biomarker assays that can be carried out in the clinic or laboratory each have strengths and weaknesses, and further validation studies or development of new assays will be needed before they can replace or fully complement urinary cytology. For routine use, these assays should reach quality criteria established by guidelines for the development of accurate biomarker tests [43]. Therefore urologists and scientists should contribute to the standardization of detection techniques and conduct prospective randomized trials to determine the most appropriate urinary biomarkers for the detection of bladder cancer.
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[41] Sarosdy MF, Schellhammer P, Bokinsky G, Kahn P, Chao R, Yore L, et al. Clinical evaluation of a multi-target fluorescent in situ hybridization assay for detection of bladder cancer. J Urol 2002;168:1950 4. [42] Lokeshwar VB, Selzer MG. Urinary bladder tumor markers. Urol Oncol 2006;528 37. [43] Shariat SF, Lotan Y, Vickers A, Karakiewicz PI, Schmitz-Drager BJ, Goebell PJ, et al. Statistical consideration for clinical biomarker research in bladder cancer. Urol Oncol 2010;28:389 400.
Urine Cytology and Urinary Biomarkers Ok-Jun Lee, Ho-Won Kang and Seok Joong Yun Chungbuk National University, Cheongju, Republic of Korea
Chapter Outline Introduction Urine Cytology Normal Cytology Neoplasms of the Urinary Bladder Urinary Biomarkers
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Soluble Urinary Biomarkers Cell-Based Markers Conclusions References
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INTRODUCTION Diagnosis of bladder cancer currently depends on urine cytology and cystoscopy. Cystoscopy is the gold-standard method for diagnosis, but ideally most of patients would be preferable to avoid cystoscopy because of its invasive nature. The contact between bladder tumors and urine suggests that urine should be a valuable source of biomarkers for diagnosis of bladder cancer. However, although urinary cytology is an established diagnostic tool, only a limited number of biomarkers have yet been used in clinical practice, and it remains a challenge to urologists to develop convenient and accurate assays for measurement of biomarkers in urine. Urinary biomarkers could have a role in detection of bladder cancer, which is generally diagnosed following presentation with hematuria or voiding symptoms. Cystoscopy remains the standard tool for diagnosis of bladder cancer, but identification of urinary biomarkers with high negative-predictive value would be helpful to reduce the need for cystoscopy. Urinary biomarkers with high positive-predictive values could also be used for follow-up of patients with bladder cancer for accurate and early diagnosis of cancer recurrence or progression. Many reports have been published on experimental assessment of tumor DNA, RNA, microRNA, methylation, and proteins as urinary biomarkers for Bladder Cancer. DOI: http://dx.doi.org/10.1016/B978-0-12-809939-1.00006-0 © 2018 Elsevier Inc. All rights reserved.
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bladder cancer. However, only a few markers have progressed to clinical trials. In general, these experimental urinary markers are not close to clinical application. We therefore describe here existing urinary markers and tests, including urinary cytology, nuclear matrix protein-22 (NMP22), bladder tumor antigen (BTA), and BCLA-4.
URINE CYTOLOGY Urine cytology is an important noninvasive approach for the detection of urothelial carcinoma. The combined use of urine cytology with cystoscopic biopsy has a major role in the diagnosis of urothelial carcinoma. Despite many attempts to develop tests with better diagnostic accuracy, urine cytology remains one of the best ways to diagnose a variety of cancers of the urinary tract. Conventional urine smear has been popular as a diagnostic procedure since the 1940s [1]. An improved technique, liquid-based cytology (LBC), was developed in the 1990s as an alternative to conventional cytology for processing gynecological specimens [2]. LBC proceeds via cell enrichment to reduce levels of background inflammatory cells and blood cells, after which a uniform layer of cells is deposited on a microscope slide by an automated process, facilitating the identification of malignant cells. LBC has now become the preferred method for urine diagnosis, but differences with conventional cytology have been reported [3 5]. Although differences occur according to the LBC method that is adopted, results suggest that LBC and conventional cytology are comparable for the morphological assessment of tumor cells, and no significant improvement in sensitivity is observed with LBC [6,7]. However, the automation and excellent slide quality associated with LBC techniques are notable advantages of this approach. Regardless of the difference between conventional smear and LBC preparations, it should be noted that urine cytological diagnosis is very effective in highgrade urothelial carcinomas, but less effective in low-grade urothelial tumors. The mean sensitivity of urine cytology is B50% for detecting urothelial carcinoma (B80% in high-grade carcinomas and B25% in lowgrade lesions) [8]. To make any cytological diagnosis, it is necessary to differentiate between urothelial cancer cells and detached, normal urothelial cells, which requires an understanding of the cytological differences between these cell types.
Normal Cytology The two types of urine specimen are voided urine and instrumented urine [9]. Instrumented samples are generated from cystoscopy, bladder wash-outs, catheterization, and direct brushing (Figs. 6.1 and 6.2). Voided and instrumented samples, as well as samples that are not suitable for diagnostic purposes, have the following cytological characteristics:
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FIGURE 6.1 Normal (noncancerous) voided-urine liquid-based cytology. Most voided-urine samples reveal a mixture of urothelial cells and squamous cells. Most of the urothelial cells are basal or intermediate.
FIGURE 6.2 Liquid-based cytology of a sample prepared by bladder washing. (A) Superficial umbrella cells are large and have abundant cytoplasm and prominent nucleoli. Binucleation and multinucleation are common. (B) Basal urothelial cells are tightly clustered. These cells show round nuclei with evenly distributed granular chromatin.
1. Voided-urine samples G Samples have variable cellularity and are often sparsely cellular G Usually consist of isolated individual cells; tight clusters of urothelial cells are uncommon G Contain intermediate and superficial (umbrella) cells G Contain bland urothelial cells with homogeneous, granular, or finely vacuolated cytoplasm, round nuclei, and small nucleoli G Contain squamous cells G Contain epithelial cells of the prostate or seminal vesicles in men (rare)
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2. Instrumented samples G Samples have high cellularity G Contain clusters of urothelial cells, often quite large G Contain basal, intermediate, and superficial cells 3. Samples that are not suitable for diagnostic purposes G Samples with low cellularity G Samples with obscuring inflammation G Samples consisting of blood only G Samples with marked degenerative changes
Neoplasms of the Urinary Bladder Multiple grading schemes have been used to define urothelial neoplasms, but the 2004 classification system of the World Health Organization (WHO) and the International Society of Urological Pathology (ISUP) is now widely accepted [10,11]. Cytological diagnoses based on the WHO/ISUP 2004 classification can be divided into two groups: low-grade urothelial lesions and high-grade urothelial carcinomas. Cytological diagnosis according to the WHO/ISUP 2004 classification system defines the following types of urothelial neoplasm: 1. Low-grade urothelial lesions G Papillary urothelial neoplasm of low malignant potential (PUNLMP) G Papillary carcinoma, low grade 2. High-grade urothelial lesions G Papillary carcinoma, high grade G Carcinoma in situ (CIS)
Low-Grade Urothelial Lesions In the WHO/ISUP 2004 classification system, a PUNLMP consists of delicate papillae with little or no fusion, and its covering urothelium is usually thickening but shows minimal. Nuclei are normal in size or slightly enlarged, without notable nuclear atypia. Low-grade papillary urothelial carcinoma shows papillae that are mostly delicate and separate, with some fusion. The nuclei tend to be uniformly enlarged, with mild atypia (Fig. 6.3). The cytological features of these lesions are similar, but PUNLMPs are more difficult to recognize cytologically than low-grade carcinomas because PUNLMPs have less atypia. Diagnosis of low-grade urothelial lesions is made by the following cytological criteria: 1. Papillary fragments with fibrovascular cores rarely identified 2. Cytological features that suggest underlying tumors G Increased homogeneity of urothelial cytoplasm G Mild increase in nuclear-to-cytoplasmic ratio G Irregular nuclear membranes
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FIGURE 6.3 Features of a low-grade urothelial lesion. (A) Voided-urine liquid-based cytology shows an increased nuclear-to-cytoplasmic ratio compared with noncancerous samples and irregular nuclear outlines, but these changes are subtle. (B) Subsequent histology of a low-grade papillary urothelial carcinoma from the same patient.
High-Grade Urothelial Lesions Histologically, high-grade urothelial carcinoma is defined as a tumor with moderate-to-marked cytological atypia and disordered growth. Urothelial CIS is most often seen in association with high-grade papillary carcinoma or invasive urothelial carcinoma. De novo CIS accounts for only 1% 3% of newly diagnosed cases of bladder cancer [12,13]. Urothelial CIS is a flat and noninvasive lesion that is confined to the epithelium and shows marked cytological atypia. The cytological distinction between high-grade urothelial carcinoma and CIS is very difficult to determine, and they are considered together in the following description of cytological diagnostic features (Figs. 6.4 and 6.5): 1. 2. 3. 4. 5. 6. 7.
Increased cellularity Individual cells and cohesive groups Enlarged nuclei with marked hyperchromasia High nuclear-to-cytoplasmic ratio Irregular nuclear outlines Coarsely granular chromatin Necrosis and/or red blood cells in the background
Other Malignant Lesions Other primary cancers of the urinary tract include squamous-cell carcinoma, adenocarcinoma, urachal carcinoma, and small-cell carcinoma. The limited diagnostic accuracy of urinary cytology, and the infrequent occurrence of these tumors, means that accurate diagnosis is challenging, and they are likely to be interpreted as urothelial carcinomas. Despite these limitations, urinary cytology is valuable for diagnosis of rare tumors when combined with histological confirmation.
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FIGURE 6.4 Features of high-grade urothelial carcinoma. (A) Voided-urine liquid-based cytology shows loosely cohesive clusters and individually dispersed cells with enlarged nuclei with marked hyperchromasia. Irregular nuclear outlines are evident. (B) Histology of a high-grade papillary urothelial carcinoma from the same patient.
FIGURE 6.5 Features of urothelial carcinoma in situ. (A) Voided-urine, liquid-based cytology shows loosely cohesive urothelial cells with enlarged nuclei with coarsely granular chromatin, high nuclear-to-cytoplasmic ratios, and irregular nuclear outlines. Based on cytological features, high-grade urothelial carcinoma and carcinoma in situ are indistinguishable. (B) Histological features of carcinoma in situ from the same patient.
URINARY BIOMARKERS Urinary biomarkers have been investigated for roles as potential alternatives or adjuncts to standard tests for the initial diagnosis of bladder cancer or the identification of recurrent disease. The FDA has approved qualitative BTA Stat (Polymedco, Cortland, NY, USA), quantitative BTA TRAK (Polymedco), quantitative NMP22 (Alere, Waltham, MA, USA), qualitative NMP22 BladderChek Test (Alere), and UroVysion (Abbot Laboratoris, Abbott Park, IL, USA) for diagnosis and follow-up, whereas ImmunoCyt/UCyt (DiagnoCure, Que´bec, Canada) is approved for follow-up [14,15]. The qualitative NMP22 and BTA tests can be performed as point-of-care tests, whereas the others are performed in a laboratory. These biomarker tests are summarized in Table 6.1.
TABLE 6.1 Currently Available Urinary Biomarker Assays Urinary Biomarker
Assay Type
Sensitivity Mean (Range)
Specificity Mean (Range)
Advantages
Disadvantages
NMP22
Immunochromatic assay or sandwich ELISA
67.5% (31.0% 91.7%)
74.4% (5.1% 94.3%)
Unaffected by BCG, detects lowgrade tumors
High false-positive rate, no clearly defined cutoff value
BTA stat (complement factor H-related protein)
Dipstick immunoassay
68.7% (52.8% 89.0%)
73.7% (54.0% 93.0%)
Sensitivity and specificity
Influenced by benign genitourinary conditions
BTA TRAK
Sandwich ELISA
62.0% (17.0% 77.5%)
73.6% (50.5% 95.0%)
BLCA-4
ELISA
93% (90% 95%)
97% (95% 98%)
Sensitivity and specificity
Needs further study
UroVysion (chromosomal aneuploidy)
Multicolor, multiprobe FISH
77% (73% 81%)
98% (96% 100%)
Unaffected by BCG
Labor-intensive and expensive
ImmunoCyt
Immunocytochemistry
58.2% (38.5% 86.1%)
78.8% (73.0% 83.9%)
Survivin
Bio-dot test, ELISA
64% (35% 83%)
93% (88% 93%)
UBC (cytokeratins)
Sandwich ELISA or point-ofcare test
60.7% (48.7% 70.0%)
83.8% (72.0% 95.0%)
CYFRA 21-1 (cytokeratin fragment)
Immunoradiometric assay or electrochemiluminescent immunoassay
74.2% (69.0% 79.3%)
91.3% (88.6% 94.0%)
High interobserver variability Sensitivity and specificity
Needs further study Influenced by benign genitourinary conditions and vesical instillations
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Soluble Urinary Biomarkers Nuclear Matrix Protein-22 Nuclear matrix proteins (NMPs) form part of the internal structural framework of the cell nucleus. This nonchromatin structure supports the nuclear shape, organizes DNA, and has important roles in DNA replication, transcription, and gene expression. NMP22 is a nuclear mitotic protein that is involved in the proper distribution of chromatin to daughter cells during cellular replication [14]. Healthy individuals have low levels of urinary NMP22, whereas patients with bladder cancer may have levels that are 25-fold higher. Urinary elevation of the ubiquitous cellular protein NMP22 is associated with increased cell death [16,17]. The standard test for NMP22 is a quantitative microtiter sandwich ELISA that uses two antibodies, each of which recognizes a different epitope of the protein. This assay should be carried out in a laboratory by trained personnel, so it is not a point-of-care test. Because the NMP22 test is quantitative, it is important to note the cutoff value that is used in any particular study. Although the manufacturer’s recommended cutoff value is 10 U/mL, variable limits ranging from 3.6 U/mL to 27 U/mL have been applied, depending on the optimum sensitivity and specificity determined by the receiver operating curve. A qualitative point-of-care NMP22 assay has now become available, which involves addition of four drops of urine to a proprietary immunochromatographic-assay device; the results can be read 30 50 minutes later [18 20]. Results from various studies have demonstrated that the sensitivity of the NMP22 ELISA ranges from 47% to 100%, and is typically 60% 70%. Notably, these studies mostly involved patients without a history of bladder cancer. The sensitivity of the NMP22 assay increases with tumor size, grade, and stage. NMP22 measurement has a lower sensitivity to detect recurrent tumors than primary tumors because recurrent tumors often are smaller. The reported specificity of the ELISA assay is between 60% and 90%, depending on the cutoff value used. The NMP22 test has higher false-positive rates (33% 50%) in patients with urolithiasis, inflammation, benign prostatic hyperplasia, or urinary tract infections. Because of the high false-positive rate, the assay cannot be recommended without cystoscopy [15,21 23]. Both tests for NMP22 have been approved by the FDA for surveillance of bladder cancer, and BladderChek test is also approved for diagnosis in high-risk patients. Bladder Tumor Antigen BTA is also known as human complement factor H-related protein [24,25]. The BTA TRAK and BTA stat assays both measure levels of the protein in urine. BTA stat is a qualitative immunoassay that can be performed at the point-of-care within several minutes. BTA TRAK is a quantitative test that is
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performed in a laboratory [20]. The sensitivity of both the BTA stat and BTA TRAK tests varies with tumor grade and stage and the cutoff limit used on the test. The specificity of BTA stat and BTA TRAK tests in healthy individuals is $90%. However, these tests have lower specificity in patients with urinary tract infections, urinary calculi, nephritis, renal stones, cystitis, benign prostatic hyperplasia, hematuria, or proteinuria. Complement factor H is present in human serum at high concentrations (0.5 mg/mL), so the BTA stat test might give false-positive results in many benign conditions that cause hematuria. The manufacturer recommends that the BTA stat and BTA TRAK tests should only be used when information is available for the clinical evaluation of the patient and alongside other diagnostic procedures, and that the tests should not be used for screening [14,18,22,26,27]. The FDA has approved both BTA stat and BTA TRAK tests for use in the management of bladder cancer in combination with cystoscopy.
BLCA-4 Six NMPs that are specifically expressed in bladder cancer have been identified, including BLCA-4. Overexpression of BLCA-4 seems to increase the cellular growth rate and also causes cells to express a more tumorigenic phenotype [28]. Urinary levels of BLCA-4 are analyzed by ELISA, which has a reported sensitivity of 89% 96.4% and specificity of 95% 100%. These levels of sensitivity and specificity for detecting bladder cancer are promising but should be confirmed in a larger trial. In addition to BCLA-4, BLCA-1 is a potentially useful marker for bladder cancer that is currently under investigation [27,29,30]. Cytokeratins Cytokeratins are proteins of cytoskeletal intermediate filaments, and their main function is to enable cells to withstand mechanical stress. In humans, 20 different cytokeratin isotypes have been identified. Cytokeratins 8, 18, 19, and 20 have been associated with bladder [31]. Cytokeratin fragment 21-1 (CYFRA 21-1) is a soluble fragment of cytokeratin 19 that can currently be measured either by a solid-phase sandwich immunoradiometric assay or an electrochemiluminescent immunoassay. On the basis of a limited number of studies, these CYFRA 21-1 assays have been estimated to have sensitivities of 75% 97% and specificities of 67% 71% for the detection of bladder cancer [18]. Although CYFRA 21-1 seems to be the best cytokeratin-associated antigen for use as a urinary biomarker for bladder cancer, it does not perform well as a marker for low-stage bladder cancer, and urinary CYFRA21-1 levels are strongly influenced by benign urological diseases and therapeutic intravesical instillations [32]. The urinary bladder cancer tests UBC Rapid (Concile, Freiburg, Germany) and UBC-ELISA detects fragments of cytokeratins 8 and 18 in urine. UBC
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Rapid is a point-of-care test, whereas UBC-ELISA is a 2 hour sandwich ELISA test. The results of several retrospective cohort and case control studies have demonstrated that the sensitivities of UBC tests for detection of bladder cancer (both primary and recurrent) are between 35% and 79%. In addition to low overall sensitivity, these tests have especially low sensitivity for detection of both low-grade and low-stage tumors [33,34].
Survivin Survivin is a member of a family of proteins that regulate cell death, known as the inhibitor of apoptosis family [35]. Overexpression of survivin inhibits extrinsic and intrinsic pathways of apoptosis. In bladder cancer, survivin is expressed in urine, and its expression is associated with disease recurrence, stage, progression, and mortality [32,35]. Immunohistochemical studies have shown that survivin expression is elevated in bladder cancer tissues, and that nuclear localization of survivin may be related to disease-free survival. The survivin levels in urine have been measured by immunoblotting with the BioDot microfiltration detection system, in which urine samples are blotted as dots on nitrocellulose membranes, and survivin present in the samples is detected using a rabbit polyclonal antisurvivin antibody and standard dotblot detection reagents. In studies with limited numbers of patients with bladder cancer, the survivin dot-blot assay had 100% sensitivity. Its specificities among healthy individuals and patients with benign genitourinary conditions were 100% and 87%, respectively. Results from a limited number of studies have shown that survivin may be a useful marker for the detection of bladder cancer. However, more cohort studies are needed to evaluate this marker [18,35,36]. HA HAase Test The HA HAase test is a combination of two tests that measure urinary levels of hyaluronic acid (HA) and hyaluronidase (HAase) [37]. HA is a glycosaminoglycan that promotes tumor metastasis, and its concentration is elevated in several tumors. Small, angiogenic fragments of HA are generated when HAase degrades HA. HYAL1-type HAase is the major tumor-derived HAase secreted by tumor cells, and a molecular determinant of bladder tumor growth and invasion. The HA test is an ELISA-like assay based on the competitive-binding principle that measures urinary HA levels (ng/mL), which are then normalized to total urinary protein (mg/mL). Urinary HA levels $500 ng/mg total protein (cutoff limit) constitute a positive HA test result. The HAase test is also an ELISA-like assay that measures urinary HAase activity (mU/mL), which is normalized to total urinary protein. Urinary HAase levels $10 mU/mg total protein constitute a positive HAase test result. The HA test detects bladder cancer, regardless of the tumor grade, whereas the HAase test preferentially detects bladder tumors of grades G2
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and G3. The HA HAase test is a combination of the HA and HAase tests. For a positive HA HAase test result, either or both individual tests must be positive [18,37]. In case control and cohort studies, the sensitivity of the HA HAase test varies between 83% and 94% for detection of both primary and recurrent bladder tumors. The sensitivities of the test for detection of G1, G2, and G3 tumors are 75% 90%, 84% 100%, and 92% 100%, respectively. The overall specificity of the HA HAase test in healthy individuals, patients with benign genitourinary conditions, and patients with a history of bladder cancer varies between 77% and 84%. This test may also be useful in screening a high-risk population for bladder cancer. However, the test is not commercially available [14,18,23,29].
Cell-Based Markers uCyt Immunocytology is based on the visualization of tumor-associated antigens in urothelial carcinoma cells via binding of labeled monoclonal antibodies [38]. uCyt, formerly known as ImmunoCyt, is a commercially available immunocytological assay that detects bladder cancer cells through the use of fluorescein-labeled monoclonal antibodies M344 and LDQ10 that are directed against sulfated-mucin glycoproteins, and a Texas red-linked monoclonal antibody 19A211 against glycosylated forms of high-molecular-weight carcinoembryonic antigens [34]. The sensitivity of uCyt varies from 38% to 100%, and the specificity ranges from 75% to 90%. The uCyt assay is sensitive and reasonably specific, and is suitable for use in combination with voided-urine cytology. However, issues associated with a steep learning curve, observerdependent inference, and quality control should be addressed to improve the general acceptability of this test [18,28]. The assay has been approved by the FDA for surveillance of patients with a history of bladder cancer. DD23 DD23 is a monoclonal antibody that detects a protein dimer expressed on bladder cancer cells [39]. This immunoglobulin G1 murine monoclonal antibody resulted from the immunization of a BALB/c mouse with fresh human bladder cancer. The DD23 antibody is used in an alkaline phosphataseconjugated immunohistochemical assay and to visualize tumor cells in urine specimens. In a case control study, the sensitivity for detection of bladder cancer was 85%, with a specificity of 95% [14,18,40]. UroVysion UroVysion is a multitarget, multicolor fluorescence in situ hybridization (FISH) assay that involves staining of exfoliated cells in urine with four denatured,
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fluorescent, centromeric chromosome-enumeration probes [41]. These probes detect chromosome 3, chromosome 7, chromosome 17, and the chromosome 9p21 locus. After staining, cells are observed under a fluorescence microscope. The criteria for detection of bladder cancer by the UroVysion test are observation of five or more cells with a gain of two or more chromosomes, $10 cells with a gain of one chromosome, or $ 20% of cells with a loss of the 9p21 locus. Currently, however, no universally accepted criteria exist to determine the positivity of a FISH test. In case control studies, the sensitivity of the UroVysion test ranges from 69% to 87%. The test has a high sensitivity to detect high-grade and high-stage tumors (83% 97%) and also to detect CIS (B100%). The specificity of the UroVysion test, as reported in various studies, is high (89% 96%) in patients with a variety of benign genitourinary conditions. A potential advantage of a FISH-based method is the ability to detect occult disease not visible on urethrocystoscopy. A false-positive FISH test result can predict future recurrence within 3 12 months in 41% 89% of patients. Another advantage of FISH is that it is unaffected by Bacillus Calmette-Gue´rin (BCG) therapy and can be used for surveillance in patients who have been treated with intravesical BCG instillation. A disadvantage is that this test is laborintensive and has a learning curve for reliable operation [18,29,35,42]. The UroVysion test has been approved by the FDA both for monitoring patients with a history of bladder cancer and for detection of bladder cancer in patients with hematuria.
CONCLUSIONS Urinary cytology is undoubtedly effective for the detection of urothelial carcinomas, especially in high-grade cancer. Although technological improvements have been introduced to the process, urinary cytology has so far been limited to a mean sensitivity of B50% for the detection of urothelial carcinoma. Urinary biomarker assays that can be carried out in the clinic or laboratory each have strengths and weaknesses, and further validation studies or development of new assays will be needed before they can replace or fully complement urinary cytology. For routine use, these assays should reach quality criteria established by guidelines for the development of accurate biomarker tests [43]. Therefore urologists and scientists should contribute to the standardization of detection techniques and conduct prospective randomized trials to determine the most appropriate urinary biomarkers for the detection of bladder cancer.
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