Dysplasia and Carcinoma in Situ of the Urinary Bladder. Page Proof

Urothelial dysplasia (low-grade intraurothelial neoplasia) is recognized as a premalignant urothelial lesion in the 2004 World Health Organization (WHO) classification system. Although clarification of the diagnostic criteria of urothelial dysplasia has improved in recent years, there is still a lack of interobserver reproducibility. Active clinical follow-up is mandatory in patients with a diagnosis of urothelial dysplasia since it constitutes a marker of urothelial instability, and disease progression, in up to 19% of cases. The differential diagnosis of urothelial dysplasia is with other flat urothelial lesions with atypia, including flat urothelial hyperplasia, reactive urothelial atypia, urothelial atypia of unknown significance, and urothelial carcinoma in situ (highgrade intraurothelial neoplasia). In most cases, especially when small amounts of tissue are available, morphologic features alone may not be sufficient for diagnosis. Immunohistochemistry can be of help in selected cases, 0884-6812/15/3700-0000/$18.00/0 Science Printers and Publishers, Inc. Dysplasia and Carcinoma in Situ of the Urinary Bladder Antonio Lopez-Beltran, M.D., Ph.D., Rita C. Marques,, Rodolfo Montironi, M.D., F.R.C.Path., I.F.C.A.P., Carlos Reymundo,, Jorge Fonseca,, and Liang Cheng, M.D. and a panel of cytokeratin 20, p53, and CD44 may help in the diagnosis. The use of HER2, p16, and Racemase remains as an option pending validation. Herein, we present the pathologic features and clinical significance of urothelial dysplasia and carcinoma in situ with emphasis on differential diagnosis from common flat lesions with atypia. (Anal Quant Cytopathol Histopathol 2015;37:000 000) Keywords: bladder cancer, carcinoma in situ, diagnosis, dysplasia, intraurothelial neoplasia, pathology, reactive atypia, urinary bladder, urothelial dysplasia. Bladder cancer is a significant public health problem worldwide. It is the sixth most common cancer in men, accounting for 4.4% of all cancers. The typical cost per bladder cancer patient from diag- From the Department of Surgery and Pathology, Cordoba University Medical School, Cordoba, Spain; Champalimaud Clinical Center, Champalimaud Center for the Unknown, Lisbon, Portugal; Institute of Pathological Anatomy and Histopathology, Polytechnic University of the Marche Region, Ancona, Italy; and the Departments of Pathology and Laboratory Medicine and of Urology, Indiana University School of Medicine, Indianapolis, USA. Dr. Lopez-Beltran is, Department of Surgery and Pathology, Cordoba University Medical School, and, Champalimaud Clinical Center, Champalimaud Center for the Unknown. _Dr.?_ Marques is, Champalimaud Clinical Center, Champalimaud Center for the Unknown. Dr. Montironi is Professor, Institute of Pathological Anatomy and Histopathology, Polytechnic University of the Marche Region. _Dr.?_ Reymundo is, Department of Surgery and Pathology, Cordoba University Medical School. _Dr.? Fonseca is, Champalimaud Clinical Center, Champalimaud Center for the Unknown. _Dr.?_ Cheng is, Departments of Pathology and Laboratory Medicine, and Urology, Indiana University School of Medicine, Indianapolis. Address correspondence to: Antonio Lopez-Beltran, M.D., Ph.D., Unit of Anatomical Pathology, Department of Surgery and Pathology, University of Cordoba, Faculty of Medicine, Avda. Menendez Pidal s/n, E-14004 Cordoba, Spain (em1lobea@uco.es). Financial Disclosure: The authors have no connection to any companies or products mentioned in this article. D 1

2 Lopez-Beltran et al nosis to death was estimated to be the highest among all cancers. Such high costs are due, in part, to the high propensity for recurrence and progression characteristic of bladder cancer. 1-48 The 1998 International Society of Urological Pathology (ISUP)/WHO system was adopted in 2004 for the WHO publication Pathology and Genetics of Tumors of the Urinary System and Male Genital Organs. In addition to urothelial dysplasia (low-grade intraurothelial neoplasia), the 2004 WHO classification covers the nomenclature and histological findings of the following flat preneoplastic urothelial lesions: (1) flat urothelial hyperplasia, (2) reactive urothelial atypia, (3) urothelial atypia of unknown significance, and (4) carcinoma in situ (high-grade intraurothelial neoplasia) (Table I). 1-6 The purpose of this article is to review the clinical significance and pathologic features of urothelial dysplasia with emphasis in the differential diagnosis from other flat urothelial lesions with atypia. Urothelial Dysplasia Urothelial dysplasia, also known as low-grade intraurothelial neoplasia, is defined as abnormal urothelium with distinctive cytologic and architectural changes that do not meet all the criteria for the unequivocal diagnosis of urothelial carcinoma in situ (CIS). In these cases the urothelium demonstrates significant cytologic atypia that cannot be attributed to inflammation or a reparative process (Figure 1). 1-22 Table I Pathologic Features The thickness of the urothelium may be normal, increased, or decreased, exemplifying the many faces in which urothelial dysplasia can appear in the daily practice of uropathology (Table II). Umbrella cells are mostly present. Cytological abnormalities, including cellular crowding, loss of orderly maturation, and loss of polarity, are restricted to the basal and intermediate cell layers. Individual dysplastic cells show nuclear enlargement and occasional conspicuous nucleoli with irregular notched contours and coarse chromatin. Multiple nucleoli and nuclear overlapping may be seen. 1-48 Mitoses, when present, are generally basally located. The pathologic diagnosis of urothelial dysplasia is based primarily in nuclear and architectural features. The histologic criteria for distinguishing severe dysplasia from carcinoma in situ are unreliable in most cases, with frequent lack of reproducibility in most studies (Table II). It is also difficult to distinguish mild dysplasia from moderate dysplasia. Recognizing these limitations, it is recommended that severe dysplasia and carcinoma in situ be combined into a single category. It is also recommended that dysplasia not be further subclassified into mild or moderate dysplasia. Furthermore, the 2004 WHO classification suggests that the term mild dysplasia not be used and that flat lesions with minimal cytologic atypia and architectural disorder should be recognized within the spectrum of normal urothelium. 11-29 Immunohistochemistry Immunohistochemical features of urothelial dysplasia include aberrant cytokeratin 20 expression (Figure 1) at different levels of the urothelium, but Clinical Outcomes of Patients with Atypical Urothelial Proliferations of the Urinary Bladder Based on the 1998 WHO/ISUP Classification 2004 WHO classification Clinical significance D Reactive atypia/atypia of unknown significance None developed dysplasia, carcinoma in situ, or urothelial carcinoma Recurrence Progression Urothelial dysplasia Primary Unknown 15 19% progression to CIS Secondary 73% vs. 43% in tumors without 30 36% progression to muscle-invasive adjacent dysplasia carcinoma Urothelial CIS *Primary 50% 20 83% progression to muscle-invasive carcinoma **Secondary 37% 87% CIS = carcinoma in situ. *Recurrence or progression after BCG therapy in primary urothelial CIS. **Recurrence or progression after definitive therapy.

Volume 37, Number 0/Month 2015 Dysplasia and Carcinoma in Situ 3 LOW RESOLUTION NEED TO BE REDONE also, there is usually overexpression of p53, and high Ki-67 proliferation index. 22-34 While cytokeratin 20 (CK20) immunostaining is limited to the superficial cell layers in normal urothelium, dysplastic urothelium expresses CK20 in the superficial and intermediate cell layers. Likewise, positive Table II Pathologic Features of Flat Intraepithelial Lesions Arising in the Urothelium Figure 1 Urothelial dysplasia (A) with expression of p53 (B), CK20 (C), and mild p16 nucleo/ cytoplasmic expression (D). CD44 immunostaining, which is observed only in the basal cells in normal urothelium, is either absent entirely or focally present in basal layers of the dysplastic urothelium. In contrast, full-thickness positive membranous CD44 staining is typical of reactive urothelium. Dysplastic cells also show in- Reactive atypia Hyperplasia Dysplasia CIS Cell layers Variable >7 cells Variable Variable Polarization Slightly abnormal Normal Slightly abnormal Abnormal Cytoplasm Vacuolated Homogeneous Homogeneous Homogeneous N:C ratio Normal or slightly Normal or slightly Slightly increased Increased increased increased Nuclei Anisonucleosis Normal Normal Mild Moderate to severe Borders Regular/smooth Regular/smooth Notches/creases Pleomorphic Chromatin Fine/dusty Fine Slight hyperchromasia Coarse/hyperchromatic Chromatin distribution Even Even Even Uneven Nucleoli Large Small/absent Small/absent Large/prominent Mitotic figures Variable Absent Rare and rare Often Denudation Variable No No Variable Cytokeratin 20 Surface Surface Variable Variable Stromal microvascular proliferation Variable Variable Less prominent Often prominent D N:C = nucleus-to-cytoplasm ratio, CIS = urothelial carcinoma in situ.

4 Lopez-Beltran et al creased p53 expression, whereas p53 nuclear accumulation is undetectable or weak in the basal and parabasal cells of reactive urothelium (Table III). Ki67 immunohistochemical expression is more variable since it may be seen increased in both urothelial dysplasia and reactive urothelium. 1-12 The main differential consideration for urothelial dysplasia is with reactive atypia. Distinction may be particularly challenging in patients previously treated for carcinoma in situ using bacillus Calmette-Guérin (BCG) intravesical immunotherapy. Immunohistochemical stains such as CK20, CD44, p53, and Ki-67 in may be helpful. Likewise, a point of caution is the finding that molecular (multicolor fluorescence in situ hybridization [FISH] using the UroVysion probe set [Urovysion,, ]) and immunohistologic (expression of cytokeratin 20, high-molecularweight cytokeratin, Ki-67, p53) analyses cannot reliably solve diagnostic variation of flat intraepithelial lesions of the urinary bladder in some cases, emphasizing the fact that the diagnosis of flat lesions with atypia ultimately relies on histological characteristics of the lesion and the experience of the pathologist. 1-48 A conservative approach with repeat cystoscopy and biopsy after the inflammation has resolved is suggested in equivocal cases. Recent studies show moderate to strong and diffuse membranous staining with HER2 antibody in CIS cases, and absent to weak or/and focal HER2 expression in urothelial dysplasia and reactive conditions. 45 From the differential diagnostic standpoint, immunostaining for HER2 protein, with a Table III Immunohistochemical Features of Flat-Related Lesions high reported sensitivity (63%) and specificity (93%) for CIS, might represent a useful adjunct to aid in the delineation between CIS and urothelial dysplasia and reactive conditions of the bladder. 1-48 Additionally, molecular studies using cyclin D3 have suggested the presence of its amplification as a predictor of aggressive behavior in BCG-treated CIS. In a series of 28 cases of primary and secondary BCG-treated CIS cases, cyclin D3 gene amplification was found to be present in 29% of secondary CIS cases and absent in primary CIS cases and also in normal urothelium. Secondary CIS cases with cyclin D3 amplification were associated with lower recurrence and progression-free survival. 21 Lewis (y) antigen expression in urothelial dysplasia and reactive conditions of the bladder (patchy discontinuous expression restricted to individual cells scattered singly throughout the urothelial mucosa) differed from urothelial CIS (full thickness expression throughout the entire urothelium including the basal cell layer) in one study. Recent studies suggest that AMACR and p16 may help to differentiate reactive urothelial atypia (negative) and CIS (positive) (Table IV). PTEN loss of staining intensity has been associated with higher recurrence risk in CIS. 48 Novel markers such as GATA3, placental S100 (S100P), uroplakin III, and uroplakin II have been stated as markers of urothelial differentiation, with no benefit in separation of reactive atypia from CIS. 45,48 Clinical Significance of Urothelial Dysplasia Clinically, dysplasia occurs in two distinct clinical Flat Atypia of urothelial Reactive unknown Normal hyperplasia atypia significance Dysplasia CIS CK20 Limited to Limited to Limited to Limited to Deep layers May be full umbrella umbrella umbrella umbrella thickness cells cells cells cells CD44 Limited to Limited to Increased Increased Absent to Absent basal basal reactivity reactivity limited to cells cells in all cell in all cell some basal layers layers cells p53 Absent Absent Absent Absent Positive, Positive, frequently frequently weak staining, through usually < 10% all cell of cells layers CIS = urothelial carcinoma in situ. D

Volume 37, Number 0/Month 2015 Dysplasia and Carcinoma in Situ 5 Table IV settings: (1) de novo (primary, isolated) and (2) in patients with concurrent or previous urothelial neoplasms (secondary, concomitant). The true incidence of de novo dysplasia in the general population is unknown due to lack of large-scale screening studies. Clinical information on patients with de novo dysplasia is limited as well. Cheng et al reported 15 19% biopsy-proven cancer including carcinoma in situ, papillary urothelial carcinoma, and high-grade invasive carcinoma in patients with primary dysplasia. 8 Similarly, Zuk et al found that 15% of patients with primary urothelial dysplasia and with a mean follow-up of 4.8 years developed carcinoma in situ. 9 Secondary dysplasia is more common than is primary dysplasia and has a higher rate of progression to carcinoma than does de novo dysplasia, with estimates ranging from 30 36%. 1-40 Potential New Markers Helpful in Differential Diagnosis of Flat Urothelial Lesions Normal urothelium/ Marker reactive atypia Dysplasia CIS HER2 Absent to weak or/and Absent to weak or/and Moderate to strong diffuse focal membranous focal membranous membranous staining staining staining p16 Absent staining Focal nuclear staining Nuclear and cytoplasmatic staining in full thickness of the epithelium AMACR Absent staining (umbrella Not known Cytoplasmatic macrogranular cells may show faint intense staining ( 5% of nongranular staining) cells) CIS = carcinoma in situ. Urothelial Dysplasia: Differential Diagnosis Normal Urothelium The urinary bladder is lined by a multilayer mucosa. The thickness of this mucosa varies according to degree of distension. Normal urothelium is usually 4 7 cells thick in a contracted bladder. There are 3 cell layers: basal, intermediate, and superficial umbrella. The basal cells are small and cuboidal. Their nuclei have condensed chromatin and the cytoplasm is scant. Intermediate cells are slightly larger than basal cells. The intermediate cell layer may be up to 5 cells thick. Intermediate cell nuclei are oval and have finely stippled chromatin. They have moderate amounts of cytoplasm and distinct cytoplasmic membranes. Normal intermediate cells demonstrate apical-basal polarity with the long axis perpendicular to the basement membrane. Superficial umbrella cells are large, elliptical cells with abundant cytoplasm. Umbrella cell nuclei have condensed chromatin with prominent nucleoli and occasional binucleation. These features should not be misinterpreted as indicating dysplasia. All 3 layers of normal urothelium typically contain glycogen, which dissolves during processing, resulting in clear areas (cytoplasmic clearing). 30-48 Cytoplasmic clearing may be lost in dysplasia. Mitotic figures are usually not apparent in normal urothelium, and there is orderly maturation from basal to superficial cells. Loss of normal polarity, nuclear crowding, and loss of cytoplasmic clearing with increased eosinophilia are often indicative of intraepithelial neoplasia. 1-12 In normal urothelium the immunohistochemical profile of cytokeratin 20 (CK20), p53, Ki-67, and CD44 shows CK20 expression limited to the superficial umbrella cells. Nuclear staining for p53 is absent in normal urothelium but variably present in other flat urothelial lesions with atypia. CD44 staining is limited to the basal region. 30-46 Recent molecular studies have demonstrated generalized genetic instability in phenotypically normal urothelium from bladders with urothelial carcinoma. This observation supports the concepts of malignancy-associated changes and field change in bladder urothelial carcinogenesis. Loss of heterozygosity of chromosome 9q and mutations in FGFR3 appear important in early bladder carcinogenesis, while other gene abnormalities, such DBC1, TP53, and RB1, are involved in tumor progression. 1-26 D Flat Urothelial Hyperplasia Urothelial hyperplasia is characterized by markedly thickened mucosa with an increase in the number of cell layers, usually 10 or more. The cells do not

6 Lopez-Beltran et al show any significant cytologic abnormalities, and retain evidence of maturation from base to surface. Flat urothelial hyperplasia may be associated with inflammatory disorders, urolithiasis, dysplasia, CIS, and low-grade papillary tumors. There is no evidence to suggest that primary urothelial hyperplasia is premalignant. Likewise, molecular analyses have shown that this lesion may be clonally related to the papillary tumors in patients with known bladder cancer. Hyperplastic urothelium may be encountered within the entire spectrum of flat intraepithelial lesions with atypia (reactive atypia, dysplasia, and CIS). 12-22 The true incidence of flat hyperplasia is not known due to lack of large scale screening studies. Flat urothelial hyperplasia may occur adjacent to low-grade papillary tumors or as an isolated lesion. Isolated flat hyperplasia does not appear to have a premalignant potential. Genetic alterations in chromosome 9 are frequent in flat hyperplasia with concurrent low-grade papillary tumors. FISH studies for 9q22 (FACC) and 9p21 (p16/cdk12) have shown the same chromosome 9 deletions in hyperplasia and normal urothelium when there are coexisting low-grade papillary tumors. In contrast, alterations involving 17p13 (TP53) are uncommon in urothelial hyperplasia. 2-36 Molecular studies have also shown that flat hyperplasia adjacent to papillary tumors may show a high degree of genetic similarity to the papillary tumor cells. A possible genetic relationship between flat hyperplasia and low-grade papillary tumors has further been supported by recent molecular studies showing chromosome 9q deletions and mutations in the fibroblast growth factor receptor 3 (FGFR3) gene in both urothelial hyperplasia and concomitant low-grade papillary neoplasia. 1-48 Reactive Urothelial Atypia and Urothelial Atypia of Unknown Significance Reactive atypia is characterized by mild nuclear abnormalities occurring in association with acute or chronic inflammation. In most cases there is a history of cystitis, instrumentation, infection, stones, or previous therapy. The urothelium may be thickened, and the cells are often larger than normal. The cytoplasmic content may be increased, imparting a squamoid appearance. Nuclei are uniformly enlarged, with vesicular chromatin and prominent, centrally located nucleoli. The cells maintain polarity, and nuclear pleomorphism is generally lacking. Frequent mitoses may be present in the lower epithelial layers. Inflammatory cells in the lamina propria and infiltrating into the urothelium are commonly present. Diffuse CK5/6 reactivity in reactive urothelial atypia and negative CK5/6 reactivity in urothelial CIS may be helpful in distinguishing between these 2 entities. 1-40 The term atypia of unknown significance was introduced by the ISUP consensus group to describe lesions for which the pathologist was uncertain whether the changes were reactive or dysplastic. The degree of nuclear polymorphism and hyperchromasia present is greater than that in reactive atypia, and dysplasia cannot be definitely ruled out. The cellular changes present are disproportionate to any degree of inflammation seen. There is no evidence supporting a premalignant nature of such lesions at the present time and the use of the designation atypia of unknown significance is discouraged. 3-33 Overall, CK20 and CD44 appear to be the most useful objective markers for distinguishing reactive atypia/atypia of unknown significance from dysplasia. As mentioned earlier, CK20 expression is normally limited to the superficial umbrella cells. Reactive urothelium typically shows staining with CD44 in a diffuse membranous full-thickness pattern or with patchy basal and intermediate cell expression. This is in contrast to absence or the presence of limited reactive cells in basal layers of CD44 staining in dysplasia. It is important to note that these staining patterns are not absolute. Therefore, caution must be exercised when interpreting them, and clinical correlation with morphology is critical. 1-48 Urothelial Carcinoma in Situ Urothelial CIS, also known as high-grade intraurothelial neoplasia, is a flat lesion characterized by the presence of unequivocal cytologically malignant cells, some of which may be anaplastic (Figure 2). Full thickness cytologic atypia is not required for the diagnosis. The urothelium may be denuded, reflecting the discohesive nature of the cells, or it may be diminished in thickness, of normal thickness, or hyperplastic. Superficial (umbrella) cells may or may not be present. Marked disorganization of cells with loss of cellular polarity and decreased cellular cohesiveness is a common feature. 15-21 The tumor cells tend to be large and pleomorphic, with moderate to abundant cytoplasm, coarse and clumped chromatin, and multiple prominent nucleoli. Atypical mitotic figures D

Volume 37, Number 0/Month 2015 Dysplasia and Carcinoma in Situ 7 LOW RESOLUTION NEED TO BE REDONE may be present, often extending to the upper layers of the urothelium. The adjacent mucosa often contains lesser degrees of cytologic abnormality. The lamina propria is frequently hypervascular and inflamed. Nucleomegaly is a common finding in CIS, and it is determined by comparison to normal urothelium and stromal lymphocytes. Large nuclei in CIS are about 5 times the size of a normal lymphocyte, whereas the nuclear size of normal urothelium was approximately twice the size of lymphocytes. 15-21 Additionally, there are some morphologic variants of urothelial CIS. These include large-cell CIS, small-cell CIS, denuding and clinging pattern with dyscohesive cells, lepidic or undermining, pagetoid CIS, microinvasive CIS, and CIS with squamous or glandular differentiation. The high content of pleomorphic cells in some CIS cases argues in favor of a giant cell variant of CIS. Awareness of the histologic diversity of CIS may aid in the differential diagnosis. 11-33 Urothelial CIS shows intense aberrant CK 20, increased Ki-67 labeling, strong and diffuse membranous HER2 staining, and p53 positivity in the majority of malignant cells. The neoplastic cells are uniformly negative for CD44 immunostaining. 47 Figure 2 Urothelial carcinoma in situ (A) showing immunohistochemical expression of p53 (B), CK 20 (C), and p16 (nucleo/ cytoplasmic) (D). Therapy-induced Reactive Changes in the Urothelium Several systemic or intravesical antineoplastic agents, such as thiotepa (triethylenethiophosphoramide), mitomycin C, cyclophosphamide, ketamine, BCG, and radiation therapy produce atypical urothelial changes that can mimic dysplasia or cancer, histologically. Reactivation of polyomavirus infection can also be an important mimic of urothelial dysplasia or CIS. 30-46 Mitomycin C and thiotepa induce exfoliation, epithelial denudation, multinucleation, cytoplasmic vacuolization, and the appearance of bizarre, nonmalignant nuclei in the superficial layers of the urothelium. A marked necroinflammatory process with a histiocytic response extending deep into the bladder wall and eosinophilic cystitis may be seen with Mitomycin C. In addition, both Mitomycin C and thiotepa destroy the tips of the papillae of papillary urothelial carcinoma. These truncated papillae, when associated with denudation and inflammation, may be mistaken for carcinoma in situ or dysplastic changes instead of residual papillary urothelial carcinoma. 30-46 Cyclophosphamide therapy may induce stromal fibrosis, vascular intimal thickening, mural fibrin D

8 Lopez-Beltran et al deposition in vessels, vascular ectasia, and epithelial necrosis. Regenerative changes with binucleated and multinucleated cells, often with large bizarre nuclei, may be mistaken for malignancy. Cyclophosphamide may also induce reactivation of polyomavirus infection, causing marked nuclear atypia in the surface urothelium, a lesion that mimics urothelial dysplasia/cis. Patients receiving ketamine may present reactive urothelial changes that can mimic urothelial dysplasia/cis. 42 In BCG-treated bladders, reactive, mostly reparative changes may develop as a result of acute and chronic inflammation, reactive epithelial atypia, and granulomatous reaction deep in the bladder wall. It is important to keep in mind that CIS may recur after BCG therapy and that this may be seen mainly in Brunn s nests. Radiation therapy produces a variety of bladder lesions, beginning with acute cystitis and hyperemia with edema of the lamina propria approximately 3 6 weeks after therapy. The urothelium shows cytoplasmic and nuclear vacuolization, karyorrhexis, stromal hyalinization, thrombosis of blood vessels, stromal cell atypia, and fibroblast proliferation. Bizarre multinucleated cells with enlarged nuclei, smudged chromatin, and degenerative changes are usually present in radiation atypia and are not seen in dysplasia/cis. Pseudocarcinomatous epithelial hyperplasia, a lesion seen occasionally after radiation and/or chemotherapy, should be distinguished from flat lesions with atypia. 33-45 Pathologists should be aware of these diagnostic pitfalls and exercise caution when evaluating urothelial atypia following treatment. If a distinction between treatment-induced atypia and dysplasia/ CIS cannot be made, a conservative approach with follow-up repeat biopsy is indicated after inflammation has subsided. Conclusion Flat urothelial lesions with atypia comprise a spectrum of morphologic changes ranging from reactive atypia to urothelial dysplasia or carcinoma in situ. Differentiating these lesions is important because of different clinical outcome and therapy. Currently, differential diagnosis relies on histopathological evaluation of samples and the experience of the pathologist. Combined use of CK20, p53, and CD44 may allow discrimination between reactive and neoplastic flat lesions in difficult cases. References 1. Lopez-Beltran A, Cheng L, Andersson L, Brausi M, de Matteis A, Montironi R, Sesterhenn I, van det Kwast KT, Mazerolles C: Preneoplastic non-papillary lesions and conditions of the urinary bladder: An update based on the Ancona International Consultation. Virchows Arch 2002;440:3-11 2. Hartmann A, Moser K, Kriegmair M, Hofstetter A, Hofstaedter F, Knuechel R: Frequent genetic alterations in simple urothelial hyperplasias of the bladder in patients with papillary urothelial carcinoma. Am J Pathol 1999;154:721-727 3. Hartmann A, Schlake G, Zaak D, Hungerhuber E, Hofstetter A, Hofstaedter F, Knuechel R: Occurrence of chromosome 9 and p53 alterations in multifocal dysplasia and carcinoma in situ of human urinary bladder. Cancer Res 2002;62:809-818 4. Epstein JL, Amin MB, Reuter VR, Mostofi FK: The World Health Organization/International Society of Urologic Pathology consensus classification of urothelial (transitional cell) neoplasms of the urinary bladder. Bladder Consensus Conference Committee. Am J Surg Pathol 1998;22:1435-1438 5. Cheng L, Cheville JC, Neumann RM, Bostwick DG: Flat intraepithelial lesions of the urinary bladder. Cancer 2000;88: 625-631 6. Emerson RE, Cheng L: Immunohistochemical markers in the evaluation of tumors of the urinary bladder: A review. Anal Quant Cytol Histol 2005;27:301-316 7. McKenney JK, Desai S, Cohen C, Amin MB: Discriminatory immunohistochemical staining of urothelial carcinoma in situ and non-neoplastic urothelium: An analysis of cytokeratin 20, p53, and CD44 antigens. Am J Surg Pathol 2001;25: 1074-1078 8. Cheng L, Cheville JC, Neumann RM, Bostwick DG: Natural history of urothelial dysplasia of the bladder. Am J Surg Pathol 1999;23:443-447 9. Zuk R, Rogers H, Martin J, Baithun S: Clinicopathological importance of primary dsyplasia of bladder. J Clin Pathol 1988;41:1277-1280 10. Lopez-Beltran A, Montironi R, Vidal A, Scarpelli M, Cheng L: Urothelial dysplasia of the bladder: Diagnostic features and clinical significance. Anal Quant Cytol Histol 2013;35: 121-129 11. McKenney JK, Gomez JA, Desai S, Lee MW, Amin MB: Morphologic expressions of urothelial carcinoma in situ: A detailed evaluation of its histologic patterns with emphasis on carcinoma in situ with microinvasion. Am J Surg Pathol 2001;25:356-362 12. Mazzucchelli R, Barbisan F, Stramazzotti D, Montironi R, Lopez-Beltran A, Scarpelli M: Chromosomal abnormalities in macroscopically normal urothelium in patients with bladder pt1 and pt2a urothelial carcinoma: A fluorescence in situ hybridization study and correlation with histologic features. Anal Quant Cytol Histol 2005;27:143-151 13. Lopez-Beltran A, Luque RJ, Mazzuchelli R, Scarpelli M, Montironi R: Changes produced in the urothelium by traditional and newer therapeutic procedures for bladder cancer. J Clin Pathol 2002;55:641-647 14. Lopez-Beltran A: Bladder treatment: Immunotherapy and Chemotherapy. Urol Clin North Am 1999;26:535-554 D

Volume 37, Number 0/Month 2015 Dysplasia and Carcinoma in Situ 9 15. Mazzucchelli R, Cheng L, Lopez-Beltran A, Scarpelli M, Montironi R: Classification and grading of noninvasive and invasive neoplasms of the urothelium. Anal Quant Cytol Histol 2012;34:111-119 16. Mazzucchelli R, Cheng L, Lopez-Beltran A, Scarpelli M, Montironi R: Clinicopathological significance of lymphovascular invasion in urothelial carcinoma. Anal Quant Cytol Histol 2012;34:173-179 17. Lopez-Beltran A: Rare lesions and tumors of the urinary bladder: Selected issues. Tumori. 2012;98:274-277 18. Lopez-Beltran A, Jimenez RE, Montironi R, Patriarca C, Blanca A, Menendez CL, Algaba F, Cheng L: Flat urothelial carcinoma in situ of the bladder with glandular differentiation. Hum Pathol 2011;42:1653-1659 19. Montironi R, Cheng L, Scarpelli M, Mazzucchelli R, Lopez- Beltran A: How much do you know about benign, preneoplastic, non-invasive and invasive neoplastic lesions of the urinary bladder classified according to the 2004 WHO scheme? Diagn Pathol 2011;6:31 20. Lopez-Beltran A, Algaba F, Berney DM, Boccon-Gibod L, Camparo P, Griffiths D, Mikuz G, Montironi R, Varma M, Egevad L: Handling and reporting of transurethral resection specimens of the bladder in Europe: A web-based survey by the European Network of Uropathology (ENUP). Histopathology 2011;58:579-585 21. Lopez-Beltran A, Ordóñez JL, Otero AP, Blanca A, Sevillano V, Sanchez-Carbayo M, Muñoz E, Cheng L, Montironi R, de Alava E: Cyclin D3 gene amplification in bladder carcinoma in situ. Virchows Arch 2010;457:555-561 22. Cheng L, Davidson DD, Maclennan GT, Williamson SR, Zhang S, Koch MO, Montironi R, Lopez-Beltran A: The origins of urothelial carcinoma. Expert Rev Anticancer Ther 2010;10:865-880 23. 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;18:401-410 24. Williamson SR, Montironi R, Lopez-Beltran A, MacLennan GT, Davidson DD, Cheng L: Diagnosis, evaluation and treatment of carcinoma in situ of the urinary bladder: The state of the art. Crit Rev Oncol Hematol 2010;76:112-126 25. Cheng L, Zhang S, Davidson DD, MacLennan GT, Koch MO, Montironi R, Lopez-Beltran A: Molecular determinants of tumor recurrence in the urinary bladder. Future Oncol 2009; 5:843-857 26. Hodges KB, Lopez-Beltran A, Davidson DD, Montironi R, Cheng L: Urothelial dysplasia and other flat lesions of the urinary bladder: Clinicopathologic and molecular features. Hum Pathol 2010;41:155-162 27. Lacy S, Lopez-Beltran A, MacLennan GT, Foster SR, Montironi R, Cheng L: Molecular pathogenesis of urothelial carcinoma: The clinical utility of emerging new biomarkers and future molecular classification of bladder cancer. Anal Quant Cytol Histol 2009;31:5-16 28. Lopez-Beltran A: Bladder cancer: Clinical and pathological profile. Scand J Urol Nephrol Suppl 2008;218:95-109 29. Lopez-Beltran A, Cheng L, Mazzucchelli R, Bianconi M, Blanca A, Scarpelli M, Montironi R: Morphological and molecular profiles and pathways in bladder neoplasms. Anticancer Res 2008;28:2893-2900 30. Lopez-Beltran A, Alvarez-Kindelan J, Luque RJ, Blanca A, Quintero A, Montironi R, Cheng L, Gonzalez-Campora R, Requena MJ: Loss of heterozygosity at 9q32-33 (DBC1 locus) in primary non-invasive papillary urothelial neoplasm of low malignant potential and low-grade urothelial carcinoma of the bladder and their associated normal urothelium. J Pathol 2008;215:263-272 31. Montironi R, Lopez-Beltran A, Scarpelli M, Mazzucchelli R, Cheng L: Morphological classification and definition of benign, preneoplastic and non-invasive neoplastic lesions of the urinary bladder. Histopathology 2008;53:621-633 32. Lopez-Beltran A, Montironi R: Non-invasive urothelial neoplasms according to the most recent WHO classification. European Urology 2004;46:170-176 33. Lopez Beltran A, Luque RJ, Moreno A, Bollito E: The pagetoid variant of bladder urothelial carcinoma in situ: A clinicopathological study of 11 cases. Virchows Arch 2002;441: 148-153 34. Lopez Beltran A, Cheng L, Andersson L, Brausi M, Van Der Kwast TH, Sesterhenn I, Mazzerolles C: Preneoplastic nonpapillary lesions and conditions of the urinary bladder: An update based on the Ancona international consultation. Virchows Arch 2002;440:3-11 35. Gunia S, Kakies C, Erbersdobler A, Koch S, May M: Scoring the percentage of Ki67 positive nuclei is superior to mitotic count and the mitosis marker phosphohistone H3 (PHH3) in terms of differentiating flat lesions of the bladder mucosa. J Clin Pathol 2012;65:715-720 36. Gunia S, Koch S, Hakenberg OW, May M, Kakies C, Erbersdobler A: Different HER2 protein expression profiles aid in the histologic differential diagnosis between urothelial carcinoma in situ (CIS) and non-cis conditions (dysplasia and reactive atypia) of the urinary bladder mucosa. Am J Clin Pathol 2011;136:881-888 37. Gunia S, Kakies C, May M, Koch S, Erbersdobler A: Lewis(y) antigen (blood group 8, BG8) is a useful marker in the histopathological differential diagnosis of flat urothelial lesions of the urinary bladder. J Clin Pathol 2011;64:672-676 38. Murata S, Iseki M, Kinjo M, Matsuzaki O, Moriuchi A, Ohtani H, Sakurai T, Satake T, Tsuzuki T: Molecular and immunohistologic analyses cannot reliably solve diagnostic variation of flat intraepithelial lesions of the urinary bladder. Am J Clin Pathol 2010;134:862-872 39. Zieger K, Marcussen N, Borre M, O/rntoft TF, Dyrskjo/t L: Consistent genomic alterations in carcinoma in situ of the urinary bladder confirm the presence of two major pathways in bladder cancer development. Int J Cancer 2009;125: 2095-2103 40. Edgecombe A, Nguyen BN, Djordjevic B, Belanger EC, Mai KT: Utility of cytokeratin 5/6, cytokeratin 20, and p16 in the diagnosis of reactive urothelial atypia and noninvasive component of urothelial neoplasia. Appl Immunohistochem Mol Morphol 2012;20:264-271 41. Siegel R, Naishadham D, Jemal A: Cancer statistics, 2012. CA Cancer J Clin 2012;62:10-29 42. Shirai T, Fukushima S, Hirose M, Ohshima M, Ito N: Epithe- D

10 Lopez-Beltran et al lail lesions of the urinary bladder in 313 autopsy cases. Jpn J Cancer Res 1987;78:1073-1080 43. Chan TY, Epstein JI: Radiation or chemotherapy cystitis with pseudocarcinomatous features. Am J Surg Pathol 2004;28: 909-913 44. Oxley JD, Cottrell AM, Adams S, Gillatt D: Ketamine cystitis as a mimic of carcinoma in situ. Histopathology 2009;55:705-708 45. Jung S, Wu C, et al: The role of immunohistochemistry in the diagnosis of flat urothelial lesions: A study using CK20, CK5/6, P53, Cd138, and Her2/neu. Ann Diagn Pathol 2014; 18:27-32 46. International Agency for Research on Cancer/World Health Organization: Globocan 2012: Estimated Cancer Incidence, Mortality and Prevalence Worldwide in 2012. Available at http://globocan.iarc.fr/pages/fact_sheets_cancer.aspx. Accessed 47. Amin MB, Trpkov K, Lopez-Beltran, et al: Best practices recommendations in the application of immunohistochemistry in the bladder lesions. Report from the international society of urologic pathology consensus conference. Am J Surg Pathol 2014;38:20-34 48. Sfakianos JP, Lin GL, et al: The role of PTEN tumor suppressor pathway staining in carcinoma in situ of the bladder. Urol Oncol 2014;32:657-662 D