—  SHORT COURSE #07  —

An Approach to the Diagnosis of Bladder Lesions in Biopsy and Transurethral Resection Specimens

Section 3 - Flat Urothelial Lesions

Mahul B. Amin, MD
Jesse K. McKenney, MD


Case 2

History:
52-year-old man with a history of high-grade papillary urothelial carcinoma who now presents with atypical cells on urine cytology. Cystoscopy revealed hyperemic mucosa.

Diagnosis:
Urothelial carcinoma in situ

Flat Urothelial Lesions

Normal Urothelium:
Urothelium, the dominant type of epithelium lining the urinary bladder, ureters, and renal pelvis has unique characteristics that separate it from all other epithelia. It is a multilayered epithelium, which typically contains longitudinal nuclear grooves in some of the cells, that matures to form very large surface cells known as "umbrella cells." Umbrella cells may have nuclear atypia, which should not be misconstrued to be dysplastic. Because of the unique nature of this mucosa, the term "urothelial" has been adopted. The urothelial cells typically have a somewhat linear organization streaming upward, perpendicular to the basement membrane.

There has been overuse of the diagnosis "mild dysplasia" for flat lesions with normal cytology, and a minimally disordered architectural pattern; vagaries of staining and fixation may also impart hyperchromasia to benign nuclei. Flat lesions with benign cytology and minimal disorder should not be designated as mild dysplasia but rather as normal.

Flat Urothelial Hyperplasia: [1, 2]
Flat urothelial hyperplasia historically has been defined as urothelium greater than seven cells layers thick. In practice, counting the number of urothelial cell layers is not reproducible, as urothelial cells do not line up in precise rows.

Under the WHO 2004, flat urothelial hyperplasia is defined as a markedly thickened mucosa without cytological atypia and with normal polarity. Rather than requiring a specific number of cell layers, marked thickening is needed to diagnose flat hyperplasia. This lesion may be seen in the flat mucosa adjacent to low-grade papillary urothelial lesions. When seen by itself, there is no data suggesting that it has premalignant potential.

Hyperplastic lesions with a papillary architecture that were previously described as "papillary hyperplasia" are assigned a papillary diagnosis (such as PUNLMP) under the WHO 2004.

Flat Lesions with Atypia

Background:
The diagnosis of flat urothelial lesions has been complicated by a lack of uniformity because of the use of multiple classification systems, often with marked differences in histologic criteria. The history of the concept of intraepithelial neoplasia in the urinary bladder and the evolution of the WHO/ISUP classification have been previously reviewed in detail and will not be repeated here. [15] Suffice it to say that the WHO 2004 classification of flat urothelial lesions with atypia [5] provides well-defined histologic criteria for stratifying lesions within the full spectrum of cytologic atypia and provides a means to compare studies between institutions, a practice that could not be undertaken previously. The diagnostic categories include reactive atypia, atypia of unknown significance, dysplasia (low-grade intraurothelial neoplasia), and carcinoma in situ (high-grade intraurothelial neoplasia).

Diagnostic Approach:
Evaluation of cold cup random biopsies which are routinely obtained in patients with non-invasive papillary tumors on surveillance involves attention to several histologic parameters. It is the constellation of the presence or absence of some of these parameters which help to arrive at the appropriate diagnosis for a given case.

Histologic parameters useful in the evaluation of flat lesions with atypia
  • Thickness of urothelium

  • Polarity

  • Cytoplasmic clearing

  • Nuclear size

  • Nuclear crowding

  • Nuclear borders including notches

  • Nuclear chromatin distribution

  • Nucleoli

  • Mitoses

  • Accompanying inflammation

  • Neovascularity and inflammation at the base of the lesion

Reactive Atypia: [4, 5]
Consists of nuclear abnormalities occurring in acutely or chronically inflamed urothelium. In reactive atypia, nuclei are uniformly enlarged, with small, central prominent nucleoli. Mitotic figures may be frequent. A history of instrumentation, stones, or therapy is often present. In the absence of appreciable nuclear hyperchromasia, pleomorphism, and irregularity in the chromatin pattern, the lesion should not be considered neoplastic. If denudation is present, the residual lining cells should be small basal cells lacking significant nuclear atypia.

Atypia of Unknown Significance: [4, 5]
In some cases it is difficult to differentiate between reactive and neoplastic atypia. There may be a greater degree of pleomorphism and/or hyperchromasia, out of proportion to the extent of the inflammation, such that dysplasia can not be ruled out with certainty. These cases should be designated as "atypia of unknown significance" so that the patients may be followed more closely and re-evaluated once the inflammation subsides.

Dysplasia (Low-grade Intraurothelial Neoplasia): [4, 5, 6, 7, 8, 21, 24]
Dysplastic urothelium was defined as "appreciable cytologic and architectural changes felt to be preneoplastic, yet falling short of the diagnostic threshold for transitional cell carcinoma in situ (CIS)." It has been proposed previously that bladder intraepithelial lesions, like intraepithelial lesions of the cervix, be graded on the basis of level of involvement of atypical cells (i.e., mild dysplasia for lesions showing atypia confined to the lower one-third, moderate dysplasia for atypia up to the middle-third and so on). [6] Evidence from morphological observations in the animal model system of Wistar rats exposed to N-butyl-N (4 hydroxybutyl) nitrosamine emphasizes the pitfall in defining urothelial dysplasia through criteria and terminology employed for cervical carcinoma. [7] The bladders were studied at regular intervals during the development of carcinoma after exposure to the above carcinogen. In the controlled environment of carcinogenesis, atypia progressed quantitatively and qualitatively (the urothelium did not develop progressive changes starting at the basal layer as seen in the uterine cervix)., stressing the importance of cytological features rather than histologic pattern (i.e.- the level of atypia). Anecdotal evidence supports a similar progression in humans.

In dysplasia, the thickness of the urothelium is usually normal (four to seven layers) but may be increased or decreased. Flat lesions with a benign cytology and minimal disorder should be considered within the spectrum of "normal." [4] Dysplastic lesions show some loss of polarity (normal cells are columnar to oval with nuclei perpendicular to the basement membrane) evidenced by crowding and more rounded to polygonal cells with nuclei parallel to the long axis. Nuclear atypia is evident but is not severe enough to merit a diagnosis of carcinoma in situ. There is often loss of increased cytoplasmic eosinophilia, nucleomegaly, irregularity of nuclear contours with notching of cell borders and mild alteration of chromatin distribution. Nucleoli are usually not conspicuous; only a minor degree of pleomorphism is allowable in dysplasia and the mitotic activity is variable though usually not in the higher layers. The lamina propria is usually unaltered but may contain increased inflammation and/or neovascularity. Comparison with more normal appearing urothelium, if present, may help in assessing features like nucleomegaly, cytoplasmic features, and loss of polarity. Denudation is not typically a feature of dysplasia. A diagnosis of primary dysplasia (no prior history or concomitant urothelial neoplasia) should be made with caution.

Because of problems with interobserver reproducibility, [3] the lack of a uniform definition, and confusing reports in the literature which often combine moderate and severe dysplasia (the latter currently regarded as CIS), the natural history of bladder dysplasia in humans is poorly understood. Dysplastic lesions are typically seen in bladders with urothelial neoplasia and are uncommon in patients without it. In patients with bladder tumors, the presence of dysplasia in the adjacent mucosa also places them at higher risk for recurrence and progression. [27]

Carcinoma In Situ (High-grade Intraurothelial Neoplasia): [4, 5, 10]
Carcinoma in situ (CIS) is a flat lesion of the urothelium that is a documented precursor of invasive cancer in some cases. The lesion is characterized by the presence of cells with large, irregular, hyperchromatic nuclei that may be either present in the entire thickness of the epithelium or only a part of it. The WHO 2004/ISUP classification expands the previous definition of carcinoma in situ; there need not be full thickness cytologic atypia and an umbrella cell layer may still be present (i.e.- CIS encompasses lesions which in the past were designated as severe dysplasia or marked atypia). The urothelium may be denuded, reflecting the discohesive nature of the cells; it may be diminished in thickness, or even hyperplastic. There may be alteration or complete loss of polarity, marked crowding, pleomorphism, and frequent mitoses; the lamina propria is frequently hypervascular and inflamed. The nuclear anaplasia is generally obvious, although a spectrum of cytologic atypia may exist. Additionally, there are varied cytologic and architectural patterns of CIS. [9]

Morphologic expressions of CIS
  • Large cell CIS with pleomorphism

  • Large cell CIS without pleomorphism

  • Small cell CIS *

  • Clinging CIS

  • Cancerization of normal urothelium
    • Pagetoid CIS

    • Undermining/Overriding
* Does not imply neuroendocrine differentiation

Although the spectrum of morphologic patterns is described below to aid in the recognition of CIS, they should not be included in pathology reports, as there is no known prognostic significance to subtyping. We simply use the WHO 2004 diagnostic term "urothelial carcinoma in situ" because these additional terms would probably only serve to confuse treating urologists. The most easily recognized pattern of CIS is large cell CIS with pleomorphism. The neoplastic cells show considerable loss of polarity and nucleomegaly with marked variation in nuclear size and shape and obvious nuclear hyperchromasia; the cells retain abundant eosinophilic cytoplasm. The neoplastic cells in other examples of CIS may be rather monomorphic (large cell CIS without pleomorphism). These lesions may mimic reactive urothelial atypia because of the uniformity of the cells with conspicuous eosinophilic cytoplasm; however, they have marked nucleomegaly and the clumped, irregular chromatin distribution characteristic of CIS. Some authors have suggested that comparison to stromal lymphocytes may aid in the assessment of nuclear size, particularly in monomorphic cases, as the nuclei of CIS are often 3-6 times the size of stromal lymphocytes. Small cell CIS has nuclear features identical to large cell CIS, but there is very scant cytoplasm. Although the cells appear smaller because of decreased cytoplasm, the nuclei are markedly enlarged with nuclear chromatin abnormalities. It should be emphasized that this is simply a descriptive term and does not imply neuroendocrine differentiation. Clinging CIS is characterized by a partially denuded urothelium with a patchy, usually single layer of residual urothelial cells meeting the morphologic criteria for CIS.

CIS may also show cancerization of adjacent normal urothelium in two patterns: 1) pagetoid spread with the presence of clusters or isolated single cells with features of CIS within normal urothelium and, 2) undermining or overriding of the normal urothelium by CIS of any pattern. CIS may show cell dropout secondary to necrosis forming small, gland-like spaces.

Adjunctive Immunohistochemistry in Flat Lesions

Although studies have shown distinct, contrasting patterns of immunoreactivity in morphologically straightforward cases of CIS, reactive atypia, and normal urothelium as shown below, the utility of the panel in morphologically ambiguous cases or dysplasia (with regard to predicting the biologic significance) has not yet been studied. [11, 12]

Immunoprofiles for CIS, Reactive Atypia, and Normal Urothelium [11, 12]

CK 20 p53 CD44
CIS Cytoplasmic staining in full thickness (81%) Strong nuclear reactivity in full thickness (57%) Non-reactive; loss of normal basal staining
Reactive atypia Reactive only in umbrella cells Non-reactive* Diffuse membranous staining in full thickness
Normal urothelium Reactive only in umbrella cells Non-reactive* Membranous staining in basal cell layer only

* Weak, background staining is frequently identified

Problems and Pitfalls in the Diagnosis of Flat Lesions with Atypia
(i) Innate vagaries of normal urothelium and histologic sectioning: The thickness of the normal urothelium varies with the state of distention of the bladder (2 to 4 cell layers when dilated and 5 to 7 layers when contracted). If the sections are thick, the urothelium may appear hyperchromatic and this artifact compounded with tangential sectioning may result in changes felt to represent dysplasia. The urothelium of the renal pelvis, urethra and the bladder neck is usually composed of slightly larger cells, which have diminished cytoplasmic clearing and hence may be interpreted as dysplasia.

(ii) Inflammatory atypia: The presence of acute or significant chronic inflammation, particularly in an intraurothelial location warrants caution in the interpretation of dysplasia or carcinoma in situ, although it must be borne in mind that inflammatory atypia may coexist with dysplasia or in situ carcinoma.

(iii) Therapy associated atypia: Upon cursory examination the changes could easily be mistaken for intraepithelial neoplasia, but the presence of abundant cytoplasm, nuclear chromatin degeneration, multinucleation, prominent nucleoli and involvement of mainly the superficial cells are key features to associate the changes with chemotherapy or radiation.

(iv) Extensive denudation: Trauma due to instrumentation, prior therapy, and carcinoma in situ are the main conditions associated with denudation. The presence of a few atypical single cells clinging to the surface is sometimes the only sign of carcinoma in situ ("denuding cystitis") - deeper sections through the block may be helpful. In the absence of atypical cells, the findings of extensive denudation, particularly that associated with neovascularity and chronic inflammation in the lamina propria must be included in the report and correlation with urine cytology findings may be suggested.

(v) Truncated papillae of treated papillary carcinoma: Mitomycin C and thiotepa therapy destroys the tips of the papillae of papillary transitional carcinoma because they act as surface abrasives. When these truncated papillae are seen in areas associated with denudation and inflammation they may be mistaken for carcinoma in situ or dysplastic changes. [13]

(vi) Carcinoma in situ involving von Brunn nests - overdiagnosis of invasion: Carcinoma in situ can involve von Brunn nests resulting in the presence of neoplastic cells within the superficial lamina propria. In the presence of inflammation, the basement membrane may be obscured and distorted, simulating invasion.

(vii) Carcinoma in situ with microinvasion - underdiagnosis of invasion: A histologically subtle, but clinically potentially ominous situation arises when carcinoma in situ is associated with microinvasion. The urologist is most often unsuspecting of invasive disease on the basis of cystoscopic evaluation and the pathologist may fail to recognize single cell invasion or small clusters of invasion that may be camouflaged by background inflammation. Desmoplasia or retraction artifacts are useful in recognizing invasion. [9, 28]

(viii) Polyoma virus infection: Infection of immunocompromised patients with the human polyoma virus, which is usually a non-pathogenic virus, results in large homogeneous inclusions in enlarged nuclei of urothelial cells. The findings are more frequently seen in urine cytology but may rarely be seen in biopsies, in both specimens malignancy (carcinoma in situ) is mimicked. [16]

Clinical and Biological Relevance of Dysplasia andCarcinoma In Situ
Dysplasia is usually a histologic diagnosis seen most commonly in patients with bladder neoplasia, in whom the incidence is 22 to 86%. The incidence approaches 100% in patients with invasive carcinoma. Little is known about de novo dysplasia due to lack of screening in the general population. Also, it is usually cystoscopically and clinically silent although it may appear as a slightly raised and mildly erythematous patch. The patients are predominantly middle-aged men presenting occasionally with irritative bladder symptoms with or without hematuria. Zuk et al found that 13% (2/15) patients with primary dysplasia progressed to carcinoma in situ. [17] Murphy and Miller in a cytology study showed that only 5% (1/19) patients developed bladder cancer in 10 years. [18] Data from studies by Smith et al [19], Althausen et al [20], Cheng et al [21], and Mostofi and Sesterhenn [27] suggest that dysplasia in patients with transitional cell carcinoma appears to be a marker for progression (increased recurrences or future invasion). It is difficult to compare these studies, however, because different thresholds were employed for diagnoses of dysplasia.

The clinical course and biologic potential of carcinoma in situ is better understood chiefly because carcinoma in situ is more often symptomatic and may be detected by cystoscopy, rendering it recognizable in a primary or de novo form. Most patients present with frequency, dysuria, nocturia and suprapubic fullness; hematuria is typically microscopic. [23, 24] The bladder at cystoscopy appears erythematous or granular although no abnormality may be present. The biologic potential of carcinoma in situ is somewhat unpredictable, although the current dogma states that it is a precursor to invasive carcinoma. The aggregate evidence in the literature suggests that approximately half the patients will develop invasive carcinoma within 5 years. Weinstein et al has referred to the enigmatic natural history of carcinoma in situ as carcinoma paradoxicum. [25] Host - tumor interactions and tumor cell properties are among the several factors determining the biologic potential. From a clinical perspective extent of disease (focal, multifocal or extensive), involvement of prostatic urethra, and response to therapy are the principal determinants of clinical outcome. Patients with primary (de novo) carcinoma in situ are more likely to have no evidence of disease (62% versus 45%), are less likely to progress (28% versus 59%), or die of disease (7% versus 45%) when compared to carcinoma in situ occurring in patients with bladder neoplasia. [26]

As the WHO/ISUP classification becomes embraced by the urology and oncology community, pathologists may be consulted regarding management of patients based on the new classification. Urologists are familiar with the clinical and pathological aspects of carcinoma in situ and have well accepted treatment regimens for the disease. The subject of dysplasia is an evolving issue, and hence the management varies between different centers. Since dysplasia appears to be a marker for urothelial instability, and hence disease progression, patients with this diagnosis should at least be closely followed-up with regular urine cytology and/or cystoscopy and biopsy.

Selected References for Flat Urothelial Lesions
  1. Taylor DC, Bhagavan BS, Larsen MP, Cox JA, Epstein JI. Papillary urothelial hyperplasia. A precursor to papillary neoplasms. Am J Surg Pathol 1996;20;1481-88.

  2. Oberman EC, Junker K, Stoehr R, Dietmaier W, Zaak d, Schubert GE, Hofstaedter F, Knuechel R, Hartmann A. Frequent genetic alterations in flat urothelial hyperplasias and comcomitant papillary bladder cancer as detected by CGH, LOH, and FISH analyses. J Pathol 2003;199:50-7.

  3. Amin MB, Grignon DJ, Eble JN, Reuter VE, Ro JY, Srigley JR, Weiss MA, Young RH: Intraepithelial lesions of the urothelium: An interobserver reproducibility study with proposed terminology and histologic criteria. Mod Pathol 1997;10:69A,

  4. Epstein JI, Amin MB, Reuter VR, Mostofi FK: The World Health Organization/International Society of Urological Pathology consensus classification of urothelial (transitional cell) neoplasms of the urinary bladder. Am J Surg Pathol 1998;22:1435-1448.

  5. Sauter G, Algaba F, Amin MB, Busch C et al: Non-invasive urothelial tumours. In: Epstein JI, Eble JN, Sesterhenn I, Sauter G (eds): World Health Organization Classification of Tumours Pathology and Genetics: Tumours of the Urinary System and Male Genital Organs. IARC Press, Lyon, 2004; pp 110.

  6. Ayala AG, Ro JY: Premalignant lesions of urothelium and transitional cell tumors, in Young R.H. (ed): Contemporary Issues in Surgical Pathology: Pathology of the Urinary Bladder. Churchill Livingstone, New York, NY, 65-101, 1989.

  7. Murphy WM, Soloway MS (eds): Developing carcinoma (dysplasia) of the urinary bladder, in Sommers SC, Rosen PP (eds): Pathology Annual, 17. Norwalk, CT, Appleton-Century-Crofts, 197-217, 1982.

  8. Amin MB , Murphy WM, Reuter VE, et al: Controversies in the pathology of transitional cell carcinoma of the urinary bladder, in Rosen PP, Fechner RE (eds): Reviews in Pathology, Chicago, IL, ASCP Press, 1-38, 1996.

  9. McKenney JK, Gomez JA, Desai S, Lee MW, Amin MB: Morphologic expressions of urothelial carcinomas in situ: a detailed evaluation of its histologic patterns with emphasis on carcinoma in situ with microinvasion. Am J Surg Pathol 25:356-362, 2001.

  10. Amin MB, Young RH: Intraepithelial lesions of the urinary bladder with a discussion of the histogenesis of urothelial neoplasia. Semin Diag Pathol 14:84-97, 1997.

  11. 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;25:1074-78.

  12. Harnden P, Eardley I, Joyce AD et al. Cytokeratin 20 as an objective marker of urothelial dysplasia. Br J Urol 1996;78:870-5.

  13. Murphy WM, Soloway MS, Finebaum PJ: Pathologic changes associated with topical chemotherapy for superficial bladder cancer. J Urol 1980;126:461-465.

  14. Amin MB , Gomez JA, Young RH: Urothelial transitional cell carcinoma with endophytic growth patterns. A discussion of patterns of invasion and problems associated with carcinoma-in-situ. J Urol 1976;116:575-580.

  15. Amin MB , McKenney JK: An approach to the diagnosis of flat intraepithelial lesions of the urinary bladder using the World Health Organization/International Society of Urological Pathology consensus classification system. Adv Anat Pathol 2002;9:222-232.

  16. Koss LG: "Diagnostic cytology and its histopathologic basis," 4th Edition. Philadelphia: JB Lippincott, 1992.

  17. Zuk RJ, Rogers HS, Martin JE, et al: Clinicopathological importance of primary dysplasia of bladder. J Clin Pathol 1988;41:1277-1280.

  18. Murphy WM, Miller AW: Bladder cancer, in Javadpour NE (ed): Baltimore, MD, William & Wilkins, 100-122, 1984.

  19. Smith G, Elton RA, Beynon LL: Prognostic significance of biopsy results of normal-looking mucosa in cases of superficial bladder cancer. Br J Urol 1983;55:665-669.

  20. Althausen AF, Prout GRJ, Dal JJ: Noninvasive papillary carcinoma of the bladder associated with carcinoma-in-situ. J Urol 1976;116:575-580.

  21. Cheng L, Cheville JC, Neumann RM, Bostwick DG: Flat intraepithelial lesions of the urinary bladder. Cancer 2000;88:625-631.

  22. Amin MB. Urothelial dysplasia. In: World Health Organization Classification of Tumours. Pathology and Genetic Tumors of the Urinary System and Male Genital Organs. Editors: Eble JN, Sauter G, Epstein J, Sesterhenn I; IARC Press, Lyon, 2004; pages 111-112.

  23. Lamm DL: Carcinoma in situ. Urol Clin North Am 19:499-508, 1992.

  24. Murphy WM, Soloway MS: Urothelial dysplasia. J Urol 1982;127:849- 854.

  25. Weinstein RS, Miller AW, Pauli BU: Carcinoma in situ: Comments on the pathobiology of a paradox. Urol Clin of North America vol 7, No. 3, 1980.

  26. Orozco RE, Martin AA, Murphy WM: Carcinoma in situ of the urinary bladder: Clues to host involvement in human carcinogenesis. Cancer 1994;76115-122.

  27. Mostofi FK, Sesterhenn IA. Pathology of epithelial tumors and carcinoma in situ of bladder: Bladder cancer. Progress Clin Bio Res 1984;162A:55-74.

  28. Jiminez RE, Keane TE, Hardy H, Amin MB. pT1 urothelial carcinoma of the bladder: criteria for diagnosis, pitfalls, and clinical implications. Adv Anat Pathol 2000;7:13-25.