A Newly Developed Uroplakin II Antibody With Increased Sensitivity in Urothelial Carcinoma of the Bladder

Transcription

1 A Newly Developed Uroplakin II Antibody With Increased Sensitivity in Urothelial Carcinoma of the Bladder Laura L. Hoang, PhD; David E. Tacha, PhD; Weimin Qi, MD, PhD; Charlie Yu, MD; Ryan E. Bremer, PhD; Joseph Chu, BS; Thomas S. Haas, DO; Liang Cheng, MD Context. Uroplakin II is a 15-kDa protein component of the urothelial plaques that enhance the permeability barrier and strength of the urothelium. Studies have shown uroplakin II messenger RNA to be expressed in bladder cancer tissues and peripheral blood of patients with urothelial carcinoma. Little is known about the protein expression of uroplakin II in urothelial carcinoma, possibly because of the absence of a commercially available uroplakin II antibody. Pathologists have used the uroplakin III antibody (AU1) to identify tumors of urothelial origin; however, the use of AU1 is limited because of its poor sensitivity. Objectives. To evaluate a newly developed mouse monoclonal uroplakin II antibody (BC21) in urothelial carcinoma and to compare it with previously developed mouse monoclonal uroplakin III (BC17 and AU1). Design. Uroplakin II and III antibodies were optimized for staining using a horseradish peroxidase-polymer detection system and were visualized with 3,3 0 -diaminobenzidine. Results. BC21, BC17, and AU1 demonstrated sensitivities in urothelial carcinoma of the bladder of 79% (44 of 56), 55% (31 of 56) (P ¼.002), and 34% (19 of 56) (P,.001), respectively. Subsequently, the increased staining sensitivity and intensity of BC21, compared with BC17, was validated in a larger study (134 of 174; 77% and 94 of 174; 54%, respectively) (P,.001). BC21 was found to be highly specific when evaluated in various normal and neoplastic tissues, including prostatic and renal carcinomas. Conclusions. The mouse monoclonal uroplakin II antibody (BC21) demonstrated superior sensitivity and specificity in urothelial carcinoma, compared with uroplakin III (BC17 and AU1), suggesting its advantages in the differential diagnosis of urothelial carcinoma and in the detection of tumors of unknown origin. (Arch Pathol Lab Med. 2014;138: ; doi: / arpa oa) Bladder cancer is the sixth most commonly occurring carcinoma in the United States, with new cases expected to be diagnosed in This cancer is particularly associated with high recurrence and progression rates. About 70% of patients with superficial bladder cancer will experience tumor recurrence, and 10% to 15% of that subpopulation will eventually progress to muscle invasion. 2 Accepted for publication September 13, From the Department of Research and Development, Biocare Medical, LLC, Concord, California (Drs Hoang, Tacha, Qi, Yu, and Bremer and Mr Chu); the Department of Pathology, Mercy Hospital and Trauma Center, Mercy Health System, Janesville, Wisconsin (Dr Haas); and the Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis (Dr Cheng). Dr Tacha is a founder, chief scientific officer, and stockholder of Biocare Medical, LLC. Drs Hoang, Qi, Yu, and Bremer and Mr Chu are employees of Biocare Medical. Dr Haas serves as a consultant for Biocare Medical. Dr Cheng is a member of the Board of Advisory of Biocare Medical. The authors have no other relevant financial interest in the products or companies described in this article. Presented in part at the annual meeting of the United States and Canadian Academy of Pathology; March 6th, 2013; Baltimore, Maryland. Reprints: David Tacha, PhD, Department of Research and Development, Biocare Medical, LLC, 4040 Pike Lane, Concord, CA ( dtacha@biocare.net). Early diagnosis, when the disease is still at a localized stage, increases the chance of successful treatment. The survival rate for in situ urinary bladder cancer is 97%. 3 Tissue-based biomarkers for early diagnosis of bladder cancer are a major clinical need. Urothelial carcinoma (UC) originates in the urothelium and accounts for more than 90% to 95% of all bladder tumors. Adenocarcinoma and squamous cell carcinoma make up about 1% and 5%, respectively, of bladder carcinomas. Biomarkers expressed in the urothelium, such as uroplakins, could be valuable markers of UC of the bladder. Pathologists have used uroplakin (UP) III (clone AU1) to establish the urothelial origin of the tumor; however, the use of AU1 has been limited because of its poor sensitivity. The asymmetric-unit membrane plaques of the urothelium maintain the blood-urine barrier and prevent cells from rupturing during bladder distention. The molecular constituents of those plaques comprise four transmembrane UP isoforms: UPIa, UPIb, UPII, and UPIII, which are specific differentiation products of the urothelial cells. The UPII is a 15-kDa protein component of the urothelial plaques that enhance the permeability barrier and strength of the urothelium. 4 The expression of UPII is aberrant in urothelial carcinoma of the bladder, and thus, it may be a useful marker Arch Pathol Lab Med Vol 138, July 2014 Uroplakin II in Urothelial Bladder Carcinoma Hoang et al 943

2 Figure 1. A, Western blot of uroplakin II antibody (clone BC21) (1 lg/ml) with uroplakin II protein (left lanes) and uroplakin III protein (right lanes). B, Western blot of uroplakin III antibody (clone BC17) (1 lg/ml) with uroplakin II protein (left lanes) and uroplakin III protein (right lanes). for the diagnosis of bladder carcinoma. 5 Wu et al 5 reported that UPII mrna might be a promising diagnostic marker for bladder carcinoma and micrometastases of the bladder carcinoma in the pelvic lymph nodes. The UPII messenger RNA (mrna) was detected in 19 of 19 (100%) bladder tumor tissue specimens and 15 of 16 (93.8%) pelvic lymph node samples with metastasis. On the other hand, UPII mrna was detected in only 6 of 66 cases (9%) of pelvic lymph node samples without metastasis. Therefore, positive expression of UPII mrna may indicate nodal metastases from bladder carcinoma. It is important to determine the nodal metastases in patients with bladder carcinoma after radical cystectomy because that subpopulation of patients urgently needs postoperative chemotherapy to survive. The authors concluded that the detection of UPII mrna might improve clinical outcome following radical cystectomy by providing helpful information in the diagnosis and management of bladder carcinoma. It is desirable, therefore, to develop a UPII antibody to detect UPII protein expression in the tissues of patients with UC. Studies have shown UPII mrna to be expressed in both bladder tissues and peripheral blood of patients with primary and metastatic UC, suggesting its potential role as a biomarker for UC The clinical usefulness of UPII was recognized from these studies based solely on its mrna data. Additional investigations characterizing the protein localization of UPII in bladder carcinoma were warranted, especially because protein molecules are typically more stable than mrna molecules. One study employed a pan-up antibody, which reacted with UPIb, UPII, and UPIII isoforms, to demonstrate the persistent expression of UP in advanced UC; however, the specific UPII protein level could not be determined using that pan-up antibody. 11 Little is known about the protein expression of UPII in UC, possibly because of the absence of a specific UPII antibody. A clear need exists for a sensitive and specific UPII antibody for use in cancer diagnosis. The UPIII antibodies have been previously developed as markers for carcinomas of urothelial origin. Our research goal was to evaluate the sensitivity and specificity of a newly developed mouse monoclonal UPII antibody (BC21) in UC and to compare it with previously developed mouse monoclonal UPIII (BC17) and UPIII (AU1) antibodies. MATERIALS AND METHODS BC21 Production and Purification A mouse monoclonal UPII antibody (BC21) (Biocare Medical, Concord, California) was developed by immunizing BALB/c mice with a recombinant human UPII protein, corresponding to amino acids 26 to 155, obtained by Escherichia coli expression. The immune reactivity to UPII was assessed by direct enzyme-linked immunosorbent assay on the recombinant UPII protein. Mice with the highest titer were chosen for developing hybridomas by cell fusion. A hybridoma clone demonstrating the best reactivity to UPII on human tissues was chosen and designated BC21. The isotype of the BC21 clone was determined to be mouse immunoglobulin (Ig) G1/j. The BC21 antibody was produced by large-scale tissue culture of the hybridoma cells and by ascites in BALB/c mice. The supernatant and ascites fluid were collected, and the antibody was purified with a protein-a affinity column. Western Blotting The specific reactivity of BC21 to human UPII protein and BC17 to human UPIII protein was demonstrated by Western blotting. 944 Arch Pathol Lab Med Vol 138, July 2014 Uroplakin II in Urothelial Bladder Carcinoma Hoang et al

3 Table 1. Comparison of Uroplakin II (UPII; Clone BC21) and Uroplakin III (UPIII; Clones BC17 and AU1) on Urothelial Carcinoma Antibody Grade The recombinant UPII and UPIII proteins were subjected to protein gel electrophoresis, using 4% to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis with Tris-glycine buffer, and were transferred onto nitrocellulose filters in Tris-glycine buffer. Proteins on the blots were visualized by incubating BC21 or BC17 for 60 minutes at room temperature after blocking with blocking buffer, followed by incubating with peroxidase-conjugated goat anti-rabbit immunoglobulins. Tissue Specimens Positive A preliminary comparison between UPII (BC21) and UPIII (BC17 and AU1) was performed using a tissue microarray (TMA) with 56 cases of UC. A second TMA of 118 cases of UC (with confirmed diagnosis, grade, and stage) and various normal and neoplastic tissues were tested with BC21 in comparison with BC17. The final diagnostic sensitivity for UPII and UPIII was based on the combined data from 2 groups (56 cases and 118 cases, n ¼ 174). The TMA used in the 56 case study had a single, 1.5-mm core for each case. The TMA used in the 118 case study had a single, 1-mm core for each case. The UPII was also tested on TMAs and whole tissue of various normal tissue types (n ¼ 37) and neoplastic tissues (n ¼ 461) for specificity. The TMAs were either constructed in house or purchased commercially. Immunohistochemistry Negative UPII (BC21), n ¼ 56 I, II, III 44 (79) 12 (21) UPIII (BC17), n ¼ 56 I, II, III 31 (55) 25 (45) UPIII (AU1), n ¼ 56 I, II, III 19 (34) 37 (66) Sections of formalin-fixed, paraffin-embedded tissues (5 lm) were deparaffinized in xylene substitute and rehydrated via graded ethanol, followed by blocking of endogenous peroxidases with 3% hydrogen peroxide solution. A standard immunohistochemistry technique was performed using a prediluted mouse monoclonal BC21 (API3051AA, Biocare Medical), a prediluted mouse monoclonal BC17 (API3023AA, Biocare Medical), and a prediluted mouse monoclonal AU1 (PM377AA, Biocare Medical). Heatinduced antigen retrieval was performed using a modified citratebuffer formulation in a pressure cooker (Reveal Decloaking Chamber, Biocare Medical) at 1258C for 30 seconds. Antibodies were incubated at room temperature for 30 minutes. Detection was performed with an IgG mouse antibody probe for 10 minutes, followed by a horseradish peroxidase polymer conjugate for 10 minutes (MACH 4, Biocare Medical). Visualization was achieved Table 2. Comparison of Uroplakin II (UPII; Clone BC21) and Uroplakin III (UPIII; Clone BC17) on Urothelial Carcinoma Antibody Grade Positive Negative UPII, n ¼ 174 a I, II, III 134 (77) 40 (23) UPIII, n ¼ 174 a I, II, III 94 (54) 80 (46) UPII, n ¼ 48 I 42 (88) 6 (13) UPIII, n ¼ 48 I 26 (54) 22 (46) UPII, n ¼ 82 II 67 (82) 15 (18) UPIII, n ¼ 82 II 48 (59) 34 (41) UPII, n ¼ 42 III 23 (55) 19 (45) UPIII, n ¼ 42 III 18 (43) 24 (57) a There were two cases with no grade identified. Table 3. Comparison of Uroplakin II (UPII; Clone BC21) and Uroplakin III (UPIII; Clone BC17) on Urothelial Carcinoma (n ¼ 94) Antibody Score 1 þ, Score 2 þ, Score 3 þ, Score 4 þ, UPII 22 (23) 24 (26) 19 (20) 29 (31) UPIII 57 (61) 17 (18) 15 (16) 5 (5) with 3,3 0 -diaminobenzidine to produce a brown precipitate at the site of the antibody binding. For each tested antibody, a case was deemed positive if more than 1% of tumor cells were stained. Conversely, less than 1% of tumor cells staining determined a case as negative. For cases that were both UPII þ and UPIII þ (94 of 174; 54%), a semiquantitative scoring system for staining percentage was applied: 1 þ, less than 25%; 2 þ, 26% to 50%; 3 þ, 51% to 75%; and 4 þ, more than 76% of cells stained. The UPII and UPIII negative cases were not scored. The McNemar test was used to compare the sensitivity of BC21 versus BC17 and AU1 on the same TMAs. Statistical significance was accepted at P,.05. RESULTS Western Blot for Mouse Monoclonal UPII (BC21) and UPIII (BC17) Antibodies Selective binding of UPII antibody to UPII protein was demonstrated by Western blot (Figure 1, A and B). The UPII antibody did not cross-react with the UPIII protein, proving its outstanding specificity in identifying the UPII protein (Figure 1, A). Conversely, the UPIII antibody did not bind the UPII protein but recognized only the UPIII protein (Figure 1, B). On Western blot (Figure 1), the UPII antibody appeared bigger than its 15-kDa molecular weight because it was fused with a glutathione-s-transferase tag, which was used in protein purification. The glutathione-s-transferase tag is about 26-kDa. The UPIII antibody also appeared bigger than its 47-kDa molecular weight on the blot for the same reason. UPII Immunohistochemistry In a preliminary comparison using a small TMA with 56 cases of UC, UPII (BC21) outperformed the previously developed UPIII (BC17 and AU1) in staining sensitivity (Table 1). In all grades, BC21, BC17, and AU1 demonstrated sensitivities of 79% (44 of 56), 55% (31 of 56) (P ¼.002, BC21 versus BC17), and 34% (19 of 56) (P,.001, BC21 versus AU1), respectively (Table 1). The UPII (BC21) was a more sensitive marker for UC and stained more tumor cells in each case than the UPIII (BC17 or AU1) did. To confirm the superior staining sensitivity of BC21 compared with BC17, the two antibodies were further compared using another 118 specimens of UC. The final diagnostic sensitivity for UPII and UPIII was based on the combined data from the 2 groups (56 cases and 118 cases, n ¼ 174). In all grades, UPII identified 134 specimens as positive (77%), compared with 94 specimens (54%) determined to be positive with UPIII (P,.001). In grade II specimens, UPII and UPIII demonstrated sensitivities of 82% (67 of 82) and 59% (48 of 82) (P,.001), respectively. In grade III specimens, UPII achieved a sensitivity of 55% (23 of 42), which was higher than the 43% (18 of 42) positive with UPIII (Table 2); however, statistical significance was Arch Pathol Lab Med Vol 138, July 2014 Uroplakin II in Urothelial Bladder Carcinoma Hoang et al 945

4 Table 4. Uroplakin II Antibody Staining of Various Neoplastic Tissues (n ¼ 461) Tissue Types Positive, No (%) Negative, No (%) Prostatic adenocarcinoma, n ¼ 88 1 a (1) 87 (99) Lung adenocarcinoma and squamous cell carcinoma, n ¼ 20 0 (0) 20 (100) Renal carcinoma (clear cell, papillary, and various other phenotypes), n ¼ 72 0 (0) 72 (100) Upper urinary tract (ureter and renal pelvis) urothelial carcinoma, n ¼ 3 3 (100) 0 (0) Colon adenocarcinoma, n ¼ 63 0 (0) 63 (100) Brain carcinoma (astrocytoma), n ¼ 13 0 (0) 13 (100) Lymphoma (B cell, T cell, and other various phenotypes), n ¼ 25 0 (0) 25 (100) Melanoma (classic), n ¼ 19 0 (0) 19 (100) Ovarian carcinoma (serous and endometrioid), n ¼ 11 0 (0) 11 (100) Seminoma, n ¼ 14 0 (0) 14 (100) Breast-infiltrating ductal carcinoma, n ¼ 74 0 (0) 74 (100) Adrenal carcinoma (pheochromocytoma), n ¼ 2 0 (0) 2 (100) Thyroid carcinoma (papillary carcinoma), n ¼ 2 0 (0) 2 (100) Pancreatic carcinoma (adenocarcinoma, islet cell tumor, and various phenotypes), n ¼ 10 0 (0) 10 (100) Head and neck carcinomas (squamous cell carcinomas), n ¼ 10 0 (0) 10 (100) Soft tissue carcinoma (sarcomas), n ¼ 10 0 (0) 10 (100) Hepatocellular carcinoma, n ¼ 10 0 (0) 10 (100) Cervical carcinoma (squamous cell carcinoma and other phenotypes), n ¼ 10 0 (0) 10 (100) Bladder adenocarcinoma, n ¼ 1 0 (0) 1 (100) Bladder squamous cell carcinoma, n ¼ 4 0 (0) 4 (100) a The single positive case could be metastatic urothelial carcinoma that spread to prostate. not reached in grade III (P ¼.13). In many comparisons, UPII stained more tumor cells than UPIII did. Of the 94 UC cases (54%) that were positive by both UPII and UPIII, 29 cases (31%) had a score of 4 þ when stained with UPII, whereas only 5 cases (5%) had a score of 4 þ with UPIII. There were 22 cases (23%) having a score of 1 þ when stained with UPII, whereas most cases (57 of 94; 61%) had a score of 1 þ, as determined with UPIII. A comparison of UPII and UPIII staining on the 94 cases of UC is summarized in Table 3. Compared with grade I and II tumors, grade III tumors showed a decrease in staining sensitivity for both UPII and UPIII. The UPII identified 88% (42 of 48), 82% (67 of 82), and 55% (23 of 42) in grades I, II, and III tumors, respectively (Table 2). Likewise, UPIII identified 54% (26 of 48), 59% (48 of 82), and 43% (18 of 42) in grade I, II, and III tumors, respectively (Table 2). The UPII antibody was found to be highly specific when evaluated on a variety of neoplastic tissues (n ¼ 461) (Table 4). Almost all tumors exhibited 100% negative staining with UPII, with the exception of the following cases: 1 case (1 of 88; 1%) of prostatic carcinoma stained positive with UPII, which was likely caused by metastatic UC that had spread to the prostate, and 3 cases (3 of 3; 100%) of upper urinary tract UC stained positive with UPII, demonstrating that UPII also stains upper urinary tract carcinomas (Table 4). Because UPII only stained UC and not renal carcinoma, it may be useful in differentiating between those 2 tumors. The UPII did not stain any other tumors, indicating its high specificity. In addition, BC21 (Table 4) and BC17 (data not shown) demonstrated negative staining in both adenocarcinoma (n ¼ 1) and squamous cell carcinoma (n ¼ 4) of the bladder. The UPII was evaluated in the US Food and Drug Administration 37 normal tissue types (Table 5). Bladder and ureter were the only normal tissues to stain positive. Such staining is expected, considering the known expression of UPII in normal urothelium. The UPII did not stain any other normal tissues, thus demonstrating its high specificity. The mouse monoclonal UPII antibody exhibited improved sensitivity compared with mouse UPIII (BC17 and AU1). The UPII and UPIII immunoreactivity were observed in both membranous and cytoplasmic patterns. Figure 2 shows staining comparisons between BC21 and both BC17 and AU1 in serial sections of the same UC specimen, demonstrating the greater sensitivity of BC21. Figure 2, A through C, shows a case of grade I UC with positive staining by all 3 clones; however, BC21 stained the most tumor cells, whereas AU1 stained the fewest tumor cells. In a grade II case (Figure 2, D through F), staining with BC21 exhibited strong membrane and cytoplasmic expression, whereas BC17 staining was minimal and AU1 staining results were negative. Figure 2, G through I, shows a grade III case that also exhibited strong staining with BC21; in contrast, BC17 and AU1 staining results were negative. Finally, the grade III case shown in Figure 2, J through L, displayed strong staining with BC21 and BC17, but only limited staining with AU1. COMMENT The UPII antibody exhibited increased staining intensity and sensitivity across more tumor cells than the UPIII antibodies did, probably because the 2 different uroplakin isoforms have distinct roles in the formation of urothelial plaques. The difference in their function is not fully known; however, mice lacking the upk2 gene showed no urothelial plaque formation, whereas mice lacking the upk3 gene still retain small urothelial plaques. 12 If UPII and UPIII do indeed exhibit nonoverlapping functions, determination of either isoform may not be sufficient for the most effective diagnosis of UC. In some cases, much stronger staining was observed for UPIII than for UPII; however, UPII and UPIII þ staining was not observed in the same case. Because UPII and UPIII were both highly specific for UC of the bladder, the combination of the 2 antibodies in an antibody cocktail may further increase the sensitivity for detection of UC of the bladder. The UPII antibody was found to be highly specific to urothelium when evaluated in various normal and neoplastic tissues. Urothelium consists of 3 to 6 layers of cells that line most of the urinary tract, including the renal pelvis, ureters, bladder, and parts of the urethra. The urothelium maintains its exceptional blood-urine barrier and apical surface strength by having unique features, called asymmetric unit membrane plaques of the urothelium. The molecular 946 Arch Pathol Lab Med Vol 138, July 2014 Uroplakin II in Urothelial Bladder Carcinoma Hoang et al

5 Table 5. Uroplakin II Antibody Staining of 37 US Food and Drug Administration Normal Tissue Types (n ¼ 121) Normal Tissue Positive Adrenal gland, n ¼ 3 0 (0) Bladder, n ¼ 7 5 (71) Bone marrow, n ¼ 1 0 (0) Eye, n ¼ 2 0 (0) Breast, n ¼ 3 0 (0) Cerebellum, n ¼ 3 0 (0) Cerebral cortex, n ¼ 3 0 (0) Fallopian tube, n ¼ 3 0 (0) Esophagus, n ¼ 3 0 (0) Stomach, n ¼ 3 0 (0) Small intestine, n ¼ 3 0 (0) Colon, n ¼ 3 0 (0) Rectum, n ¼ 3 0 (0) Heart, n ¼ 3 0 (0) Kidney, n ¼ 16 0 (0) Liver, n ¼ 5 0 (0) Lung, n ¼ 3 0 (0) Ovary, n ¼ 3 0 (0) Pancreas, n ¼ 5 0 (0) Parathyroid, n ¼ 1 0 (0) Pituitary gland, n ¼ 2 0 (0) Placenta, n ¼ 3 0 (0) Prostate, n ¼ 5 0 (0) Skin, n ¼ 2 0 (0) Spinal cord, n ¼ 2 0 (0) Spleen, n ¼ 2 0 (0) Striated muscle, n ¼ 4 0 (0) Testis, n ¼ 3 0 (0) Thymus, n ¼ 3 0 (0) Thyroid, n ¼ 4 0 (0) Tonsil, n ¼ 3 0 (0) Ureter, n ¼ 3 3 (100) Uterus (cervix), n ¼ 3 0 (0) Uterus (endometrium), n ¼ 3 0 (0) Tongue, n ¼ 1 0 (0) Epiglottis, n ¼ 1 0 (0) Blood vessels, n ¼ 1 0 (0) constituents of those plaques comprise 4 transmembrane UP isoforms: UPla, UPlb, UPII, and UPIII. Those UPs feature markedly asymmetric mass distributions, with the luminal domain being considerably larger than the cytoplasmic domain, thus explaining the asymmetry of the unit membrane plaques. 13 The asymmetric unit membrane plaques are insoluble to most substances found in the urine or blood, thereby contributing to the impermeability of the urothelium to toxic chemicals in the urine, such as ammonia and urea. 14 This study showed that the UPII mouse monoclonal antibody is specific to the urothelium of both bladder and upper urinary tract. This tissue specificity may be due to the role played by UPII in forming the asymmetric unit membrane plaques of the urothelium. A small number of normal bladder (n ¼ 7), bladder adenocarcinoma (n ¼ 1), and bladder squamous cell carcinoma (n ¼ 4) tested in this study (Tables 4 and 5) may not represent the full spectrum of diseases. Further investigation is warranted. Pathologists have used AU1 to establish the urothelial origin of tumors; however, the use of AU1 is limited because of its poor sensitivity. Kaufmann et al 15 first demonstrated an overall sensitivity of 57% (38 of 67) for AU1 staining of primary and metastatic urothelial carcinomas. That sensitivity was determined using a cutoff value of 1% of tumor cells staining positive for AU1 as the criteria for identifying a case as positive by AU1 staining. The sensitivity of AU1 routinely observed in clinical diagnosis (where higher cutoff values, such as between 5% and 10% of tumor cells staining, which may be the standard practice) is much lower than that reported by Kaufmann et al 15 Consequently, the conclusion among many practicing pathologists is that AU1 is not sufficiently sensitive to be a useful marker in the diagnosis of UC. The sensitivity reported by Kaufmann et al 15 has not, to our knowledge, been reproduced or validated in the literature in the 12 years since its publication, as would be typical for a diagnostically useful marker. It is generally known among pathologists that the poor sensitivity of AU1 prevents its use as a reliable marker for UC of the bladder. More-sensitive antibodies for the detection of UP are desired. The loss of UPIII expression has been observed in highgrade bladder carcinoma and is associated with a poor prognosis. Matsumoto et al 16 reported that UPIII expression was significantly decreased in bladder tumor tissues compared with normal control tissues, with loss of its expression in high-grade and muscle-invasive cases. Importantly, the loss of UPIII expression was associated with a decrease in cancer-specific survival. Similarly, Ohtsuka et al 17 also found lower UPIII expression in grade III, compared with grades I and II, urothelial tumors, suggesting a correlation between decreased UPIII expression and the level of aggressiveness of the bladder carcinoma. Our study showed that UPIII and UPII protein levels were lower in grade III, compared with grades I and II, bladder tumors (Table 2). The loss of UPII and UPIII expression in highgrade tumors suggested their potential prognostic value in UC of the bladder. Moreover, because the expression levels of both UPII and UPIII were reduced in high-grade tumors, as compared with low-grade tumors, the enhanced sensitivity of UPII improves the ease of interpretation. This study reported that 1 prostatic carcinoma case out of 88 prostatic carcinomas (1%) stained positively with UPII (Table 4) and was likely metastatic UC that had spread to the prostate. Shen et al 18 reported that prostatic carcinoma involvement by UC was detected in a high percentage of cases, such as 69 of 214 cases (32%). Because metastatic UC into the prostate is not uncommon, it is reasonable to speculate that 1 of 88 cases (1%) of prostatic carcinoma could be metastatic UC. The poor sensitivity of AU1 encouraged development of BC17. One study using clone AU1 showed that UPIII mrna was often expressed in the absence of the UPIII protein in advanced UC. 6 Our study demonstrated that BC17 is a more-sensitive antibody than AU1 for UC detection, providing a close match between UPIII mrna and UPIII protein expression by immunohistochemistry. Moreover, in metastatic UC, UPII mrna was expressed in 70% of cases, when compared with 50% expression of UPIII mrna. 6 These findings led us to explore developing a UPII antibody. We have presented cases in which a pathologist could definitively identify the presence of UC with the UPII antibody, which would not have been possible with the lesssensitive UPIII antibodies or ambiguous results would have been obtained with UPIII antibodies, which may have led to equivocal diagnoses that lack the confidence or fail to classify the case, whereas UPII expression offered clear and unambiguous results (Figure 2). The UPII provided strong, clear staining of UC, which may allow a pathologist to make a definitive diagnosis of UC, allowing a patient to receive the most appropriate treatment expeditiously. Arch Pathol Lab Med Vol 138, July 2014 Uroplakin II in Urothelial Bladder Carcinoma Hoang et al 947

6 Figure 2. A, Uroplakin II (clone BC21); B, uroplakin III (clone BC17); and C, uroplakin III (clone AU1) staining on serial sections of a grade I urothelial carcinoma case. D, Uroplakin II (clone BC21); E, uroplakin III (clone BC17); and F, uroplakin III (clone AU1) staining on serial sections of a grade II urothelial carcinoma case. G, Uroplakin II (clone BC21); H, uroplakin III (clone BC17); and I, uroplakin III (clone AU1) staining on serial sections of a grade III urothelial carcinoma case. J, Uroplakin II (clone BC21); K, uroplakin III (clone BC17); and L, uroplakin III (clone AU1) staining on serial sections of a grade III urothelial carcinoma case (original magnifications 3400 [A through C], 3200 [D through F], and 3100 [G through L]). 948 Arch Pathol Lab Med Vol 138, July 2014 Uroplakin II in Urothelial Bladder Carcinoma Hoang et al

7 The newly developed UPII antibody used in this study was a mouse monoclonal antibody, an in vitro diagnostic product that was optimized for immunohistochemistry application on bladder tissues. Currently, existing UPII antibodies are polyclonal and have not yet been characterized for immunohistochemistry application but are research-use only products, tested only for Western blotting, and suitable for Western blotting, immunoprecipitation, immunofluorescence, and solid-phase enzymelinked immunosorbent assay only. In summary, the mouse monoclonal UPII (BC21) demonstrated increased sensitivity in UC when compared with mouse monoclonal UPIII antibodies (BC17 and AU1). This antibody exhibited superior specificity and sensitivity, thus making it useful in the identification of tumors of urothelial origin. The highly sensitive and specific UPII may serve as a promising tissue-based biomarker in the differential diagnosis of UC and in the evaluation of tumors of unknown origin, specifically in cases of metastatic bladder carcinoma that has spread to the prostate or to other organs. Additional investigations evaluating the association of UPII with UC recurrence, progression, and treatment outcome are warranted. The combination of UPII and UPIII could also serve as powerful prognostic factors for UC. The authors would like to thank Yi Yun Lou, MD, from Biocare Medical, for the construction of the in-house neoplastic-tissue TMAs; and Mr Shelley Gofstein, HT(ASCP), from Biocare Medical, for histology support. References 1. American Cancer Society, Cancer Facts & Figures Atlanta, GA: American Cancer Society; Frantzi M, Makridakis M, Vlahou A. Biomarkers for bladder cancer aggressiveness. Curr Opin Urol. 2012;22(5): Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, CA Cancer J Clin. 2010;60(5): Wu XR, Kong XP, Pellicer A, Kreibich G, Sun TT. Uroplakins in urothelial biology, function, and disease. Kidney Int. 2009;75(11): Wu X, Kakehi Y, Zeng Y, Taoka R, Tsunemori H, Inui M. Uroplakin II as a promising marker for molecular diagnosis of nodal metastases from bladder cancer: comparison with cytokeratin 20. J Urol. 2005;174(6): Olsburgh J, Harnden P, Weeks R, et al. Uroplakin gene expression in normal human tissues and locally advanced bladder cancer. J Pathol. 2003; 199(1): Lu JJ, Kakehi Y, Takahashi T, et al. Detection of circulating cancer cells by reverse transcription-polymerase chain reaction for uroplakin II in peripheral blood of patients with urothelial cancer. Clin Cancer Res. 2000;6(8): Li SM, Zhang ZT, Chan S, et al. Detection of circulating uroplakin-positive cells in patients with transitional cell carcinoma of the bladder. J Urol. 1999; 162(3, pt 1): Yuasa T, Yoshiki T, Isono T, Tanaka T, Okada Y. Molecular cloning and expression of uroplakins in transitional cell carcinoma. Adv Exp Med Biol. 2003; 539(pt A): Okegawa T, Kinjo M, Nutahara K, Higashihara E. Value of reverse transcription polymerase chain assay in peripheral blood of patients with urothelial cancer. J Urol. 2004;171(4): Huang HY, Shariat SF, Sun TT, et al. Persistent uroplakin expression in advanced urothelial carcinomas: implications in urothelial tumor progression and clinical outcome. Hum Pathol. 2007;38(11): Kong XT, Deng FM, Hu P, et al. Roles of uroplakins in plaque formation, umbrella cell enlargement, and urinary tract diseases. J Cell Biol. 2004;167(6): Moll R, Wu XR, Lin JH, Sun TT. Uroplakins, specific membrane proteins of urothelial umbrella cells, as histological markers of metastatic transitional cell carcinomas. Am J Pathol. 1995; 147(5): Lewis SA. Everything you wanted to know about the bladder epithelium but were afraid to ask. Am J Physiol Renal Physiol. 2000;278(6):F867 F Kaufmann O, Volmerig J, Dietel M. Uroplakin III is a highly specific and moderately sensitive immunohistochemical marker for primary and metastatic urothelial carcinomas. Am J Clin Pathol. 2000;113 (5): Matsumoto K, Satoh T, Irie A, et al. Loss expression of uroplakin III is associated with clinicopathologic features of aggressive bladder cancer. Urology. 2008;72(2): Ohtsuka Y, Kawakami S, Fujii Y, et al. Loss of uroplakin III expression is associated with a poor prognosis in patients with urothelial carcinoma of the upper urinary tract. BJU Int. 2006;97(6): Shen SS, Lerner SP, Muezzinoglu B, Truong LD, Amiel G, Wheeler TM. Prostatic involvement by transitional cell carcinoma in patients with bladder cancer and its prognostic significance. Hum Pathol. 2006;37(6): Arch Pathol Lab Med Vol 138, July 2014 Uroplakin II in Urothelial Bladder Carcinoma Hoang et al 949