Urinary immunocytology—Promise or nonseller? A review with an opinion

Urologic Oncology: Seminars and Original Investigations 32 (2014) 383–390

Review article

Urinary immunocytology—Promise or nonseller? A review with an opinion Malte Böhm, M.D.a,b,*, Martin Schostak, M.D.a, Oliver W. Hakenberg, M.D.c a

Urologische Universitätsklinik, Otto-von-Guericke Universität, Magdeburg, Germany b Urologie in den Dillkliniken, Dillenburg, Germany c Urologische Universitätsklinik Rostock, Rostock, Germany

Received 12 July 2013; received in revised form 18 October 2013; accepted 5 November 2013

Abstract Urine cytology is considered a valid diagnostic method of urological and nephrological diagnosis and follow-up, whereas immunohistochemistry is an indispensable adjunct to histopathology. The combination of both—urinary immunocytology—has, so far, only attained a marginal role. This review gives a state-of-the-art update of urinary markers and relevant epitopes, elucidates some methodological pitfalls, and gives an outlook on the promise of urinary immunocytology today. It suggests that morphological urine cytology should be amended by immunology in a mutual quest of urologists and pathologists to improve the diagnostic power of urine cytology. The cost-effectiveness of the method is considered. This review also sheds light on the age-old dispute among pathologists about the nature of urothelial carcinoma that is reflected in the frequent and controversial reclassifications of the disease. r 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). Keywords: Urine cytology; Urinary immunocytology; Immunocytochemistry

1. Introduction When Papanicolaou and Marshall [1] introduced urine cytology as a diagnostic procedure in cancers of the urinary tract, they initiated a development that rendered urine cytology a valid diagnostic method of urological and urooncological diagnosis and follow-up. Panurothelial assessment, high specificity and sensitivity, cost-effectiveness, and a stable and easy workflow are some of its advantageous features. When Köhler and Milstein [2] described the first monoclonal antibody, they sparked an explosion in the field of immunology. A wide variety of diseases became understandable and immunological analysis has become an indispensable adjunct to histopathologic diagnosis. The combination of these 2 approaches—urinary immunocytology—however, has a marginal role only so far. This review gives a state-of-the-art update of relevant epitopes, elucidates Corresponding author. Tel.: þ49-2771-850651; fax: þ49-2771-850654. E-mail address: [email protected] (M. Böhm).

*

some reasons and methodological pitfalls, and gives an outlook on the promise of urinary immunocytology today. 2. The urologist’s approach to urinary immunocytology Clinical experience suggests that urothelial carcinomas are—unlike colorectal carcinomas, e.g.—not well characterized as a continuum from benign to undifferentiated carcinoma. They can rather be divided into 2 groups. Although a larger (470% of patients) group of low-grade tumors have a tendency to recur but progress rarely, a smaller group of high-grade carcinomas progress and metastasize early and are fatal if not diagnosed and treated early and with determination. The second group is the one that deserves and attracts most scientific attention. Morphological urine cytology has stood the test of time and is consistently considered the gold standard of urinebased diagnosis of urothelial carcinoma with unmatched specificity and sensitivity for the detection of high-grade tumors [3]. Its value in various clinical settings has

http://dx.doi.org/10.1016/j.urolonc.2013.11.002 1078-1439/r 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

384

M. Böhm et al. / Urologic Oncology: Seminars and Original Investigations 32 (2014) 383–390

frequently been evaluated and corroborated, and the World Health Organization classification of 2004 has been taken into account [4]; for more recent reports see Refs. [5,6]. The limitations of conventional morphology-based urine cytology have been lined out (for reviews see Refs. [5–9]), and responsible cytologists have formulated clear guidelines for its use [10]. Morphometric approaches can add some diagnostic information [11], but they are time consuming or expensive (Quanticyt) and have thus been implemented in few centers only. For high-grade tumors, the quest for additional powerful markers has been virulent in urology for many years. Many promising markers have been suggested to complement or replace urine cytology, so far with little or no additional diagnostic power. The hype of the 1990s has long cooled down because published results were difficult to reproduce. So far, no urine marker has been able to achieve the specificity of urine cytology let alone replace it. Some urine markers gained Food and Drug Administration approval on the basis of studies designed to highlight their performance and major drawbacks, such as excessive false-positive rates were sometimes ignored as was the case with nuclear matrix protein 22 (NMP22). The sobering criticism that ensued has not deterred some urine markers from being widely marketed, and caution is warranted when employing a new diagnostic test [12]. Several immunocytology-based urine tests are commercially available that rely on antibodies against cell surface antigens that are commonly expressed by urothelial carcinoma cells. The immunocytological urine test with the widest distribution is the Immunocyt/uCytþ. For the remainder of cases, urinary immunocytology has largely been limited to detect and identify rare and episodic tumors of the genitourinary tract.

3. The pathologist’s approach to urinary immunocytology In pathology, immunocytology is an established adjunct to morphology in many disorders, both malignant and benign, which improves the diagnostic accuracy and also allows the identification of markers both for prognosis and for targeted therapy. Its use in detecting metastatic carcinoma [13] or neuroblastoma [14,15] cells in blood or bone marrow is established while being aware of methodological limitations. In the diagnosis of effusions that often contain few and distorted cells, immunocytology is an accepted pivotal tool [16,17]. Current cytopathology textbooks attribute immunocytology only a marginal role when diagnosing urothelial carcinoma [9,16,18]. Current reviews on immunocytochemistry [19] and on urothelial immunohistochemistry [20] also do not specifically mention urinary immunocytology. It appears that immunocytology of urine, however promising from a theoretical point of view and however

much is being published on the issue, has not (yet) gained much influence, neither in urology nor in pathology. 4. What is the bottom line? When reviewing the literature, it appears that the markers, genes, and immunological epitopes considered relevant for urothelial cancer are differently viewed by pathologists and urologists (author [P ¼ pathology, U ¼ urology]): Netto (P) [21], Gakis et al. (U) [22], Burger et al. (U) [23], Lindemann-Docter et al. (P) [24], Nawroth et al. (P,U) [25], Skoog and Tani (P) [19], Sullivan et al. (P) [26], Protzel and Hakenberg (U) [27], Bolenz and Loten (U) [28], van Rhijn et al. (U) [29], and Vrooman and Witjes (U) [30]. These reviews are discussed in Ref. [31], and partly in Refs. [32,33]. 5. Commercially available tests using urinary immunocytology ImmunoCyt/uCytþ uses 2 fluorescein-coupled antibodies, M344 and LDQ10, directed against 2 sulfated glycoproteins and Texas Red–coupled antibody 19a211 directed against glycosylated high-molecular-weight carcinoembryonic antigen. When using it as a reflex test in patients with atypical urine cytology, a negative result of ImmunoCyt predicts a negative cystoscopy [34]. Schmitz-Dräger et al. [36] published several studies on the value of ImmunoCyt in patients with asymptomatic hematuria [35,36]. Although not an immunological technique, fluorescence in situ hybridization can detect genetic instability in urothelial carcinomas that correlates with a more aggressive behavior of the tumor. The UroVysion test uses centromeric DNA probes of chromosomes 3, 7, and 17 plus a probe of chromosomal region 9p21, which are labeled with distinct fluorescent dyes. With the UroVysion test, additional diagnostic information can be expected in some carcinomas of the upper urinary tract, admittedly a difficult field to tackle [24,37]. Both ImmunoCyt/uCytþ and UroVysion perform well and have been recommended as confirmatory tests [19,26,34,38,39]. If cystoscopy is negative or equivocal, this approach can avoid biopsies and thus save money [40]. Nonetheless commercially available immunocytological urine systems have not gained widespread acceptance in clinical routine. This is attributed to costs as well as the considerable time consumption associated with their use. Calculation of the cost of the various tests depends— among others—on the viewing angle and the system of remuneration. Estimates are from several hundred USD for the UroVysion (of which 90 USD are for reagents), approximately 100 USD for the bladder tumor antigen (BTA)/bladder tumor antigen stat (BTA stat) (30 USD for reagents), and 60 USD for urine cytology (approximately 5

M. Böhm et al. / Urologic Oncology: Seminars and Original Investigations 32 (2014) 383–390

USD for reagents). A systematic review of costeffectiveness in the United Kingdom suggests that for the initial diagnosis of bladder cancer, the strategy of urine cytology and white light cystoscopy is less costly (GBP 1043 per patient) than the combination cystoscopy, ImmunoCyt/uCytþ, and photodynamic diagnostics (GBP 2370 per patient), but also less effective in terms of life years (11.59 vs. 11.66) [41]. Additional immunohistochemistry stains cost about 15 to 20 USD per antibody. This renders the strategy of standard urine cytology plus immunocytochemistry, when needed, versatile, cost-effective, and responsive.

6. What novel markers and epitopes might be of interest? Hundreds of cellular pathways have been identified, and this trend is unbroken [42]. A list of 500 pathway maps can be accessed via www.qiagen.com/pathway-central. Many targets relevant for the malignant potential of tumors have thus been identified and many therapeutics directed at such targets are being introduced, an approach termed “targeted therapy” (for a review relating to urothelial carcinomas see Refs. [28,43–45]). Targeted diagnostics should keep pace with this development also in urothelial cancer to direct the most appropriate therapy to every patient [46]. Microscopy is taking leaps toward superresolution, optical sectioning, and 3D-microscopy. Cytology is ideal for these approaches, because cells are isolated, i.e., the amount of data to collect and to compute are limitable. Most superresolution approaches rely on immunostaining. This review focuses on markers and pathways that can be addressed by immunological analyses and thus complement morphological urine cytology. This approach bears the potential to improve urine cytological diagnosis. A number of tests have been suggested that use soluble markers and detect them with no information of their cellular origin. Most of the proliferation-associated markers lack specificity because benign conditions, such as inflammation and stones, are also related with cell proliferation. By omitting the morphological information of the origin of the marker from benign vs. malignant cells, the distinction between benign and malignant conditions becomes difficult. Soluble markers are not discussed in this review for this reason. Cell adhesion molecules may not be ideal epitopes for urinary immunocytology because cells must detach from their tissue origin before they are shed into the urine and become accessible to urine cytology. Alteration of cell adhesion molecules on the protein level is associated with this process irrespective of the malignant potential of the shed cells. Cytokeratin stains are helpful [47]. This includes pancytokeratin (clones AE1–AE3 and MNS116, e.g., DAKO) and cytokeratin 20 (CK-20), a marker of umbrella cells [48], and also cytokeratin fragments (CYFRA), in particular fragments

385

8, 18, 19, and 21 to 1 [49]. CYFRA 8 and 18 are detected by the urinary bladder cancer-Rapid (UBC Rapid) test, and CYFRA 8, 18, and 19 are used in the tissue plasminogen activator test, but both tests omit information of cellular origin and morphology. CK-20-positive atypical urothelial cells are indicators of low-grade urothelial carcinomas of the lower [50,51] and upper urinary tract [52]. Urothelial carcinomas stain positive for placental S100, GATA3, high-molecular-weight cytokeratins (CK-7 and CK-20, in particular), uroplakin III, and p63. Expression of both CK-20 and Ki-67 correlates with poor prognosis [53,54]. A review of human keratins has been published [55]. Fibroblast growth factor (FGF) and its receptor (FGFR) are markers of genetic stability [56–58]. Mutation of FGFR3 appears to be associated with low-grade tumors and good prognosis [59–61], mutation of p53 [62,63] and immunhistochemical overexpression of MIB-1 with highgrade tumors and poorer prognosis. From this a “molecular tumor grade” has been suggested [59]. A similar approach uses the genes FGFR3, PIK3CA, KRAS, HRAS, NRAS, TP53, CDKN2A, and TSC 1 to distinguish low-grade from high-grade and non–muscle-invasive from muscle-invasive carcinomas [64]. The transformation from FGFR3associated superficial to p53-associated invasive tumors may be mediated via p63 and p73 [65], and mutation and overexpression of FGFR3 are associated with less malignant urothelial carcinomas in a population of Jordan [60]. The significance of the pathway phosphatidylinositol-3kinase/Akt/mammalian target of rapamycin (mTOR) [66,67] and of the pathway mTOR/AMPK/OGG1 [68] in the oncogenesis of urothelial carcinomas have been elucidated. Staining of molecules of the mTOR pathway and of hypoxia-inducible factor could become relevant for personalized medicine [69], as medication interacting with this pathway is available. Transcription factors and nuclear matrix proteins may be good targets to address the aggressiveness and metastatic potential of cancer. The significance of cyclin-dependent kinase 4 and miR-195 [70], serine-threonine-kinase Pim-1 [71], and fatty acid synthase [72] have been addressed. Immunohistochemical expression of serine-threonine-kinases aurora A and aurora B is elevated in poorly differentiated urothelial carcinomas [73]. Carbonic anhydrase 9 upregulation appears to indicate worse prognosis in transitional cell carcinoma [74]. BLCA-4 overexpression appears to indicate poor prognosis [75]. Overexpression of fascin, an effector of the ribosomal S6 kinase 2-cAMP response elementbinding protein signaling pathway, correlates with the invasion of urothelial carcinomas and can be detected immunologically in urine cytology [76]. CXC-chemokine receptor-4 upregulation is associated with muscle invasion in bladder cancer, and cells from muscle-invasive urothelial carcinomas can be stained with its fluorescence-coupled antagonist TY14003 and detected in urine cytology [77]. With regard to oncoproteins and tumor suppressors probably a combination of candidates yields additional diagnostic information, such as a combination of cell

386

M. Böhm et al. / Urologic Oncology: Seminars and Original Investigations 32 (2014) 383–390

cycle–regulating proteins p53, pRb, p21, and p27 [78]. Expression of p21 can—unlike other cell cycle–associated proteins—together with clinical parameters, tumor size, and accompanying carcinoma in situ helps predict the tendency for recurrence of non–muscle-invasive bladder carcinomas [79]. Immunocytochemistry of p16 (INK4a) can improve the detection of low-grade [80] and high-grade [81] urothelial carcinomas. The combination of appropriate markers can be viewed as a step toward a personalized medicine [46]. Factors of angiogenesis and p53 are involved in the oncogenesis of urothelial carcinoma but not with their invasion [82]. Expression of tumor suppressors (p53 and p21waf1) and factors of angiogenesis (vascular endothelial growth factor and endoglin/CD105) can predict the tendency for recurrence of non–muscle-invasive bladder cancer [83]. Here, the technique used can directly be transferred to urine cytology. Urinary immunocytology of p53 and epidermal growth factor receptor was able to detect malignancy [84], but the combination of p53 with glutathione-S-transferase was not [85]. Minichromosome maintenance protein-2 is associated with cell proliferation, and urinary immunocytology has been used as an adjunct to urine cytology [86]. LIM-and-SH3-domain-protein-1 (LASP-1) is an actinbinding protein involved in cell migration and regulation of the cytoskeleton. Its detection in urine has been propagated as a prognostic soluble urinary marker [87,88], but LASP-1 immunocytology is feasible.

A number of tests have been suggested to improve the detection of low-grade urothelial carcinoma. Staining for CD44 is negative in contrast to normal urothelium and reactive atypia [20], and absent expression of variant CD44v6 expression is an independent adverse predictor of urothelial bladder cancer recurrence and overall survival [89]. Fluorescence staining of hydrogen peroxide indicates the urothelial production of reactive oxygen species. This helps detect low-grade urothelial carcinomas in urine cytology [90]. Immunohistochemical expression of vimentin distinguishes low-grade transitional cell carcinomas (negative) from reactive renal tubular cells (positive) [91]. Last, but not least, urinary immunocytology for prostatespecific antigen and high-molecular-weight cytokeratins have been used to define the origin of malignant cells in urine [92]. This short synopsis of some of the more promising epitopes must be incomplete and subject to constant updating as research progresses. At this point, studies on urinary immunocytology are scarce. Often data on performance or diagnostic power are not provided or not yet available. For this reason, the authors have refrained from a ranking. Time will tell which cocktail of candidates will generate additional diagnostic information beyond the morphological categories “low grade” and “high grade.” Some of the markers mentioned have already been used in urine cytology, and these are compiled in the Table. Others have been used on tissue sections, but bear the

Table Epitopes that have been used in urinary immunocytology. FISH is included because it ultilizes cellular information Test/epitope

References

Finding

Sensitivity (%)

Specificity (%)

Cytology FISH, UroVysion ImmunoCyt/uCytþ

Tilki et al. [103] Parker et al. [47] Proctor et al. [33] Sullivan et al. [26] Goodison et al. [32]

Reviews

12–90 30–100 50–100

78–100 69–96 62–85

p16INK4a CXCR4 Fascin CK-20

Alameda et al. [80] Nishizawa et al. [77] McKnight et al. [76] Mai et al. [52] Srivastava et al. [104] Morsi et al. [105] Golijanin et al. [106] Bhatia et al. [51] Ikeda et al. [84] Oǧuztüzün et al. [85] Saeb-Parsy et al. [86]

Detection of low-grade tumors Detection of high-grade tumors Predicts invasiveness Detection of urothelial carcinomas

Detection of malignancy Not helpful Detection of bladder cancer

67–75 N/A N/A 27 70 80 82 N/A 95 None 60–86

46–83 N/A N/A 100 71 78 77 N/A N/A None 52–90

83 N/A 35 30 59 50 174 14 108 124 497

Reactive oxygen species (ROS) cytology PAX2

Shimada et al. [90]

Increases sensitivity of cytology

64–100

97

50

Herlitz et al. [97]

N/A

N/A

N/A

PSA, HMW-CK

Mai et al. [92]

Differentiation of benign nephrogenic adenoma (þ) from TCC () Differentiation of malignant cells in urine, PCA vs. TCC

N/A

N/A

N/A

p53 and EGFR p53 and GST MMP-2

n

CXCR4 ¼ CXC-chemokine receptor-4; EGFR ¼ epidermal growth factor receptor; FISH ¼ fluorescence in situ hybridization; GST ¼ glutathione S-transferase; HMW-CK ¼ high-molecular-weight cytokeratins; MMP-2 ¼ matrix metalloproteinase-2; PAX2 ¼ paired box 2; PCA ¼ prostate cancer; PSA ¼ prostate-specific antigen; TCC ¼ transitional cell carcinoma.

M. Böhm et al. / Urologic Oncology: Seminars and Original Investigations 32 (2014) 383–390

potential for transfer to urine cytology. The quest is to implement urinary immunocytology of these (and other) epitopes into the diagnosis and treatment of urothelial cancer. It is suggested that morphological urine cytology be complemented by immunocytology in a concerted effort by both urologists and pathologists to improve the diagnostic power of urine cytology. It is apparent that the epitopes considered relevant in urothelial cancer [21,33,93], i.e., those that bear potential as prognostic markers, are different from those used in most of the bladder cancer tests that are currently being marketed. This may be in part to explain their modest performance. Possibly, an altered viewing angle on the underlying pathways to urothelial malignancy that are being meticulously compiled by pathologists [44] may help define more powerful markers of urothelial malignancy. The authors maintain that urinary immunocytology is a tool to achieve this. 7. Nonmalignant conditions: Immunocytology or immunocytochemistry can help in the diagnosis of viral urinary tract infections that today often go undiagnosed or maldiagnosed as “dysuria” or “pelvic dysfunction” and are hence maltreated. Simian vacuolating virus 40 immunostaining is a good standard to detect infection with polyoma virus (BK-virus) in patients with kidney transplants [94]. It complements the detection of decoy cells in these patients. Concomitant infected urothelial carcinomas—kidney transplant recipients have a higher risk of being diagnosed with urothelial carcinomas—are difficult to differentiate [95], but diagnostic accuracy can be improved in these cases with additional staining (proliferating cell nuclear antigen, p53, and retinoblastoma) [96]. Staining for renal transcription factor PAX2 differentiates between benign nephrogenic adenoma (positive) and transitional cell carcinoma (negative) [97]). Immunostaining of CD3-positive cells in urine is an indication of transplant rejection [98]. In urine cytology, virus-induced alterations can be difficult to distinguish from malignancy-associated alterations [99], which trouble even experienced cytologists. This results in a relevant interobserver variability, because of both interfering difficult diagnoses such as viral infections [99] and insufficiently defined morphological criteria [100]. Immunocytology for virus-specific epitopes could help here. 8. Complementation is the key A histopathologist would not base a diagnosis on an immunostaining alone but rather in conjunction with the morphology. She or he would not use either hematoxylin and eosin staining or immunostaining, but rather start with hematoxylin and eosin and complement it with immunohistochemistry as needed. Likewise, a urine cytologist

387

should not found his diagnosis on one marker vs. the other. Possibly this is a reason why so many potential urinary markers of malignancy have failed clinical application: They were designed to replace rather than to complement conventional urine cytology, i.e., they do not use the morphology of the urothelial cells and the information that can be mined from it to fortify the test result. By not using morphological information, they weaken their diagnostic power. A complementing approach appears promising in gynecological cytology [101]. Accordingly, the combination of cytomorphologic analysis as the initial screening test with the cell-based ImmunoCyt/uCytþ and UroVysion as reflex or confirmatory tests has been recommended [19,26,34,39] share this view. Complementation implies that cell-based rather than soluble markers be used, because only cell-based markers allow the comparison with morphology. Here, immunostains give an idea not only of the presence or absence of an epitope but also of its amount and of its intracellular distribution. The workflow of immunocytology is not much different from immunohistochemistry, the latter being a versatile and cost-effective adjunct to histological diagnosis. Urinary immunocytology bears the potential to become just a valuable adjunct to the cytological diagnosis of urine. The impulse of this review is to utilize the accumulated expertise in immunostaining to improve the power of urine cytology. When standard urine cytology (which is performed by many urologists) is complemented by immunocytology (a primary focus of pathologists), this may also further improve the interdisciplinary cooperation between these 2 fields of medicine benefiting the patient. It may also shed more light on the emerging molecular genetic mechanisms of bladder cancer tumorigenesis [44]. With this in mind urinary immunocytology may contribute 3-fold to patient care: (1) it may add diagnostic power, (2) it may contribute to the scientific understanding of urothelial disorders, notably cancers, possibly better than a consensus-oriented approach, and (3) it may help resolve an age-old dispute among histopathologists whether lowmalignant papillary lesions need to be classified as “carcinoma” vs. “papilloma” or “PUNLMP” on the one hand, and whether flat lesions are to be termed “carcinoma in situ” implying malignant potential on the extreme vs. “denuding cystitis” on the other hand. The quest is to identify the “true” cancers [102], i.e., those that kill the patient, and one should always be aware that a diagnosis is not so much to satisfy the cytologist but to aid the clinician and to guide the patient. Cytology reports should, notwithstanding a detailed description of the findings (for a suggestion see Ref. [26]), come as near as possible to 1 of 3 conclusions that entail clinical implication: (1) negative or no malignancy, patient needs no further follow-up; (2) equivocal or dubious, a control investigation is warranted; and (3) suspicious of malignancy, further investigations are required to confirm the diagnosis, locate the tumor, and

388

M. Böhm et al. / Urologic Oncology: Seminars and Original Investigations 32 (2014) 383–390

initiate treatment. The second category will probably benefit most from an immunocytological approach. A point raised in Ref. [102] is the clinical setting in which urine cytology is employed. In a community care or screening setting most patients do not have urothelial carcinomas, and the challenge is to not miss a lesion. Here it is possibly better to overinterpret, i.e., to aim for high sensitivity and trade it for a lower positive predictive value. In a cancer center or referral setting where the diagnosis has already been made in many patients, the opposite may be preferential. 9. Conclusion All this implies that a novel, wider approach to urinary immunocytology is warranted. The combination of morphology and immunology bears the potential to fortify the diagnostic power of urine cytology, as it has done in histopathology. The identification of relevant epitopes and the delineation of adequate technique should become a mutual quest of urologists and pathologists, and a combined effort may lead to both a better understanding of the nature of urothelial carcinoma and improved patient care. References [1] Papanicolaou GN, Marshall VF. Urine sediment smears as a diagnostic procedure in cancers of the urinary tract. Science 1945; 101:519–20. [2] Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495–7. [3] Niedworok C, Rembrink V, Hakenberg OW, Börgermann C, Rossi R, Schneider T, et al. Stellenwert der Urinzytologie in der Diagnostik von high-grade-Urotheltumoren. Urologe 2009;48:1018–24. [4] Tschirdewahn S, Boergermann C, Becker M, Szarvas T, Rübben H, vom Dorp F. Urinzytologische Diagnostik vor dem Hintergrund der neuen histopathologischen Klassifikation. Urologe 2009;48:615–8. [5] Tschirdewahn S, vom Dorp F, Rübben H, Hakenberg OW. Exfoliative Urinzytologie in der Behandlung des Harnblasenkarzinoms. Urologe 2011;50:292–6. [6] Hüppe P, Wawroschek F. Methodik und aktueller Stellenwert der Mikrohämaturiediagnostik. Urologe 2011;50:287–91. [7] Fernández MI, Parikh S, Grossman HB, Katz R, Matin SF, Dinney CP, et al. The role of FISH and cytology in upper urinary tract surveillance after radical cystectomy for bladder cancer. Urol Oncol 2012;30:821–4. [8] Messer J, Shariat SF, Brien JC, Herman MP, Ng CK, Scherr DS, et al. Urinary cytology has a poor performance for predicting invasive or high-grade upper-tract urothelial carcinoma. BJU Int 2011;108:701–5. [9] Wijkström H, Du Reitz B, Tolf A. Urinary cytology, cytometry, and new approaches in the assessment and monitoring of urothelial cancer. Droller MJ, editor Urothelial tumors—American Cancer Society atlas of clinical oncology. Hamilton, Canada: BC Decker Inc.; 2004. [10] Rathert P. Urinzytologie beim Harnblasenkarzinom: Kritische Wertung. Urologe A 2003;42:908–11. [11] Vom Dorp F, Pal P, Tschirdewahn S, Rossi R, Börgermann C, Schenck M, et al. Correlation of pathological and cytologicalcytometric grading of transitional cell carcinoma of the urinary tract. Urol Int 2011;86:36–40. [12] Scales CD Jr., Dahm P, Sultan S, Campbell-Scherer D, Devereaux PJ. How to use an article about a diagnostic test. J Urol 2008;180:469–76.

[13] Schlimok G, Funke I, Holzmann B, Göttlinger G, Schmidt G, Häuser H, et al. Micrometastatic cancer cells in bone marrow: in vitro detection with anti-cytokeratin and in vivo labeling with anti-17-1 A monoclonal antibodies. Proc Natl Acad Sci U S A 1987; 84:8672–6. [14] Cheung IY, Barber D, Cheung N-KV. Detection of microscopic neuroblastoma in marrow by histology, immunocytology, and reverse transcription-PCR of multiple molecular markers. Clin Cancer Res 1998;4:2801–5. [15] Beiske K, Burchill SA, Cheung IY, Hiyama E, Seeger RC, Cohn SL, et al. Consensus criteria for sensitive detection of minimal neuroblastoma cells in bone marrow, blood and stem cell preparations by immunocytology and QRT-PCR: recommendations by the International Neuroblastoma Risk Group Task Force. Br J Cancer 2009;100: 1627–37. [16] Shidham VB. Appendix II: immunocytochemistry of effusions— processing and commonly used immunomarkers. Shidham VB, Atkinson BF, editor Cytopathologic diagnosis of serous fluids. Philadelphia: W.B. Saunders; 2007:237–58. [17] Metzgeroth G, Kuhn C, Schultheis B, Hehlmann R, Hastka J. Diagnostic accuracy of cytology and immunocytology in carcinomatous effusions. Cytopathology 2008;19:205–11. [18] Atkinson BF, Silverman JF. Atlas of difficult diagnoses in cytopathology, 1st ed. Philadelphia: W.B. Saunders: 1998. [19] Skoog L, Tani E. Immunocytochemistry: an indispensable technique in routine cytology. Cytopathology 2011;22:215–29. [20] Hodges KB, Lopez-Beltran A, Emerson RE, Montironi R, Cheng L. Clinical utility of immunohistochemistry in the diagnoses of urinary bladder neoplasia. Appl Immunohistochem Mol Morphol 2010;18: 401–10. [21] Netto GJ. Molecular biomarkers in urothelial carcinoma of the bladder: are we there yet? Nat Rev Urol 2012;9:41–51. [22] Gakis G, Schwentner C, Todenhöfer T, Stenzl A. Current status of molecular markers for prognostication and outcome in invasive bladder cancer. BJU Int 2012;110:233–7. [23] Burger M, vom Dorp F. Nutzung von Markersystemen in der Behandlung des Harnblasenkarzinoms. Urologe 2011;50:303–8. [24] Lindemann-Docter K, Gaisa NT, Smeets D, Knüchel-Clarke R. Uringebundene Marker mit besonderer Berücksichtigung der Fluoreszenz-in-situ-Hybridisierung (FISH). Urologe 2011;50:297–302. [25] Nawroth R, Hartmann A, Olbert PJ, Merseburger AS, Stöhr R, Knüchel R, et al. Blasenkarzinom—Update: was gab es Neues auf dem Jahreskongress 2010 der Deutschen Gesellschaft für Urologie in Düsseldorf? Urologe 2011;50:221–6. [26] Sullivan PS, Chan JB, Levin MR, Rao J. Urine cytology and adjunct markers for detection and surveillance of bladder cancer. Am J Transl Res 2010;2:412–40. [27] Protzel C, Hakenberg OW. Molekulare Marker in Diagnostik und Therapie des Urothelkarzinoms. Urologe 2010;49:1415–24. [28] Bolenz C, Lotan Y. Translational research in bladder cancer: from molecular pathogenesis to useful tissue biomarkers. Cancer Biol Ther 2010;10:407–15. [29] van Rhijn BWG, van der Poel HG, van der Kwast TH. Cytology and urinary markers for the diagnosis of bladder cancer. Eur Urol Suppl 2009;8:536–41. [30] Vrooman OP, Witjes JA. Urinary markers in bladder cancer. Eur Urol 2008;53:909–16. [31] Böhm M, vom Dorp F, Schostak M, Hakenberg OW für den Arbeitskreis Urinzytologie der DGU. Urinzytologie - Update 2013: eine systematische Übersicht der neueren Literatur. Urologe 2013;52: 1207–24. [32] Goodison S, Rosser CJ, Urquidi V. Bladder cancer detection and monitoring: assessment of urine- and blood-based marker tests. Mol Diagn Ther 2013;17:71–84. [33] Proctor I, Stoeber K, Williams GH. Biomarkers in bladder cancer. Histopathology 2010;57:1–13.

M. Böhm et al. / Urologic Oncology: Seminars and Original Investigations 32 (2014) 383–390 [34] Odisho AY, Berry AB, Ahmad AE, Cooperberg MR, Carroll PR, Konety BR. Reflex immunoCyt testing for the diagnosis of bladder cancer in patients with atypical urine cytology. Eur Urol 2013;63: 936–40. [35] Cha EK, Tirsar LA, Schwentner C, Christos PJ, Mian C, Hennenlotter J, et al. Immunocytology is a strong predictor of bladder cancer presence in patients with painless hematuria: a multicentre study. Eur Urol 2012;61:185–92. [36] Schmitz-Dräger BJ, Tirsar LA, Schmitz-Dräger C, Dorsam J, Ebert T, Bismarck E. Rolle der Immunzytologie in der Abklärung von Patienten mit schmerzloser Makrohämaturie. Urologe 2010;49:741–6. [37] Mian C, Mazzoleni G, Vikoler S, Martini T, Knüchel-Clarke R, Zaak D, et al. Fluorescence in situ hybridisation in the diagnosis of upper urinary tract tumours. Eur Urol 2010;58:288–92. [38] Todenhöfer T, Hennenlotter J, Esser M, Mohrhardt S, Tews V, Aufderklamm S, et al. Combined application of cytology and molecular urine markers to improve the detection of urothelial carcinoma. Cancer Cytopathol 2013;121:252–60. [39] Dimashkieh H, Wolff DJ, Smith TM, Houser PM, Nietert PJ, Yang J. Evaluation of UroVysion and cytology for bladder cancer detection: a study of 1835 paired urine samples with clinical and histologic correlation. Cancer Cytopathol 2013;121:591–7. [40] Gayed BA, Seideman C, Lotan Y. Cost-effectiveness of fluorescence in situ hybridization in patients with atypical cytology for the detection of urothelial carcinoma. J Urol 2013;190:1181–6. [41] Mowatt G, Zhu S, Kilonzo M, Boachie C, Fraser C, Griffiths TR, et al. Systematic review of the clinical effectiveness and costeffectiveness of photodynamic diagnosis and urine biomarkers (FISH, ImmunoCyt, NMP22) and cytology for the detection and follow-up of bladder cancer. Health Technol Assess 2010;14:1–331. [42] Knüchel-Clarke R, Dahl E, Gaisa NT, Schwamborn K, LindemannDocter K, Henkel C. Stand des Wissens zur molekularen Pathologie des Urothelkarzinoms. Pathologe 2010;31:234–8. [43] Niegisch G, Koch A, Knievel J, Schulz WA, Albers P. Signaltransduktion im Urothelkarzinom: wie genau kennen wir die Ziele für eine zielgerichtete Therapie? Urologe 2010;49:1401–5. [44] McConkey DJ, Lee S, Choi W, Tran M, Majewski T, Lee S, et al. Molecular genetics of bladder cancer: emerging mechanisms of tumor initiation and progression. Urol Oncol 2010;28:429–40. [45] Castillo-Martin M, Domingo-Domenech J, Karni-Schmidt O, Matos T, Cordon-Cardo C. Molecular pathways of urothelial development and bladder tumorigenesis. Urol Oncol 2010;28:401–8. [46] Grossman HB. Are biomarkers for bladder cancer beneficial? J Urol 2010;183:11–2. [47] Parker J, Spiess PE. Current and emerging bladder cancer urinary biomarkers. Sci World J 2011;11:1103–12. [48] Melissourgos ND, Kastrinakis NG, Skolarikos A, Pappa M, Vassilakis G, Gorgoulis VG, et al. Cytokeratin-20 immunocytology in voided urine exhibits greater sensitivity and reliability than standard cytology in the diagnosis of transitional cell carcinoma of the bladder. Urology 2005;66:536–41. [49] Hakenberg OW, Fuessel S, Richter K, Froehner M, Oehlschlaeger S, Rathert P, et al. Qualitative and quantitative assessment of urinary cytokeratin 8 and 18 fragments compared with voided urine cytology in diagnosis of bladder carcinoma. Urology 2004;64:1121–6. [50] Lin S, Hirschowitz SL, Williams C, Shintako P, Said J, Rao JY. Cytokeratin 20 as an immunocytochemical marker for detection of urothelial carcinoma in atypical cytology: preliminary retrospective study on archived urine slides. Cancer Detect Prev 2001;25:202–9. [51] Bhatia A, Dey P, Kumar Y, Gautam U, Kakkar N, Srinivasan R, et al. Expression of cytokeratin 20 in urine cytology smears: a potential marker for the detection of urothelial carcinoma. Cytopathology 2007;18:84–6. [52] Mai KT, Teo I, Robertson SJ, Marginean EC, Islam S, Yazdi HM. Immunostaining as a diagnostic aid in cytopathologic study of upper urinary tract urothelial carcinoma. Acta Cytol 2009;53:611–8.

389

[53] Ye YK, Bi XC, He HC, Han ZD, Dai QS, Liang YX, et al. CK20 and Ki-67 as significant prognostic factors in human bladder carcinoma. Clin Exp Med 2010;10:153–8. [54] Bertz S, Otto W, Denzinger S, Wieland WF, Burger M, Stöhr R, et al. Combination of CK20 and Ki-67 immunostaining analysis predicts recurrence, progression, and cancer-specific survival in pT1 urothelial bladder cancer. Eur Urol 2014;65:218–26. [55] Moll R, Divo M, Langbein L. The human keratins: biology and pathology. Histochem Cell Biol 2008;129:705–33. [56] Kompier LC, Lurkin I, van der Aa MN, van Rhijn BW, van der Kwast TH, Zwarthoff EC. FGFR3, HRAS, KRAS, NRAS and PIK3CA mutations in bladder cancer and their potential as biomarkers for surveillance and therapy. PLoS One 2010;5:e13821. [57] Wild PJ, Fuchs T, Stoehr R, Zimmermann D, Frigerio S, Padberg B, et al. Detection of urothelial bladder cancer cells in voided urine can be improved by a combination of cytology and standardized microsatellite analysis. Cancer Epidemiol Biomarkers Prev 2009;18:1798–806. [58] Zuiverloon TC, van der Aa MN, van der Kwast TH, Steyerberg EW, Lingsma HF, Bangma CH, et al. Fibroblast growth factor receptor 3 mutation analysis on voided urine for surveillance of patients with low-grade non-muscle-invasive bladder cancer. Clin Cancer Res 2010;16:3011–8. [59] van Rhijn BW, Zuiverloon TC, Vis AN, Radvanyi F, van Leenders GJ, Ooms BC, et al. Molecular grade (FGFR3/MIB-1) and EORTC risk scores are predictive in primary non-muscle-invasive bladder cancer. Eur Urol 2010;58:433–41. [60] Bodoor K, Ghabkari A, Jaradat Z, Alkhateeb A, Jaradat S, Al-Ghazo MA, et al. FGFR3 mutational status and protein expression in patients with bladder cancer in a Jordanian population. Cancer Epidemiol 2010;34:724–32. [61] Serizawa RR, Ralfkiaer U, Steven K, Lam GW, Schmiedel S, Schuz J, et al. Integrated genetic and epigenetic analysis of bladder cancer reveals an additive diagnostic value of FGFR3 mutations and hypermethylation events. Int J Cancer 2011;129:78–87. [62] Goebell PJ, Groshen SG, Schmitz-Dräger BJ. International studyinitiative on bladder cancer. p53 immunohistochemistry in bladder cancer—a new approach to an old question. Urol Oncol 2010;28:377–88. [63] Cheng L, Zhang S, MacLennan GT, Williamson SR, Lopez-Beltran A, Montironi R. Bladder cancer: translating molecular genetic insights into clinical practice. Hum Pathol 2011;42:455–81. [64] Lindgren D, Frigyesi A, Gudjonsson S, Sjodahl G, Hallden C, Chebil G, et al. Combined gene expression and genomic profiling define two intrinsic molecular subtypes of urothelial carcinoma and gene signatures for molecular grading and outcome. Cancer Res 2010;70: 3463–72. [65] Sayan AE, D'Angelo B, Sayan BS, Tucci P, Cimini A, Ceru MP, et al. p73 and p63 regulate the expression of fibroblast growth factor receptor 3. Biochem Biophys Res Commun 2010;394:824–8. [66] Ching CB, Hansel DE. Expanding therapeutic targets in bladder cancer: the PI3K/Akt/mTOR pathway. Lab Invest 2010;90:1406–14. [67] Chen M, Gu J, Delclos GL, Killary AM, Fan Z, Hildebrandt MA, et al. Genetic variations of the PI3K-AKT-mTOR pathway and clinical outcome in muscle invasive and metastatic bladder cancer patients. Carcinogenesis 2010;31:1387–91. [68] Habib SL, Kasinath BS, Arya RR, Vexler S, Velagapudi C. Novel mechanism of reducing tumourigenesis: upregulation of the DNA repair enzyme OGG1 by rapamycin-mediated AMPK activation and mTOR inhibition. Eur J Cancer 2010;46:2806–20. [69] Tickoo SK, Milowsky MI, Dhar N, Dudas ME, Gallagher DJ, Al-Ahmadie H, et al. Hypoxia-inducible factor and mammalian target of rapamycin pathway markers in urothelial carcinoma of the bladder: possible therapeutic implications. BJU Int 2011;107:844–9. [70] Lin Y, Wu J, Chen H, Mao Y, Liu Y, Mao Q, et al. Cyclin-dependent kinase 4 is a novel target in micoRNA-195-mediated cell cycle arrest in bladder cancer cells. FEBS Lett 2012;586:442–7.

390

M. Böhm et al. / Urologic Oncology: Seminars and Original Investigations 32 (2014) 383–390

[71] Guo S, Mao X, Chen J, Huang B, Jin C, Xu Z, et al. Overexpression of Pim-1 in bladder cancer. J Exp Clin Cancer Res 2010;29:161. [72] Sugino T, Baba K, Hoshi N, Aikawa K, Yamaguchi O, Suzuki T. Overexpression of fatty acid synthase in human urinary bladder cancer and combined expression of the synthase and Ki-67 as a predictor of prognosis of cancer patients. Med Mol Morphol 2011;44:146–50. [73] Bufo P, Sanguedolce F, Tortorella S, Cormio L, Carrieri G, Pannone G. Expression of mitotic kinases phospho-aurora A and aurora B correlates with clinical and pathological parameters in bladder neoplasms. Histol Histopathol 2010;25:1371–7. [74] Klatte T, Belldegrun AS, Pantuck AJ. The role of carbonic anhydrase IX as a molecular marker for transitional cell carcinoma of the bladder. BJU Int 2008;101:45–8. [75] Zhao Q, Shen WH, Chen ZW, Zhou ZS, Ji HX. High expression level of BLCA-4 correlates with poor prognosis in human bladder cancer. Int J Clin Exp Pathol 2012;5:422–7. [76] McKnight R, Cohen C, Siddiqui MT. Fascin stain as a potential marker of invasiveness in carcinomas of the urinary bladder: a retrospective study with biopsy and cytology correlation. Diagn Cytopathol 2011;39:635–40. [77] Nishizawa K, Nishiyama H, Oishi S, Tanahara N, Kotani H, Mikami Y, et al. Fluorescent imaging of high-grade bladder cancer using a specific antagonist for chemokine receptor CXCR4. Int J Cancer 2010;127:1180–7. [78] Shariat SF, Chade DC, Karakiewicz PI, Ashfaq R, Isbarn H, Fradet Y, et al. Combination of multiple molecular markers can improve prognostication in patients with locally advanced and lymph node positive bladder cancer. J Urol 2010;183:68–75. [79] Behnsawy HM, Miyake H, Abdalla MA, Sayed MA, Ahmed Ael F, Fujisawa M. Expression of cell cycle-associated proteins in non–muscle-invasive bladder cancer: correlation with intravesical recurrence following transurethral resection. Urol Oncol 2011;29: 495–501. [80] Alameda F, Juanpere N, Pijuan L, Lloveras B, Gimeno J, Baro T, et al. Value of p16 (INK4a) in the diagnosis of low-grade urothelial carcinoma of the urinary bladder in urinary cytology. Cancer Cytopathol 2012;120:276–82. [81] Tauber S, Brunken C, Vierbuchen M. Vorkommen einer p16INK4aExpression in Zytologien der Harnblase: ein neuer Weg zur Früherkennung und Verlaufskontrolle beim Urothelkarzinom. Urologe 2011;50: 1130–3. [82] Ayala de la Pena F, Kanasaki K, Kanasaki M, Tangirala N, Maeda G, Kalluri R. Loss of p53 and acquisition of angiogenic microRNA profile are insufficient to facilitate progression of bladder urothelial carcinoma in situ to invasive carcinoma. J Biol Chem 2011;286: 20778–87. [83] Agrawal U, Mishra AK, Salgia P, Verma S, Mohanty NK, Saxena S. Role of tumor suppressor and angiogenesis markers in prediction of recurrence of non–muscle-invasive bladder cancer. Pathol Oncol Res 2011;17:91–101. [84] Ikeda S, Funakoshi N, Suzuki K. Usefulness of epidermal growth factor receptor and p53 cocktail immunostaining for differential diagnosis with urine cytology. Acta Cytol 2009;53:29–35. [85] Oǧuztüzün S, Sezgin Y, Yazıcı S, Fırat P, Ozhavzalı M, Ozen H. Expression of glutathione-S-transferases isoenzymes and p53 in exfoliated human bladder cancer cells. Urol Oncol 2011;29: 538–44. [86] Saeb-Parsy K, Wilson A, Scarpini C, Corcoran M, Chilcott S, McKean M, et al. Diagnosis of bladder cancer by immunocytochemical detection of minichromosome maintenance protein-2 in cells retrieved from urine. Br J Cancer 2012;107:1384–91. [87] Ardelt P, Grunemay N, Strehl A, Jilg C, Miernik A, Kneitz B, et al. LASP-1, a novel urinary marker for detection of bladder cancer. Urol Oncol 2013;31:1591–8.

[88] Payton S. Bladder cancer: LASP-1-a promising urine marker for detection of bladder cancer. Nat Rev Urol 2012;9:240. [89] Klatte T, Seligson DB, Rao JY, Yu H, de Martino M, Garraway I, et al. Absent CD44v6 expression is an independent predictor of poor urothelial bladder cancer outcome. J Urol 2010;183:2403–8. [90] Shimada K, Fujii T, Anai S, Fujimoto K, Konishi N. ROS generation via NOX4 and its utility in the cytological diagnosis of urothelial carcinoma of the urinary bladder. BMC Urol 2011;11:22. [91] Ohsaki H, Hirakawa E, Nakamura M, Norimatsu Y, Kiyomoto H, Haba R. Expression of vimentin and high-molecular-weight cytokeratin (clone 34ssE12) in differentiating reactive renal tubular cells from low-grade urothelial carcinoma cells in voided urine. Cytopathology 2011;22:247–52. [92] Mai KT, Ahmed I, Robertson SJ, Belanger EC, Veinot JP, Islam S. Immunocytochemical study of urine cytological preparations from secondary prostatic adenocarcinoma involving the urinary bladder. Diagn Cytopathol 2008;36:715–20. [93] Lughezzani G, Burger M, Margulis V, Matin SF, Novara G, Roupret M, et al. Prognostic factors in upper urinary tract urothelial carcinomas: a comprehensive review of the current literature. Eur Urol 2012:100–14. [94] Courtade-Saïdi M, Aziza J, Collin L, d'Aure D. Les difficultés diagnostiques liées aux polyomavirus en cytologie urinaire. Ann Pathol 2010;30:176–81. [95] Loghavi S, Bose S. Polyomavirus infection and urothelial carcinoma. Diagn Cytopathol 2011;39:531–5. [96] Pillai KR, Jayasree K, Pisharody R, Abraham EK. Decoy cells in the urine cytology of a renal transplant recipient: an immunohistochemical study. Indian J Pathol Microbiol 2010;53:347–50. [97] Herlitz LC, Tong GX, Hamele-Bena D, Greenebaum E. Nephrogenic adenoma identified on urine cytology using PAX-2 immunostaining. Diagn Cytopathol 2008;36:47–9. [98] Mihovilović K, Kardum-Skelin I, Ljubanović M, Sabljar-Matovinović M, Vidas Z. Urine immuncytology as a noninvasive diagnostic tool for acute kidney rejection: a single center experience. Coll Antropol 2010;34:63–7. [99] Glatz K, Willi N, Glatz D, Barascud A, Grilli B, Herzog M, et al. An international telecytologic quiz on urinary cytology reveals educational deficits and absence of a commonly used classification system. Am J Clin Pathol 2006;126:294–301. [100] May M, Brookman-Amissah S, Roigas J, Hartmann A, Störkel S, Kristiansen G, et al. Prognostic accuracy of individual uropathologists in noninvasive urinary bladder carcinoma: a multicentre study comparing the 1973 and 2004 World Health Organisation classifications. Eur Urol 2010;57:850–8. [101] Reuschenbach M, Clad A, von Knebel Doeberitz C, Wentzensen N, Rahmsdorf J, Schaffrath F, et al. Performance of p16INK4acytology, HPV mRNA, and HPV DNA testing to identify high grade cervical dysplasia in women with abnormal screening results. Gynecol Oncol 2010;119:98–105. [102] Murphy WM. What's the trouble with cytology? J Urol 2006;176: 2343–6. [103] Tilki D, Burger M, Dalbagni G, Grossman HB, Hakenberg OW, Palou J, et al. Urine markers for detection and surveillance of nonmuscle-invasive bladder cancer. Eur Urol 2011;60:484–92. [104] Srivastava R, Arora VK, Aggarwal S, Bhatia A, Singh N, Agrawal V. Cytokeratin-20 immunocytochemistry in voided urine cytology and its comparison with nuclear matrix protein-22 and urine cytology in the detection of urothelial carcinoma. Diagn Cytopathol 2012;40: 755–9. [105] Morsi MI, Youssef AI, Hassouna ME, El-Sedafi AS, Ghazal AA, Zaher ER. Telomerase activity, cytokeratin 20 and cytokeratin 19 in urine cells of bladder cancer patients. J Egypt Natl Canc Inst 2006;18:82–92. [106] Golijanin D, Shapiro A, Pode D. Immunostaining of cytokeratin 20 in cells from voided urine for detection of bladder cancer. J Urol 2000;164:1922–5.