Expression of cytokeratin 20 in urinary cytology of patients with bladder carcinoma
1998; Wiley; Volume: 82; Issue: 2 Linguagem: Inglês
10.1002/(sici)1097-0142(19980115)82
ISSN1097-0142
AutoresAmi Klein, Ruth Zemer, Victor Buchumensky, Ronen Klaper, Israel Nissenkorn,
Tópico(s)Gastrointestinal disorders and treatments
ResumoCancerVolume 82, Issue 2 p. 349-354 Original ArticleFree Access Expression of cytokeratin 20 in urinary cytology of patients with bladder carcinoma† Ami Klein Ph.D., Corresponding Author Ami Klein Ph.D. Laboratory of Molecular Biology, Sapir Medical Center, Sackler School of Medicine, Tel Aviv University, Kfar Saba, IsraelDepartment of Laboratories, Sapir Medical Center, Kfar Saba 44281, Israel.===Search for more papers by this authorRuth Zemer M.Sc., Ruth Zemer M.Sc. Laboratory of Molecular Biology, Sapir Medical Center, Sackler School of Medicine, Tel Aviv University, Kfar Saba, IsraelSearch for more papers by this authorVictor Buchumensky M.D., Ph.D., Victor Buchumensky M.D., Ph.D. Department of Urology, Sapir Medical Center, Sackler School of Medicine, Tel Aviv University, Kfar Saba, IsraelSearch for more papers by this authorRonen Klaper B.Sc., Ronen Klaper B.Sc. Laboratory of Molecular Biology, Sapir Medical Center, Sackler School of Medicine, Tel Aviv University, Kfar Saba, IsraelSearch for more papers by this authorIsrael Nissenkorn M.D., Israel Nissenkorn M.D. Department of Urology, Sapir Medical Center, Sackler School of Medicine, Tel Aviv University, Kfar Saba, IsraelSearch for more papers by this author Ami Klein Ph.D., Corresponding Author Ami Klein Ph.D. Laboratory of Molecular Biology, Sapir Medical Center, Sackler School of Medicine, Tel Aviv University, Kfar Saba, IsraelDepartment of Laboratories, Sapir Medical Center, Kfar Saba 44281, Israel.===Search for more papers by this authorRuth Zemer M.Sc., Ruth Zemer M.Sc. Laboratory of Molecular Biology, Sapir Medical Center, Sackler School of Medicine, Tel Aviv University, Kfar Saba, IsraelSearch for more papers by this authorVictor Buchumensky M.D., Ph.D., Victor Buchumensky M.D., Ph.D. Department of Urology, Sapir Medical Center, Sackler School of Medicine, Tel Aviv University, Kfar Saba, IsraelSearch for more papers by this authorRonen Klaper B.Sc., Ronen Klaper B.Sc. Laboratory of Molecular Biology, Sapir Medical Center, Sackler School of Medicine, Tel Aviv University, Kfar Saba, IsraelSearch for more papers by this authorIsrael Nissenkorn M.D., Israel Nissenkorn M.D. Department of Urology, Sapir Medical Center, Sackler School of Medicine, Tel Aviv University, Kfar Saba, IsraelSearch for more papers by this author First published: 31 October 2000 https://doi.org/10.1002/(SICI)1097-0142(19980115)82:2 3.0.CO;2-YCitations: 63 † Presented in part at a meeting of the American Urological Association, New Orleans, Louisiana, April 1997 (Abstract 1326) AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract BACKGROUND Of the 20 known cytokeratins, CK-19 is expressed in normal urothelium, whereas the recently identified CK-20 is expressed in urothelial carcinoma cells but not in normal urothelial cells. The aim of this study was to examine whether CK-20 expression could serve as a noninvasive test in which malignant urothelial cells in urine are detected and monitored. METHODS In the current study, the authors used reverse transcriptase-polymerase chain reaction (RT-PCR) methods to determine the expression of CK-20 in cells separated from the urine of patients with bladder carcinoma. Cells were obtained from the urine of 87 patients divided into the following 2 groups: 1) 14 healthy volunteers without any known history of transitional cell carcinoma (TCC), and 2) 73 patients with hematuria suspected for TCC of the bladder. For control purposes, CK-20 expression was examined in cells of 1) bladder carcinoma tumors of 5 patients, 2) blood of either patients with bladder carcinoma (n = 5) or healthy controls (n = 5), and 3) three different cell lines. RNA of the various cell pellets was extracted and RT-PCR was performed with CK-20 and CK-19 primers (CK-19 was used as a marker for normal epithelial cells). RESULTS CK-20 amplification band (370 bp) was obtained with mRNA extracted from TCC cells of either bladder tumor or HT-29 line (a CK-20 colon carcinoma line). Sensitivity of the method was found to be 91%, whereas specificity was 67%. Among the 7 false-positive cases, 3 showed atypia, 3 hyperplasia, and 1 metaplasia, and 2 underwent previously successful TCC tumor removals, suggesting that the CK-20 test also responded to premalignant lesions. No false-positive cases were found in the healthy control group. No other preparation, including blood of the patients of with TCC, showed the CK-20 amplification band. CONCLUSIONS These results indicate that CK-20 is a potential biomarker for noninvasive detection of bladder carcinoma by assaying uroepithelial cells from the voided urine specimen with RT-PCR. Cancer 1998;82:320-330. © 1998 American Cancer Society. Cytokeratins comprise a multigene family of 20 related polypeptides (CKs 1-20). They are constituents of the intermediate filaments (IFs) of epithelial cells, which are expressed in various combinations, depending on the epithelial type and the degree of differentiation. Of these, CK-19, for example, is a widely distributed CK that is expressed in various epithelia, including normal epithelium of the bladder.1, 2 Mol et al.3 used immunohistochemistry techniques to measure expression of a new cytokeratin, CK-20, which is expressed in gastrointestinal epithelium, uroepithelial cells from patients with bladder carcinoma, and Merkel cells. However, despite the fact that malignant cells generally retain the IFs of their progenitors, normal urothelial cells do not express the CK-20 gene.2 These findings emphasize the possibility that CK-20 is a specific biomarker for detecting bladder carcinoma in voided urinary specimens. The enhanced sensitivity and specificity of the reverse transcriptase-polymerase chain reaction (RT-PCR) method make it capable of detecting small quantities of mRNA. Burchill et al.4 and Smith et al.5 have used it to detect tumor cells in blood samples of patients with either melanoma (tyrosinase-mRNA) or neuroblastoma (tyrosine hydroxylase-mRNA). Saito6 has used the RT-PCR method to detect point mutation in H-ras. In the current study, we investigated the possibility of using RT-PCR to detect CK-20-mRNA as a marker for malignancy in cells isolated from the urine of patients with bladder carcinoma. CK-19 was used as a marker for normal epithelial cells. MATERIALS AND METHODS Cells for mRNA extractions were obtained from 1) bladder carcinoma tumors of 5 patients with transitional cell carcinoma (TCC), 2) blood of patients with metastasizing bladder carcinoma (whose urine CK-20 tests were positive) (n = 5) or blood of healthy controls (n = 5), 3) ovarian carcinoma line SH-63, 5) lung carcinoma line HTB-168, 4) CK-20-expressing colon carcinoma line HT-29, 5) urine of patients suspected for bladder carcinoma (n = 73, ages 44-84 years, 61 males, 12 females, and 6) urine of healthy controls (n = 14, ages 27-62 years, 10 males, 4 females). Informed consent was obtained from the subjects. Biopsy materials from bladder tumors were minced with phosphate-buffered saline through a metal grid (60 mesh), and cells were centrifuged at 1200 RPM (200 × g) and washed twice. Blood (10 mL) was treated several times with 3 volumes of hemolyzing buffer followed by sedimentation of the cells at 1200 RPM (200 × g) and extraction of RNA. Urine specimens (100 mL each) were obtained after the overnight accumulation had been voided. They were collected from the 14 healthy volunteers, who had no known history of TCC in the urinary tract, and from the 73 patients whose samples were provided in a blind test format. RNA from the various fresh cell pellets was immediately extracted with phenol and guanidinium thiocyanate (Tri Reagent, MRC, Cincinnati, OH) and stored at -80 °C in 75% ethanol. Synthesis of cDNA was carried out with reverse transcriptase (AMV-RT - 5 U/5 μg RNA), incubated for 1 hour at 42 °C in buffer (50 mM Tris, 40 mM KCl, 7 mM MgCl2, 1 mM dithiothreitol (DTT), and 0.1 mg/mL bovine serum albumin) containing 0.5 mM deoxynucleotide triphosphates (dNTP), 20 U RNasine (Promega, Madison, WI), and random primers (1 μmol) (final volume of 25 μL, containing 10 μL of total RNA). To the reverse transcriptase solution was added 75 μL of PCR mixture containing 0.3 mM dNTP, 67 mM K3PO4, 6.7 mM MgCl2, 1 mM DTT, and 133 μM activated calf thymus DNA containing 1 U of Taq polymerase (Promega). PCR of CK-20 was processed with a sense primer that lay in exon 1 (CAGACACACGGTGAACTATGG) and an antisense primer that lay in exon 3 (GATCAGCTTCCACTGTTAGACG).7 Primers for PCR of CK-19 were those described by Burchill et al. (sense GCGGGACAAGATTCTTGGTG, antisense CTTCAGGCCTTCGATCTGCAT).7 PCR was carried out under the following conditions: denaturation for 1 minute at 94 °C, annealing for 2 minutes at 55 °C, primer extension for 2 minutes at 72 °C, 40 cycles. RT-PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining against PCR size markers (Promega). The results were documented with a Polaroid camera. Appearance of 370 bp band presented positive CK-20 results. Specificity of the RT-PCR products was examined by Southern blotting onto a nylon membrane (Hybond N+, Amersham, Buckinghamshire, UK). Filters were hybridized with a 32P-labeled oligonucleotide probe of CK-20 (terminal transferase tailing), the sequence of which lay between each primer set. RESULTS Figure 1 (16K) shows the RT-PCR results obtained with mRNA of bladder carcinoma, blood of a patient with bladder carcinoma, and cell lines of lung and ovarian carcinoma. Except for the TCC tumor and HT-29 line (HT-29 results are not presented in the figure), no preparation-including blood of the patient with metastasis (Lane 5)-showed the CK-20-370 bp amplification band. The CK-20 band was confirmed by Southern hybridization with CK-20 exon 2 probe (Fig. 4 (15K)). Figure 1Open in figure viewerPowerPoint Reverse transcriptase-polymerase chain reaction products analysis of CK-20 (370 bp band) by agarose gel electrophoresis and ethidium bromide staining is shown. Lane 1: markers; Lane 2: lung carcinoma line; Lane 3: ovarian carcinoma line; Lane 4: transitional cell carcinoma (TCC) of the bladder; Lane 5: blood of a patient with bladder TCC. Figure 2Open in figure viewerPowerPoint Reverse transcriptase-polymerase chain reaction products analysis of CK-20 by agarose gel electrophoresis and ethidium bromide staining is shown. Sources of mRNA were cells isolated from the tumors of three patients with transitional cell carcinoma of the bladder, their urine, and urine samples from controls. Figure 3Open in figure viewerPowerPoint A reverse transcriptase-polymerase chain reaction products analysis shows CK-20 (370 bp band) and CK-19 (214 bp band) obtained with cells isolated from urine of either healthy subjects or patient with transitional cell carcinoma of the bladder. Lane 1: markers; Lane 2: CK-19 from a healthy subject; Lane 3: CK-20 from a healthy subject; Lane 4: CK-19 from a patient with bladder carcinoma; Lane 5: CK-20 from a patient with bladder carcinoma. Figure 4Open in figure viewerPowerPoint Specificity of reverse transcriptase-polymerase chain reaction (RT-PCR) is shown. (a) An RT-PCR products analysis shows CK-20 (370 bp band) separated on agarose gel electrophoresis and stained with ethidium bromide (Lanes 1-3: TCC tumors; Lanes 4-6: urine of patients with superficial TCC tumors; Lanes 7-11: urine of healthy subjects). (b) Southern blot analysis results with CK-20 32P-labeled probe show hybridization in the first 6 lanes only. RT-PCR carried out with mRNA of urine cells showed that the CK-20-370 bp band characterized patients with TCC; no false-positive tests were detected with uroepithelial preparations from the 14 healthy subjects (Fig. 2 (11K)). All cell preparations, separated from urine of patients with TCC or from healthy controls, expressed CK-19 (214 bp band, Fig. 3 (5K)), which indicated the presence of epithelial cells and the stability of RNA. Figure 4 (15K) reveals the specificity of the RT-PCR CK-20 bands obtained by Southern blotting with CK-20 32P-labeled probe. Hybridization with the probe was obtained with cells from either TCC tumor or patient's urine and not from healthy controls. Of the 73 TCC suspected patients, 48 were diagnosed positively with histologic and cytologic examinations (superficial tumors 0.5-4 cm, with a median size of 1.2 cm) (Table 1), whereas 55 were diagnosed positively with the urine CK-20 test. Comparing the results obtained from the urine CK-20 test with the histologic and cytologic results showed no significant differences between the groups (χ2 = 0.1423, P = 0.7060) (Table 2). The sensitivity of the method was found to be 91%, and the specificity was 67%. Of the seven false-positive cases, three showed atypia, three hyperplasia, and one metaplasia, and two patients had a known history of TCC (Table 3). Excluding the two patients with previous tumors, the specificity was 74% in the limited number of asymptomatic controls. Taking into account the fact that 14 of the subjects were males and 5 females, the specificity regarding gender was 71% and 80%, respectively. The cytologically and histologically negative group with negative CK-20 comprised 7 asymptomatic subjects, 1 with lymphoma, 4 with infection, 1 with severe atypia, and 1 with metaplasia. No false-positive cases were found in the healthy control group (the combined specificity of the control and negative groups was 80%). Table 1. T Classifications and Grades of Histologically Diagnosed TCC No. of patients T Classification Grade 1 Grade 2 Grade 3 Ta 10 22 T1 6 7 T2 2 1 TCC: transitional cell carcinoma. Table 2. χ2 Table of Histology, Cytology, and Urine CK-20 Results (χ2 = 0.1423, P = 0.7060) Negative Positive Total Histology and cytology 20 53 73 Urine CK-20 18 55 73 Total 38 108 146 Table 3. Histology and Cytology of 7 Patients with CK-20 False-Positive Results Patient no. Cytology Histology 1 Atypia Chr. infl. 2 N Hyperplasia 3 N Hyperplasiaaa These patients had a known history of transitional cell carcinoma. 4 Chr. infl. Metaplasia 5 Chr. infl. Naa These patients had a known history of transitional cell carcinoma. 6 Atypia Hyperplasia 7 Atypia Chr. infl. N: normal; Chr. infl.: chronic inflammation. a These patients had a known history of transitional cell carcinoma. The CK-20 results of five TCC patients were negative (the classification of all five of these cases was Ta; three of them were Grade 1 and two Grade 2, and lesion sizes ranged from 0.5 to 1.5 cm). DISCUSSION The screening and monitoring of patients for bladder malignancy is usually performed by urinary cytology, which detects high grade TCC in the majority of patients with carcinoma of the bladder. Flow cytometry for ploidy analysis, despite its low specificity, offers greater sensitivity than cytology. However, problems with fixation, storage, and interobserver variance have limited the widespread use of this method.8, 9 Consequently, a need arose for additional, more sensitive techniques for the detection and prognosis of bladder malignancies.10 Recently, Mao et al.11 described a microsatellite analysis method for detecting primary bladder carcinoma in urine. Kaempfer et al.12 described interleukin-2 expression as a prognostic parameter and predictive indicator of relapse of bladder carcinoma. Bonner et al.13 used quantitative fluorescence image analysis to detect TCC cells in voided urine samples. Our current report describes an additional method for detecting TCC in urine. Malignant cells generally retain the intermediate filaments of their progenitor cell type. For this reason, cytokeratins have been used to characterize neoplastic cells of epithelial origin.14-17 The differential expression of CK-20 in bladder carcinoma is expressed in tumor cells1 and not in normal urothelial cells.2 Our current results confirm that CK-20 is expressed in TCC bladder tumors but not in "cultured cell" lines of lung and ovarian carcinoma or peripheral blood (Fig. 1 (16K)). Based partially on these results, CK-20 may be a useful biomarker for detecting bladder carcinoma. The application by Saito et al.6 of RT-PCR techniques to detect point mutations of the ras gene in uroepithelial cells that were isolated from the urine of patients with TCC indicated that RT-PCR might be a sensitive method for detecting CK-20 expression in cells isolated from urine. This is supported by our findings, summarized in Figure 2 (11K), which show the appearance of the specific PCR-370 bp band in the urine cells of patients with TCC but not in those of healthy controls. The expression of CK-19 in uroepithelial cells from controls indicates that the negative result of CK-20 was not due to the absence of epithelial cells (Fig. 3 (5K)). The patients with TCC who participated in this study had superficial tumors of sizes ranging from 0.5 to 4.0 cm. CK-20 test results were not affected by the sizes of the tumors and displayed a sensitivity for tumors smaller than 1 cm. Of 32 patients with disease classified as Ta, 27 were CK-20 positive. In the group of 21 patients who were histologically and cytologically negative for TCC, in whom suspected areas of the bladder mucosa were examined and from whom selected biopsies were taken, the CK-20 test was positive in 7 (Table 3). Despite the specificity of 67% for asymptomatic patients with hematuria, no false-positive results were observed for the 14 asymptomatic healthy controls. These results support the observation that CK-20 is highly specific. As for the asymptomatic patients with hematuria, cytology showed atypia in 3 of them, histology showed that 3 had hyperplasia and 1 metaplasia, and 2 patients had a known history of TCC. Excluding these two patients, the specificity would have been 5/19, or 74%. A larger series of age- and gender-matched symptomatic and asymptomatic controls would be necessary to confirm the specificity and false-positive rate if assayed by RT-PCR. The expression of the CK-20 in the uroepithelial cells of two patients with a previous history of bladder carcinoma indicates that some of the false-positives may be attributed to biochemical alterations in premalignant cells.14 Our findings, on the level of cytology, histology, or both (Table 3), support occurrence of malignant changes in the uroepithelial cells of the 7 false-positive patients. This emphasizes the possibility that the CK-20 gene is expressed not only in malignant cells but in premalignant cells as well.18 In the future, cystoscopies and biopsies will determine whether the positive CK-20 and positive cytologies were false-positives or indications of early tumor detection. Bonner et al.13 showed that quantitative fluorescence image analysis allowed detection, with the human eye, of as few as 1/10,000 M-344 positive cells in a voided urine sample. Based on our own experience with PCR techniques for detecting residual hematologic diseases, the CK-20 test should be capable of detecting as few as 1/1000,000 TCC cells in a voided urine sample. Amberson and Laino19 used image cytometric DNA analysis of urine specimens as an adjunct to visual cytology. Their calculated sensitivity was 83.8% and specificity 99.3%, whereas the sensitivity of the CK-20 test was 91% as a single biomarker test. Melamed20 quantitated the DNA of uroepithelial cells from irrigation specimens by flow cytometry to improve the detection of bladder tumors. The RT-PCR test for detecting the CK-20 antigen is not invasive and is able to detect small numbers of TCC cells in voided urine samples. Regarding grading, the five false-negative results were found in both Grades 1 and 2, implying no correlation between the CK-20 test and grading. With respect to staging, one should take into consideration that 27 of the 32 patients with Ta disease and all patients with either T1 or T2 disease were CK-20 positive. Although the test detects most of the tumors, including small Ta (0.5 cm) lesions, there is a slight possibility that CK-20 expression is affected by staging. An ongoing study with secondary nested and quantitative PCR of invasive TCC of the bladder and additional studies with superficial bladder tumor will show whether this CK-20 urine test will be useful as a screening and/or prognostic test of TCC of the bladder. CK-20 analysis made with blood cells of patients with metastasizing bladder carcinoma whose urine CK-20 test was positive showed no appearance of the 370 bp PCR band. It is possible that the sensitivity of the PCR technique used was not sufficient to detect metastasizing cells. This notion was supported by our recent finding obtained with nested secondary PCR, which showed positive results in the blood cells of a patient with TCC metastasis to the liver. The current results demonstrate the possibility of using RT-PCR to measure expression of the biomarker CK-20 in uroepithelial cells and thereby detect TCC, even in the very early superficial stages prior to tumor invasion. The finding that approximately 97% of all bladder carcinoma cases in Western countries are TCC emphasizes the importance of this method as a noninvasive tool for the diagnosis, screening, and prognosis of bladder TCC. Acknowledgements The authors thanks Mrs. Hilda Deshen for her helpful assistance in the preparation of the manuscript and Ms. Mila Grankin for preparing the statistical analysis. REFERENCES 1 Moll R, Franke WW, Schiller DL. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 1982; 31: 11- 24. CrossrefCASPubMedWeb of Science®Google Scholar 2 Southgate J, Hutton KAR, Thomas DFM, Trejdesiewicz LK. Normal human urothelial cells in vitro: proliferation and induction of stratification. Lab Invest 1994; 71: 583- 94. CASPubMedWeb of Science®Google Scholar 3 Moll R, Lowe A, Laufer J, Franke WW. Cytokeratin-20 in human carcinomas: a new histodiagnostic marker detected by monoclonal antibodies. Am J Pathol 1992; 140: 427- 47. PubMedWeb of Science®Google Scholar 4 Burchill SA, Bradbury FM, Smith B, Lewis IJ, Selby P. Neuroblastoma cell detection by reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosine hydroxylase mRNA. Int J Cancer 1994; 57: 671- 5. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 5 Smith B, Selby P, Southgate J, Pittman K, Bradley C, Blair GE. Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction. Lancet 1991; 338: 1227- 9. CrossrefCASPubMedWeb of Science®Google Scholar 6 Saito S. Detection of H-ras gene point mutations in transitional cell carcinoma of human urinary bladder using polymerase chain reaction. Keio J Med 1992; 41: 80- 6. CrossrefCASPubMedGoogle Scholar 7 Burchill SA, Bradbury MF, Pittman K, Southgate J, Smith B, Selby P. Detection of epithelial cancer cells in peripheral blood by reverse transcriptase-polymerase chain reaction. Br J Cancer 1995; 71: 278- 81. CrossrefCASPubMedWeb of Science®Google Scholar 8 Walter PJ. The role of flow cytometry in the management of bladder cancer. Hematol Oncol Clin North Amer 1995; 6: 81- 98. Google Scholar 9 Wheeless LL, Badalment RA, de Vere White RW, Fradet Y, Tribukait B. Consensus review of the clinical utility of DNA cytometry in bladder cancer. Cytometry 1993; 14: 478- 81. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 10 Walter PJ. "Wildcatting" for breakthroughs in urothelial cancer detection and management: a frustrating business [editorial]. J Urol 1995; 154: 1348- 50. CrossrefPubMedWeb of Science®Google Scholar 11 Mao L, Schoenberg MP, Scicchitano M, Erozan YS, Merlo A, Schwab D, et al. Molecular detection of primary bladder cancer by microsatellites analysis. Science 1996; 271: 659- 62. CrossrefCASPubMedWeb of Science®Google Scholar 12 Kaempfer R, Gerez L, Farbstein H, Madar L, Hirschman O, Nussinovich R, et al. Prediction of response to treatment in superficial bladder carcinoma through pattern of interleukin-2 gene expression. J Clin Oncol 1996; 14: 1778- 86. CrossrefCASPubMedWeb of Science®Google Scholar 13 Bonner BR, Hemstreet GP, Fradet Y, Rao JY, Min KW, Hurst RE. Bladder cancer risk assessment with quantitative fluorescence image analysis of tumor markers in exfoliated bladder cells. Cancer 1993; 72: 2461- 9. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 14 Osborne M, Weber K. Tumor diagnosis by intermediate filament typing: a novel tool for surgical pathology. Lab Invest 1983; 48: 372- 94. PubMedWeb of Science®Google Scholar 15 Cooper D, Schermer A, Sun TT. Classification of human epithelia and their neoplasms using monoclonal antibodies to keratins: strategies, applications and limitations. Lab Invest 1985; 52: 243- 56. CASPubMedWeb of Science®Google Scholar 16 Lane EB, Alexander CM. Use of keratin antibodies in tumor diagnosis. Semin Cancer Biol 1990; 1: 165- 79. PubMedGoogle Scholar 17 Suo Z, Holm R, Nesland JM. Squamous cell carcinomas: an immunohistochemical study of cytokeratins and involucrin in primary and metastatic tumors. Histopathology 1993; 23: 45- 54. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 18 Rao JY, Hemstreet GP, Hurst RE, Bonner RB, Jones PL, Min KW, et al. Alterations in phenotypic biochemical markers in bladder epithelium during tumorigenesis. Proc Natl Acad Sci U S A 1993; 90: 8287- 91. CrossrefCASPubMedWeb of Science®Google Scholar 19 Amberson JB, Laino LP. Image cytometric deoxyribonucleic acid analysis of urine specimens as an adjunct to visual cytology in the detection of urothelial cell carcinoma. J Urol 1993; 149: 42- 5. CrossrefCASPubMedWeb of Science®Google Scholar 20 Melamed MR. Flow cytometry detection and evaluation of bladder tumors. J Occup Environ Med 1990; 32: 829- 33. CrossrefCASPubMedWeb of Science®Google Scholar Citing Literature Volume82, Issue215 January 1998Pages 349-354 FiguresReferencesRelatedInformation
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