ICUD-EAU International Consultation on Bladder Cancer 2012: Pathology

EUROPEAN UROLOGY 63 (2013) 16 35 available at www.sciencedirect.com journal homepage: www.europeanurology.com Review Bladder Cancer ICUD-EAU International Consultation on Bladder Cancer 2012: Pathology Mahul B. Amin a, *, Jesse K. McKenney b, Gladell P. Paner c, Donna E. Hansel d, David J. Grignon e, Rodolfo Montironi f, Oscar Lin g, Merce Jorda h, Lawrence C. Jenkins i, Mark Soloway i, Jonathan I. Epstein j, Victor E. Reuter g a Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA; b Department of Pathology, Cleveland Clinic, Cleveland, OH, USA; c Department of Pathology, University of Chicago, Chicago, IL, USA; d Department of Anatomic Pathology, Cleveland Clinic, Cleveland, OH, USA; e Department of Pathology and Laboratory Medicine, Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA; f Section of Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Ancona, Italy; g Department of Pathology, Memorial Sloan- Kettering Cancer Center, New York, NY, USA; h Department of Pathology, University of Miami, Miami, FL, USA; i Department of Urology, University of Miami, Miami, FL, USA; j Department of Pathology, The Johns Hopkins Hospital, Baltimore, MD, USA Article info Article history: Accepted September 27, 2012 Published online ahead of print on October 5, 2012 Keywords: Bladder cancer Pathology Markers Immunohistochemistry Staging Recommendations Abstract Context: To present a summary of the 2nd International Consultation on Bladder Cancer recommendations on the pathology of bladder cancer using an evidence-based strategy. Objective: To standardize descriptions of the diagnosis and reporting of urothelial carcinoma of the bladder and help optimize uniformity between individual pathology practices and institutions. Evidence acquisition: A detailed Medline analysis was performed for original articles addressing bladder cancer with regard to pathology. Proceedings from the last 5 yr of major conferences were also searched. Evidence synthesis: The major findings are presented in an evidence-based fashion. Large retrospective and prospective data were analyzed. Conclusions: Providing the best management for patients with bladder neoplasia relies on close cooperation and teamwork among urologists, oncologists, radiologists, and pathologists. # 2012 European Association of Urology. Published by Elsevier B.V. All rights reserved. * Corresponding author. Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, 116 N. Robertson Blvd., PACT Suite 500, Los Angeles, CA 90048, USA. Tel. +1 310 423 6627; Fax: +1 310 423 0170. E-mail address: Mahul.Amin@cshs.org (M.B. Amin). 1. Introduction We summarize the 2nd International Consultation on Bladder Cancer recommendations on the pathology of bladder cancer using an evidence-based strategy. We provide new consensus guidelines built on past endeavors, supported by a comprehensive review of the recent literature where possible and based on the best practices of an expert group of 38 international urologic pathologists from 13 countries. In this international consultation, we provide an update on (1) the knowledge of the histoanatomy of the bladder as it pertains to the diagnosis and staging of bladder cancer, (2) the prognostic significance of histologic grading by the World Health Organization (WHO) (2004)/International Society of Urological Pathology (ISUP) system, (3) the information the pathologist needs from clinicians to provide optimal pathologic reporting of bladder cancer, (4) the histologic types of bladder cancer and the variants of urothelial cancer 0302-2838/$ see back matter # 2012 European Association of Urology. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.eururo.2012.09.063

EUROPEAN UROLOGY 63 (2013) 16 35 17 with a focus on definitions for diagnosis and their implied clinicopathologic significance, (5) the role of immunohistochemistry and molecular studies, (6) the reporting of bladder cancer in transurethral resection of bladder tumor (TURBT) and cystectomy specimens, and (7) recommended nomenclature for urine cytology. 2. Evidence acquisition A detailed Medline analysis was performed for original articles addressing bladder cancer with regard to screening, diagnosis, markers, and pathology. Proceedings from the last 5 yr of major conferences were also searched. The evidence available was analyzed using the Oxford method of assigning levels of evidence (LEs) and summary recommendations. Based on these, LEs were graded as advised by the Oxford Centre for Evidence-based Medicine and integrated into each section [1]. 3. Evidence synthesis 3.1. Urinary bladder histoanatomy and impact on cancer diagnosis and staging 3.1.1. Epithelial changes 3.1.1.1. Urothelial denudation. Prior instrumentation and intravesical therapy are contributors to denudation. Urothelial carcinoma in situ (CIS) is also often associated with prominent cellular dyscohesion and exfoliation of neoplastic cells in the urine. The presence of extensive denudation in bladder biopsy samples has been associated with CIS [2] (Fig. 1). Extensive urothelial denudation in biopsy samples should be reported (grade of recommendation [GR]: C). 3.1.1.2. Urothelial hyperplasia. Urothelial hyperplasia is defined as markedly thickened urothelium in the absence of cytologic atypia and can be flat or papillary. Flat hyperplasia may be seen adjacent to low-grade papillary tumors [2]. Papillary urothelial hyperplasia is characterized by the presence of [(Fig._1)TD$FIG] Fig. 1 Urothelial carcinoma in situ with denudation. undulating hyperplastic urothelium arranged into thin mucosal papillary folds of varying heights in a noninflamed setting [2]. The lack of cytologic atypia and fibrovascular cores distinguishes this from papillary neoplasia. From a practical viewpoint, there is difficulty and controversy in diagnosing lesions that do not have fully established features of papillary neoplasia and also have accompanying cytologic atypia exceeding reactive changes. This may occur in the setting of prior treatment and in surveillance biopsies. The degree of urothelial atypia when present in the hyperplastic urothelium should be reported using the 2004 WHO/ISUP terminology for urothelial neoplasia (eg, dysplasia or CIS) (GR: C). Correlation with cystoscopy is valuable to ascertain if the lesions are papillary. 3.1.1.3. Squamous metaplasia. Nonkeratinizing squamous epithelium is present in the trigone in 75 86% of women and is a normal finding (LE: 2) [3,4]; its occurrence outside this region is considered metaplastic. The keratinizing type of squamous metaplasia usually occurs in response to chronic irritative stimuli to the bladder such as cystitis, lithiasis, diverticula, or schistosomiasis. Keratinizing squamous metaplasia can be associated with subsequent or concurrent in situ or invasive carcinomas with squamous differentiation (LE: 3) and has been suggested to be a precursor lesion for squamous cell carcinoma, and thus must be closely followed (GR: C) [5]. Pathologists should report the presence of squamous metaplasia in bladder biopsies, specifying keratinizing or not and focal or multifocal (GR: C). Current evidence does not support any form of intervention including surveillance cystoscopy for patients with focal changes, although multifocal and/or extensive lesions often require intervention due to intractable symptoms or because they precede or may be associated concurrent with neoplasia (GR: C). Spinal cord injury or paraplegic patients with squamous metaplasia must be evaluated regularly because of the increased risk for squamous cell carcinoma (GR: C). 3.1.1.4. Glandular metaplasia. Glandular metaplasia (cystitis glandularis) is a benign proliferative change that may occasionally show the presence of intestinal-type goblet cells (cystitis glandularis, intestinal type, or intestinal metaplasia). Glandular metaplasia is supposed to relate the risk of neoplastic transformation because intestinal metaplasia often coexists with adenocarcinoma of the urinary bladder [6,7]. Glandular metaplasia of the urinary bladder is not a strong risk factor for adenocarcinoma or urothelial cancer (LE: 3) [8,9]. Multifocal disease with intestinal metaplasia or that associated with dysplastic changes may warrant close clinical follow-up (GR: C). 3.1.1.5. Reactive changes. Therapeutic procedures for bladder cancer such as chemotherapy, immunotherapy, radiotherapy, photodynamic and laser treatment, previous resection, and gene therapy may produce alterations in the urothelial mucosa, and some of these changes may be mistaken for carcinoma [10] (Fig. 2). Knowledge of prior therapy is invaluable for pathologists, and clinicians should provide this information.

18 [(Fig._2)TD$FIG] EUROPEAN UROLOGY 63 (2013) 16 35 [(Fig._3)TD$FIG] Fig. 3 Invasive urothelial carcinoma involving muscularis mucosae muscle (pt1). [(Fig._4)TD$FIG] Fig. 2 Urothelial atypia after mitomycin therapy. 3.1.2. Lamina propria 3.1.2.1. Interpretation variability of (early) lamina propria invasion (pt1). There are issues with interobserver reproducibility in the diagnosis of early lamina propria (LP) invasion [11,12]. Because of the diagnostic difficulty, when early invasive urothelial carcinoma is suspected, diagnosis through examination of additional levels or in consultation with a pathology colleague is encouraged (GR: C). 3.1.2.2. Microinvasive urothelial carcinoma. Currently, there are no prospective studies or universally accepted guidelines for utilization of this terminology (LE: 4). Microinvasive urothelial carcinoma is considered present when carcinoma extends beyond the basement membrane into the LP. Stringent criteria that should be used include only focal invasion, less than one high-power field, and 0.2 cm from the nearest basement membrane (GR: C). 3.1.2.3. Substratification or substaging of lamina propria invasion (pt1). The usefulness of the subclassification or substaging of LP invasion is still controversial. Problems precluding use of the depth of invasion criteria include lack of consistent orientation with the basement membrane, specimen fragmentation, issues with reproducibility in measurement, and lack of established clinical significance. A two-tiered system identifying LP invasion up to pt1a or beyond the muscularis mucosae (MM) or vascular plexus (pt1b) and a three-tiered system identifying submucosal tumor invasion up to pt1a, in pt1b, or beyond (pt1c) the MM or vascular plexus (LE: 3) has been used [13 15]. Substaging of pt1 (Fig. 3) is currently not recommended by the WHO/ISUP consensus guidelines committee. It is recommended that pathologists provide some form of Fig. 4 Hyperplastic muscularis mucosae, haphazard type. estimate of the LP invasion in pt1 tumors (eg, focal, multifocal, extensive, etc.) (GR: C). 3.1.3. Muscularis mucosae The MM is an important histoanatomic structure to differentiate from the muscularis propria (MP), or deep muscle, when staging invasive urothelial carcinoma in the bladder. MM muscle bundles are typically thin slender bundles arranged in a single layer of interrupted, dispersed, or continuous muscle [16]. Two patterns of hyperplastic MM were recently described: (1) aggregates with haphazard (Fig. 4) orientation and irregular outlines morphologically distinct from that of MP and (2) hyperplastic compact MM with small parallel muscle fibers and regular outlines arranged singly or in small groups (Fig. 5). The latter may strongly resemble MP muscle, and the most reliable distinguishing feature from MP is on the basis of its location

[(Fig._5)TD$FIG] EUROPEAN UROLOGY 63 (2013) 16 35 19 [(Fig._6)TD$FIG] Fig. 5 Hyperplastic muscularis mucosae, compact type, that resembles muscularis propria muscle bundles. Fig. 6 Urothelial carcinoma invading muscularis propria muscle (pt2). in the LP [16], which is not always possible in limited specimens such as in TURBT (LE: 3). To avoid confusion in the interpretation of the pathology report, documentation of MM-only invasion by carcinoma is not required and should be reported as urothelial carcinoma with LP invasion (pathologic stage at least pt1) when MP is not present and urothelial carcinoma with LP invasion (pathologic stage pt1) when MP is present (GR: C). Involvement of the MM may be included in a comment to provide information on the depth/extent of invasion. It is important to document whether MP is present in a bladder specimen with high-grade pta and all invasive urothelial neoplasia (GR: B). 3.1.4. Indeterminate muscle type It may be difficult to distinguish between MM and MP muscle bundles in some TURBT samples. When indeterminate muscle bundles are observed, the presence of vascular network with thick-walled vessels may aid in recognition as MM (LE: 3) [14,16,17]. Muscle bundles indeterminate between MM and MP should be reported with terminology such as invasive urothelial carcinoma with invasion of muscle, indeterminate type to prompt the urologist for a restaging biopsy procedure (GR: C). A second biopsy may provide more definitive staging (GR: C) [18]. 3.1.5. Muscularis propria Pathologic staging is considered inadequate without detailing the presence of uninvolved MP in high-grade pta or pt1 tumors. If MP is not present, repeat resection is recommended for verification of pt stage before a subsequent management decision is made (GR: B). Several terms have been used to report MP muscle such as deep muscle, muscle proper, or detrusor muscle. Use of different terms for bladder muscles may generate confusion and are not recommended. The use of standardized nomenclature of MP muscle bundles is recommended in reporting (GR: C). MP invasion has been divided into two categories, superficial (pt2a) and deep (pt2b), based on the depth of tumor invasion. Substaging of pt2 disease (Fig. 6) and recognition of invasion beyond MP (pt3 disease) is not tenable in TURBT specimens. In cystectomy specimens, pt2 substaging is complicated by the lack of a reliable anatomic landmark to aid in their distinction (LE: 3). Currently, there are no data regarding the reproducibility of distinguishing pt2a versus pt2b among pathologists (LE: 4). Some outcome studies have shown the lack of prognostic impact and are against the use of pt2 substaging [19,20]; some other reports show the prognostic value of pt2a and pt2b subcategories [21,22]. 3.1.6. Adipose tissue Adipose tissue is frequently present in the LP and MP of the urinary bladder, usually scant in the former and abundant in the latter [23]. Involvement of adipose tissue by tumor in biopsy or TURBT specimens should not be interpreted as perivesical fat involvement or pt3 disease. Because of the consistent presence of adipose tissue (Fig. 7) in the MP, the boundary of perivesical adipose tissue from the deep (outer) MP may not be easily delineated (LE: 3) [23], and consequently, distinction between pt2b and pt3a in a cystectomy may not be straightforward (LE: 3). The prognostic significance of pt3a versus pt2 has also been questioned because it has been shown to have no significant difference in recurrence and mortality rate and incidence of lymph node involvement [24]. There is limited reliability of pt3a versus pt3b subcategorization because gross involvement of perivesical fat may not always be readily recognizable (LE: 3). Perivesical tissue reactions to the tumor or prior biopsy may be overinterpreted as extravesical fat involvement. The prognostic significance of the pt3 subcategory is controversial; some reports did not show survival differences [25 27], whereas some did show differences between pt3a and pt3b (LE: 4) [28,29].

20 [(Fig._7)TD$FIG] EUROPEAN UROLOGY 63 (2013) 16 35 3.2.2.2. Papillary urothelial neoplasm of low malignant potential. Papillary urothelial neoplasm of low malignant potential (PUNLMP) is characterized by cytologically bland/normalappearing cells with hyperplasia (increased cells per unit area and increased cell layers) on a fibrovascular core. These morphologic characteristics may cause low interobserver reproducibility, however, when distinguishing them from low-grade urothelial carcinoma [37]. In general, PUNLMP has a significantly lower rate of recurrence than either low- or high-grade papillary carcinomas and a very low rate of grade and stage progression (LE: 2). In a review of published studies, a mean tumor recurrence rate was 36%, and the stage progression rate was 3.7% [38]. 3.2.2.3. Low-grade papillary carcinoma. Low-grade papillary urothelial carcinoma shows cells with low-grade cytologic atypia (resembling urothelial dysplasia in a flat lesion) on a fibrovascular core. These tumors have a significantly higher recurrence rate than PUNLMP (LE: 2). They also have a higher rate of stage progression than PUNLMP but significantly lower than for high-grade papillary carcinoma (LE: 2). A review reveals a mean recurrence rate of 50% and a mean stage progression rate of 10% [32]. Fig. 7 Urothelial carcinoma invading adipose tissue within the muscularis propria. 3.2. Classification and grading of noninvasive urothelial neoplasms of the urinary bladder The noninvasive urothelial neoplasms may be flat, papillary (exophytic), or inverted (endophytic) depending on their growth pattern relationship with the surface of the urothelial mucosa [30]. All three growth patterns may be seen concomitantly in a single tumor (Fig. 8). 3.2.1. Flat lesions and neoplasms 3.2.1.1. Urothelial dysplasia. Urothelial dysplasia is characterized by architectural distortion and a variable degree of atypia. Overall features are those of a neoplastic atypia (clear-cut nuclear atypia) but fall short of the criteria for CIS; dysplasia is not graded further. 3.2.1.2. Urothelial carcinoma in situ. CIS is characterized by architectural disorder and distinct high-grade nuclear atypia. There is a spectrum of nuclear and architectural atypia. The development of invasion is seen in 20 30% of the cases (LE: 2) [31 33]. 3.2.2. Papillary (exophytic) neoplasms 3.2.2.1. Urothelial papilloma. Urothelial papilloma is characterized by a few fine papillary fronds lined by normalappearing urothelium. It has a low recurrence rate, and progression in such patients is rare (LE: 2) [34 36]. 3.2.2.4. High-grade papillary carcinoma. High-grade papillary carcinoma is characterized by cells with high-grade cytologic atypia (resembling CIS in a flat lesion) on a fibrovascular core. It is often associated with invasive disease at diagnosis [39 43]. These tumors not only have a risk of invasion but also have a significant risk of recurrence and progression; association with multifocality and CIS is common (LE: 2). The overall progression rate (to invasive carcinoma) ranges from 15% to 40%; more than half the cases are prone to recurrences. 3.2.3. Grading papillary urothelial neoplasms with histologic heterogeneity Grade heterogeneity is not uncommonly encountered in papillary urothelial neoplasms. The 2004 WHO (1998 ISUP/ WHO) system recommends basing the grading of heterogeneous tumor on the highest grade tumor (GR: C). When assigning the tumor grade in tumors with borderline grade histology, other tumor parameters such as multifocality, previous grade, size of lesions, frequency of recurrence, presence of concurrent CIS, and positive cytology may be factored in the grading; and the presence of a few or many of these parameters may influence upgrading to a high-grade lesion (GR: C). 3.2.4. Inverted (endophytic) neoplasms A formal grading system is not proposed for lesions with a predominant inverted growth; nor is one universally used. We recommend that for tumors with predominantly inverted growth patterns, criteria that parallel the WHO (2004)/ISUP system for exophytic tumors and flat lesions be used (shown in Table 1). 3.3. Grading of invasive urothelial carcinomas (PT1 and PT2+ tumors) Low-grade urothelial carcinomas are considered incapable of invasive behavior. It is therefore recommended when tumors are invasive that they be graded as high grade (GR: C). This rule is also applicable to the general surgical and urologic pathology community because deceptively bland variants such as the nested or small tubular variants that histologically appear low grade tend to behave like invasive high-grade tumors of a similar stage (GR: C). If for

[(Fig._8)TD$FIG] EUROPEAN UROLOGY 63 (2013) 16 35 21 Fig. 8 Examples of the spectrum of tumors in a cystectomy. (A) Whole mount section of a bladder with a preoperative diagnosis of muscle-invasive highgrade papillary carcinoma. (B) Urothelial dysplasia from the specimen shown in Figure 8A, a papillary neoplasm. (C) The neoplasm grows exophytically into the lumen of the urinary system with a papillary configuration. (D) Low-grade papillary carcinoma from the specimen shown in Figure 8C, and an inverted neoplasm. (E) The neoplasm, even though a polypoid appearance, is characterized by an epithelium that shows a noninfiltrative growth into the subepithelial connective tissue. (F) Low-grade inverted urothelial carcinoma from the specimen shown in Figure 8E. Table 1 Analogy in nomenclature for inverted lesions Flat lesions Papillary lesions * Predominant or exclusive inverted lesions * Normal Papilloma Inverted papilloma Urothelial hyperplasia Papillary neoplasm of low malignant potential Inverted papillary neoplasm of low malignant potential Urothelial dysplasia Papillary urothelial carcinoma, low grade, noninvasive Inverted papillary urothelial carcinoma, low grade, noninvasive Urothelial CIS Papillary urothelial carcinoma, high grade, noninvasive Inverted papillary urothelial carcinoma, high grade, noninvasive Papillary urothelial carcinoma, high grade, invasive Inverted papillary urothelial carcinoma, high grade, invasive CIS = carcinoma in situ. * Lesions may be both exophytic and endophytic. From Epstein et al. [45].

22 EUROPEAN UROLOGY 63 (2013) 16 35 [(Fig._10)TD$FIG] institutional disease management teams or clinical trials (eg, for some institutions and protocols in Europe), assigning grade G2 and G3 is important, the practice should continue based on respective protocols or institutional guidelines. 3.4. Urothelial carcinoma variants Variants of urothelial carcinoma are an uncommon small subset of carcinomas, and evidence therefore consists of small series and case reports (LE: 3/4). 3.4.1. Urothelial carcinoma with divergent differentiation Urothelial carcinoma has a propensity for divergent differentiation, with the most common squamous differentiation followed by glandular differentiation [44 46] (Figs. 9 and 10). To designate a bladder tumor as squamous cell or adenocarcinoma, a pure or almost pure histology is required. From a practical viewpoint, we do make note of prominent squamous or glandular differentiation in urothelial carcinoma using diagnostic terminology such as invasive urothelial carcinoma with prominent squamous (40%) and glandular (25%) differentiation [44 46]. The prognostic significance of squamous or glandular differentiation is unclear, although some studies have suggested an adverse outcome [44 46]. This difference in prognosis may not be apparent when corrected for stage of the disease. Fig. 10 Urothelial carcinoma with squamous differentiation. [(Fig._11)TD$FIG] 3.4.2. Micropapillary variant of urothelial carcinoma Micropapillary carcinoma (MPC) of the urinary tract is a special type of urothelial carcinoma that has papillary structures reminiscent of those seen in ovarian papillary serous neoplasms and typically lacks central fibrovascular cores seen within the usual papillary urothelial carcinoma [47] (Fig. 11). The percentage of the MP component has been shown to be a significant adverse prognostic factor with more MPC components, the worse prognosis [48]. However, because any amount of MPC has an impact on the prognosis, [(Fig._9)TD$FIG] Fig. 11 Micropapillary variant of urothelial carcinoma. no percentage limitation is required for the diagnosis of MPC, but the percentage of the MPC component should be reported. Most cases are muscle invasive at presentation. Recent data suggest that because of limited response to intravesical bacillus Calmette-Guérin (BCG), one group has advocated radical cystectomy for pta and pt1 tumors with MP histology [49]. However, this recommendation is not universally accepted. The prognosis is uniformly unfavorable; the 5-yr and 10-yr survival in the largest study was 51% and 24%, respectively. However, this poor prognosis is similar to that reported in high-grade, high-stage urothelial carcinoma of the usual type [50]. Fig. 9 Urothelial carcinoma with glandular differentiation. 3.4.3. Nested variant of urothelial carcinoma A variant of invasive urothelial carcinoma consisting of small irregularly distributed nests with bland cytology may simulate benign processes such as Von Brunn nests [51,52] (Fig. 12). The nested variant of urothelial carcinoma typically consists of nests of bland-appearing urothelial cells irregularly distributed within the LP and often showing

[(Fig._12)TD$FIG] EUROPEAN UROLOGY 63 (2013) 16 35 23 [(Fig._14)TD$FIG] Fig. 12 Nested variant of urothelial carcinoma, low power. Fig. 14 Sarcomatoid urothelial carcinoma. invasion of the MP. Some authors suggest that this tumor is more aggressive than the usual invasive urothelial carcinoma; others suggest that the prognosis and clinical course is similar to high-grade urothelial carcinoma stage for stage [51 53]. 3.4.4. Lymphoepithelioma-like carcinoma Lymphoepithelioma-like carcinoma of the urinary bladder is a variant of urothelial carcinoma in which there is a dense inflammatory infiltrate surrounding nests of poorly differentiated carcinoma cells, imparting a close resemblance to the well-known lymphoepitheliomas of the head and neck (Fig. 13). In pathology reports, it would be appropriate to state whether or not the tumor is pure lymphoepitheliomalike carcinoma. 3.4.5. Sarcomatoid carcinoma of the urinary bladder Sarcomatoid carcinoma of the urinary bladder is a malignant urothelial-derived carcinoma that displays both spindle [(Fig._13)TD$FIG] cell and epithelial elements (formerly carcinosarcoma). The spindle cell component usually shows high-grade morphology with nondescript architecture that often resembles malignant fibrous histiocytoma (Fig. 14). Heterologous differentiation is often present that may contain, in decreasing order of frequency, areas that resemble osteosarcoma, chondrosarcoma, rhabdomyosarcoma, liposarcoma, angiosarcoma, or a mixture of sarcomatoid histologies [54,55]. Despite the morphologic resemblance to sarcoma, it is currently recognized that these spindle cell areas are derived from an underlying epithelial malignancy, hence the revised terminology of sarcomatoid carcinoma. Compared with patients with pure urothelial carcinoma, patients with sarcomatoid carcinoma are at a greater risk for death even after adjusting for the stage at presentation. 3.4.6. Undifferentiated urothelial carcinoma with trophoblastic giant cells Trophoblastic differentiation may be seen in several settings in urothelial carcinoma: (1) serologically and immunohistochemically in otherwise typical urothelial carcinoma, or (2) morphological evidence of trophoblastic differentiation either with scattered trophoblastic giant cells in urothelial carcinoma or with a biphasic tumor indistinguishable from choriocarcinoma [56 58]. Human chorionic gonadotropin immunoreactivity in pretreatment in some studies has been a strong predictor of treatment response. Fig. 13 Lymphoepithelioma-like carcinoma. 3.4.7. Undifferentiated carcinoma (including giant cell carcinoma) Undifferentiated carcinomas include carcinomas of the urinary bladder with a phenotype composed of sheets or isolated undifferentiated cells that do not fit into urothelial, squamous, adenocarcinoma, or any other recognized category of bladder carcinoma [44,45,59 61]. Some authorities would classify these cases as large cell undifferentiated carcinoma [45,60,61]. Others may prefer to denote these lesions as high-grade urothelial carcinoma,

24 EUROPEAN UROLOGY 63 (2013) 16 35 [(Fig._15)TD$FIG] given that this is the most common cancer within the bladder and this pattern of tumor most likely represents the most poorly differentiated manifestation of conventional urothelial carcinoma [45,60,61]. Large cell undifferentiated bladder carcinoma is an aggressive variant of urothelial carcinoma; all patients had advanced stage cancer ( pt3), and 87.5% had lymph node metastases [44,45,59 62]. 3.5. Squamous cell carcinoma 3.5.1. Squamous cell carcinoma, nonschistosomal Urinary bladder squamous cell carcinoma is a primary malignancy of urothelial origin that consists almost entirely of keratin-forming squamous carcinoma. Squamous cell carcinoma comprises approximately 5% of all bladder cancers in the Western world [63]. The 5-yr disease-free survival following radical cystectomy ranges from 43% to 57% [50,64,65]. The poor outcomes associated with this disease may be attributed to the high stage at initial presentation and the relative frequency of regional lymph node metastases in 24% of patients at the time of cystectomy [64]. However, when compared with urothelial carcinoma on a stage-by-stage basis, outcomes appear similar, with a 5-yr disease-specific survival rate of 57% [50]. Squamous cell carcinoma is associated with a limited response to chemotherapy and radiation treatment. A key challenge is managing patients who present with squamous cell carcinoma on initial biopsy or transurethral resection. Due to sampling, the exclusion of a high-grade urothelial carcinoma with squamous differentiation is challenging, and this distinction can be made primarily on radical cystectomy when the entire lesion is available for histologic analysis. A second challenge is the management of squamous cell CIS lesions found on biopsy. The most concerning of all associated mucosal lesions is squamous cell carcinoma, which has a high likelihood of progression to invasive carcinoma. One study that followed in situ lesions found that five of seven patients with squamous cell CIS developed invasive carcinoma [5]. 3.5.2. Verrucous carcinoma Verrucous carcinoma is a rare subtype of squamous cell carcinoma most often related to urinary schistosomiasis infection [66,67], but it may also rarely occur in its absence [68 70]. It is a well-differentiated invasive squamous neoplasm with a broad deep border that pushes into the underlying stroma rather than demonstrating frank stromal invasion typical of a conventional squamous cell carcinoma. 3.5.3. Squamous cell carcinoma, schistosomal Urinary bladder squamous cell carcinoma consists almost entirely of keratin-forming squamous carcinoma and is associated with a precedent or concurrent infection with schistosomal species. The 5-yr overall survival for schistosomal-associated squamous cell carcinoma is approximately 50%. Fig. 15 Adenocarcinoma with enteric features. 3.6. Adenocarcinoma variants 3.6.1. Primary adenocarcinoma of the bladder Urinary bladder adenocarcinoma is a primary malignancy that shows glandular differentiation with features of enteric, mucinous, or signet-ring cell carcinoma. Five specific morphotypes have been described [71,72]: (1) Enteric tumors are acinar, cribriform, villous, or solid (Fig. 15); (2) mucinous or colloid carcinomas consist of individual cells and cell nests within lakes of mucin; (3) signet-ring cell carcinoma is restricted to those tumors that show a diffuse signet-ring morphology with varying amounts of mucin and without foci of glandular, urothelial, or squamous differentiation [73,74]; (4) adenocarcinoma not otherwise specified (NOS) applies to tumors where the architectural pattern does not conform to the preceding three morphotypes; and (5) mixed forms show two or more morphologic patterns. It has been suggested that to be included in this category, a tumor should not exhibit >75% of one tumor morphotype [72,75]. Bladder adenocarcinoma has a poor prognosis with 5-yr survivals of 11 55% [76]. Tumor stage at diagnosis has been shown to be an important prognostic feature [71,72]. Comparison showed that adenocarcinomas had the same natural history as urothelial carcinoma following radical cystectomy, but patients with adenocarcinoma were usually of a more advanced stage at the time of treatment. Morphology has been associated with outcome, with 5-yr survival rates 64% for tumors showing mixed morphology, 55% for mucinous adenocarcinomas, 51% for adenocarcinoma NOS, and 27% for signet-ring cell adenocarcinomas, although these data did not achieve statistical significance [73]. 3.6.2. Urachal carcinoma Urachal carcinomas are malignant tumors arising in urachal remnants. Although the great majority of these tumors are adenocarcinomas originating from vestigial urachal epithelium, squamous cell carcinoma, urothelial carcinoma,

EUROPEAN UROLOGY 63 (2013) 16 35 25 Table 2 Criteria for the diagnosis of urachal adenocarcinoma Wheeler and Hill [17] 1. Tumor in dome of bladder 2. Absence of cystitis cystica or cystitis glandularis 3. Predominant involvement of muscularis rather than submucosa, with intact or ulcerative bladder epithelium 4. Urachal remnant connected to neoplasm 5. Suprapubic neoplastic mass Mostofi et al. [14] Criteria additional to Wheeler and Hill [17] 1. Location in anterior wall of bladder 2. Sharp demarcation of tumor and surface epithelium 3. Extension to space of Retzius, umbilicus, or anterior abdominal wall Johnson et al. [18] 1. Tumor in bladder dome 2. Sharp demarcation between tumor and bladder epithelium 3. Exclusion of another primary (nonurachal) adenocarcinoma Gopalan et al. [4] 1. Tumor in dome/anterior wall 2. Epicenter of carcinoma in bladder 3. Absence of widespread cystitis cystica/glandularis beyond the dome/anterior bladder 4. No known primary elsewhere neuroendocrine carcinoma, and sarcomas have also been reported, albeit rarely [77]. Because of the morphologic overlap of vesical and urachal adenocarcinoma, several diagnostic criteria for urachal tumors have been proposed, as shown in Table 2. In earlier series, the prognosis of urachal carcinoma was found to be poor with a 5-yr survival of 61% [78,79]. [(Fig._17)TD$FIG] 3.6.3. Clear cell adenocarcinoma The designation of clear cell adenocarcinoma should be reserved for tumors resembling to a significant degree clear cell adenocarcinoma of the müllerian type as encountered in the female genital tract and should have one or more of the typical tubulocystic, solid, and papillary patterns as well as one or more of the typical cell types including clear cell, nonspecific cuboidal, and hobnail [80,81] (Fig. 16). The prognostic parameters are similar to those of conventional carcinoma. There is no evidence that the behavior, stage for stage, is different from the usual bladder cancer. [(Fig._16)TD$FIG] Fig. 17 Small cell carcinoma with squamous differentiation. 3.7. Neuroendocrine carcinoma of the bladder Fig. 16 Clear cell variant urothelial carcinoma, tubulocystic pattern. This neoplasm, from a woman, was associated with endometriosis. Neuroendocrine carcinoma of the bladder is a group of tumors comprising small cell carcinoma and large cell neuroendocrine carcinoma and mixed patterns [82] (Fig. 17). Histologically, small cell carcinoma shows a patternless arrangement of blue and hyperchromatic round cells, nuclear molding, smudging artifact, abundant mitoses, and geographic necrosis. Large cell neuroendocrine carcinomas have a discernible histopathologic architecture including palisading, trabecular, organoid, and nesting patterns. The cells are larger than those of small cell carcinoma, and both share a high mitotic rate [83]. Both small and large cell high-grade neuroendocrine carcinomas of the urinary bladder often present at an advanced stage, with up to 94% of cases having MP invasion or extravesical extension. Metastasis at time of presentation is not uncommon. Prognosis is influenced by the disease extent

26 EUROPEAN UROLOGY 63 (2013) 16 35 Table 3 Most commonly used immunohistochemical markers for staging of bladder cancers Pathologic issue Immunohistochemical marker Caveats Detection of microinvasion into cauterized or damaged tissue Pankeratin, keratin 8, 18 Keratin-positive myofibroblasts Residual cancer cells post-tur Pankeratin Keratin-positive myofibroblasts pt1 substaging Desmin, pankeratin pt1 versus pt2 staging Smoothelin, vimentin Overlap in staining intensity of muscularis mucosae and detrusor muscle TUR = transurethral resection. at diagnosis, use of chemotherapy, and the patient s performance status [84 86]. 3.8. Role of immunohistochemistry and ancillary diagnostics in the diagnosis, staging, prognostication, and prediction of bladder cancer in contemporary clinical practice 3.8.1. Immunohistochemical markers for the staging of bladder cancer 3.8.1.1. Determination of invasiveness of bladder cancer. The identification of invasive bladder cancer cells can be challenging, especially when the number of invading cells is small or in the presence of cautery or mechanical artifacts. Although no formal studies have been reported in the literature, it is well established that immunostaining with anticytokeratin antibodies can facilitate the diagnosis of invasion in cases with prominent inflammation obscuring the interface between epithelium and stroma. The most commonly used antibodies for this purpose are pankeratins like AE1/AE3 and antibodies against cytokeratins 8 and 18 [87], as shown in Table 3. 3.8.1.2. Immunohistochemical markers for substaging pt1 bladder cancers. There are no data to show that using immunohistochemistry for substaging improves the prediction of recurrent or progressive disease, and therefore it is not recommended for routine application. 3.8.2. Summary of molecular/genetic alterations in urothelial carcinoma Despite the extensive new information and knowledge on bladder cancer genetics, epigenetics, and gene expression, much is needed to satisfy several unanswered questions. Table 4 outlines the commonly known genetic and epigenetic aberrations in urothelial carcinoma, and Table 5 provides a summary of loss of heterozygosity and comparative genomic hybridization analyses. Much of the work points to two distinct pathways that correspond to two main groups of tumors: low-grade urothelial carcinomas and CIS/high-grade urothelial carcinomas. The role of molecular markers as diagnostic, prognostic, or predictive factors is only emerging, and Table 6 summarizes those with potential. 3.9. Recommended nomenclature for urine cytology 3.9.1. Nomenclature and reporting The Papanicolaou Society of Cytopathology Practice and Guidelines Task Force recommends a diagnostic format that mimics the Bethesda 2001 System for reporting cervical cytology format. Table 7 reviews the recommended nomenclature for urine cytology. The diagnosis of lowgrade urothelial carcinoma is rarely used due to the lack of specific criteria; therefore, most low-grade urothelial carcinomas are included in the atypical category. Regarding the atypical urothelial cells terminology, there is little Table 4 Common genetic and epigenetic aberrations in urothelial carcinoma Gene Location Role Abnormality Clinicopathologic association Diagnostic role Prognostic role TP53 17p13.1 Tumor suppressor Deletion/Mutation HGUC Y/N Y/N MDM2 12q14.3-q15 Oncogene Amplification HGUC N N HRAS* 11p15 Oncogene Activating mutations All grades and stages, up to 15% N N FGFR3 4p16.3 Oncogene Activating mutations Predominantly low grade/stage N Y/N E2F3 6p22 Oncogene Amplification Invasive HGUC N N CCND1 11q13 Oncogene Amplification All grades and stages, up to 20% N N CDKN2A 9p21 Tumor suppressor Deletion/Mutation/ All stages and grades, primarily HGUC N N Methylation RB1 13q14.2 Tumor suppressor Deletion/Mutation Invasive HGUC N Y/N ERBB2 17q21-q22 Oncogene Amplification Invasive HGUC N N MYC 8q24.21 Oncogene Amplification Invasive HGUC N N PTEN 10q23.3 Tumor suppressor Deletion/Mutation HGUC, all stages N N RASSF1A 3p21.3 Tumor suppressor Methylation Invasive HGUC, associated with progression N N FHIT 3p14.2 Tumor suppressor Deletion/Methylation HGUC, associated with progression N Y/N PTCH 9q22.3 Tumor suppressor Deletion/Mutation All grades and stages N N DBC1 9q32-33 Tumor suppressor Deletion/Methylation All grades and stages N N TSC1 9q34 Tumor suppressor Deletion/Mutation All grades and stages N N HGUC = high-grade urothelial carcinoma.

EUROPEAN UROLOGY 63 (2013) 16 35 27 Table 5 Common aberrations in urothelial carcinoma as detected by loss of heterozygosity and comparative genomic hybridization analyses LOH CGH Location Clinical association Location Type of aberration Clinicopathologic association 3p High grade/high stage 9p, 9q, 10q, 11p,Y Loss Noninvasive UC 4p High grade/high stage 1q, 17, 20q Gain Noninvasive UC 4q High grade/high stage 11q Amplification Noninvasive UC 8p High grade/high stage 2q, 4p, 4q, 5q, 6q, 8p, 9p, 9q, 10q, 11p, Loss Invasive UC 11q, 13q, 15q, 17p, 18p, 18q, Y 9q All stages/grades 1q, 3p, 3q, 5p. 6p, 7p, 8q, 10p, 17q, 19p, Gain Invasive UC 19q, 20p, 20q, Xq 11p High grade/high stage 1q22-24, 3p24-25, 6p22, 8p12, 8q22, Amplification Invasive UC 11q Recurrence 10p12-14, 10q22-23, 11q13, 12q12-21, 14q Carcinoma in situ 17q21, 20q13 CGH = genomic hybridization analysis; LOH = loss of heterozygosity; UC = urothelial carcinoma. consensus in the literature on the criteria for inclusion in this category. Nonetheless, it has been suggested that this atypical category can be subdivided into two subcategories: atypical urothelial cells of undetermined significance (AUC-US) and atypical urothelial cell, cannot rule out high-grade urothelial carcinoma or favor neoplasm. The rationale for this subclassification is that specimens diagnosed in the latter category should be referred to cystoscopy, whereas the ones diagnosed as AUC-US could be followed with repeat urines [88 90]. 3.9.2. Role of urine cytology in screening and monitoring patients with bladder cancer Urinary cytology is a simple, noninvasive, and relatively inexpensive method for detecting urothelial carcinoma [91]. It has consistently been shown to have high specificity, Table 6 Potential molecular prognostic parameters in non muscle-invasive and muscle-invasive urothelial carcinoma of bladder Emerging molecular prognostic markers Non muscle-invasive urothelial carcinoma Proliferation index (Ki-67, MIB-1, S phase) * FGFR3 mutation/overexpression (protective) * mg (FGFR#/MIB-1) * p53 inactivation/accumulation * DNA ploidy status * Multitarget FISH HRAS ERBB3, ERBB4 overexpression (protective) Loss of E-cadherin Cell cycle control: Downregulation of Rb expression Downregulation of p21 expression Downregulation of p27 expression Cyclin D3 overexpression Cyclin D1 overexpression Angiogenesis markers: VEGF overexpression HIF1-a overexpression TSP-1 overexpression Genomic and gene expression array panels Epigenetic alterations: RASSF1 promoter hypermethylation DAPK promoter hypermethylation APC promoter hypermethylation E-cadherin promoter hypermethylation EDNRB promoter hypermethylation Muscle-invasive urothelial carcinoma p53 inactivation/accumulation Alterations of Rb expression Loss of p21 expression Alteration of p16 expression Loss of E-cadherin RTK: EGFR overexpression HER2 overexpression/amplification Angiogenesis markers: VEGF overexpression HIF1-a overexpression TSP-1 overexpression mtor-akt pathway: mtor Phos S6 expression (protective) Genomic and gene expression array panels Epigenetic alterations: RASSF1 promoter hypermethylation E-cadherin promoter hypermethylation EDNRB promoter hypermethylation APC = adenomatous polyposis coli; DAPK = death-associated protein kinase; EDNRB = endothelin receptor type B; EGFR = epidermal growth factor receptor; FISH = fluorescent in situ hybridization; FGFR = fibroblast growth factor receptor; HIF = hypoxia inducible factor; mtor = mammalian target of rapamycin; TSP = thrombospondin; VEGF = vascular endothelial growth factor. * Multimarker immune expression analysis (p53, p27, Ki-67, Rb, p21).

28 EUROPEAN UROLOGY 63 (2013) 16 35 Table 7 Recommended nomenclature for urine cytology I. Adequacy statement (optional) Satisfactory for evaluation List any quality factors affecting specimen Unsatisfactory for evaluation (give reason) II. General categorization Negative for epithelial cell abnormality (see Descriptive diagnosis) Epithelial cell abnormality present (see Descriptive diagnosis) III. Descriptive diagnosis Negative for epithelial cell abnormality Infectious agents Bacterial organisms Fungal organisms Viral changes (cytomegalovirus, herpes, adenovirus, Polyomavirus) Nonspecific inflammatory changes Acute inflammation Chronic inflammation Changes consistent with xanthogranulomatous Pyelonephritis Cellular changes associated with: Chemotherapeutic agents Radiation Epithelial cell abnormalities Atypical urothelial cells (See Comment) Low-grade urothelial carcinoma High-grade urothelial carcinoma (invasive carcinoma vs carcinoma in situ) Squamous cell carcinoma Adenocarcinoma Other malignant neoplasms (specify type) IV. Other especially in detecting CIS and high-grade flat lesions [92,93]. However, it is also accepted that urine cytology has low sensitivity for low-grade urothelial carcinoma [94 104]. Diagnostic accuracy depends on diagnostic expertise [105]. 3.9.3. Role of fluorescent in situ hybridization in the screening and monitoring of bladder carcinoma UroVysion has been approved by the US Food and Drug Administration for the screening of urothelial carcinoma in patients with hematuria and monitoring for tumor recurrence in patients with a prior history of urothelial carcinoma [100,106]. The overall sensitivity and specificity of UroVysion is 72% and 83%, respectively [107]; UroVysion fluorescent in situ hybridization (FISH) has a sensitivity of 90 100% for the detection of invasive bladder cancer (pt1 4) and a specificity of 95%; for low-grade noninvasive bladder cancer, the sensitivity increases from 25% to 60 75% when compared with cytology [108]. Use of UroVysion has been advocated in equivocal urine specimens, either suspicious or atypical [109 111], due to its increased sensitivity. A negative FISH result in case of a negative or atypical cytology does not exclude low-grade urothelial neoplasia because up to 30% of these tumors are negative with the FISH assay. A positive UroVysion test has been associated with a higher rate of recurrence in patients treated with intravesical BCG for high-grade non muscle-invasive urothelial Table 8 Urinary bladder: biopsy and transurethral resection of bladder tumor Note: Use of checklist for biopsy specimens is optional. Select a single response unless otherwise indicated. Relevant clinical history Cystoscopic impression of the mucosa Indication of procedure Previous history of cancer in bladder or elsewhere in the genitourinary tract Prior therapy Procedure Biopsy TURBT Not specified Histologic type Urothelial (transitional cell) carcinoma Urothelial (transitional cell) carcinoma with percentage of squamous differentiation Urothelial (transitional cell) carcinoma with percentage of glandular differentiation Urothelial (transitional cell) carcinoma with percentage of micropapillary component Urothelial (transitional cell) carcinoma with variant histology (specify): Squamous cell carcinoma, typical Squamous cell carcinoma, variant histology (specify): Adenocarcinoma, typical Adenocarcinoma, variant histology (specify): Small cell carcinoma Undifferentiated carcinoma (specify): Mixed cell type (specify): Other (specify): Carcinoma; type cannot be determined Associated epithelial lesions (select all that apply) None identified Urothelial (transitional cell) papilloma (WHO 2004/ISUP) Urothelial (transitional cell) papilloma, inverted type Papillary urothelial (transitional cell) neoplasm, low malignant potential (WHO 2004/ISUP) Hyperplasia Dysplasia Carcinoma in situ Cannot be determined

EUROPEAN UROLOGY 63 (2013) 16 35 29 Table 8 (Continued ) Histologic grade Not applicable Cannot be determined Urothelial carcinoma (WHO 2004/ISUP) Papilloma PUNLMP Low grade High grade Urothelial carcinoma (WHO 1973) Papilloma Grade 1 Grade 2 Grade 3 Adenocarcinoma and squamous cell carcinoma GX: Cannot be assessed G1: Well differentiated G2: Moderately differentiated G3: Poorly differentiated Tumor configuration (select all that apply) Papillary Solid/nodule Flat Ulcerated Indeterminate pt1 tumors Muscularis mucosae present Thin-walled blood vessels present Depth of lamina propria invasion Type of tumor invasion Single cells Nodular Trabeculae Infiltrative Stromal reaction Stromal retraction Stromal edema Inflammation Fibroblastic proliferation Fibrosis Adequacy of material for determining muscularis propria invasion Muscularis propria (detrusor muscle) not identified Muscularis propria (detrusor muscle) present Presence of muscularis propria indeterminate Lymphovascular invasion Not identified Present Indeterminate Microscopic extent of tumor (select all that apply) Cannot be assessed Noninvasive papillary carcinoma Flat carcinoma in situ Tumor invades subepithelial connective tissue (lamina propria) Tumor invades muscularis propria (detrusor muscle) Urothelial carcinoma in situ involving prostatic urethra in prostatic chips sampled by TURBT Urothelial carcinoma in situ involving prostatic ducts and acini in prostatic chips sampled by TURBT Urothelial carcinoma invasive into prostatic stroma in prostatic chips sampled by TURBT Additional pathologic findings (select all that apply) Urothelial dysplasia (low-grade intraurothelial neoplasia) Nonneoplastic changes Inflammation/regenerative changes Therapy-related changes Cautery artifact Cystitis cystica glandularis Keratinizing squamous metaplasia Intestinal metaplasia Denuded urothelium Comment(s) ISUP = International Society of Urologic Pathology; PUNLMP = papillary urothelial neoplasm of low malignant potential; TURBT = transurethral resection of bladder tumor; WHO = World Health Organization.