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
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.
Urothelial carcinoma in situ
Flat Urothelial Lesions
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:
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
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
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.  Suffice it to say that the WHO 2004 classification of flat
urothelial lesions with atypia  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).
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
- Cytoplasmic clearing
- Nuclear size
- Nuclear crowding
- Nuclear borders including notches
- Nuclear chromatin distribution
- Accompanying inflammation
- Neovascularity and inflammation at the base of the lesion
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:
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):
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).  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.  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." 
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,  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. 
Carcinoma In Situ (High-grade Intraurothelial Neoplasia):
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. 
Morphologic expressions of CIS
* Does not imply neuroendocrine differentiation
- Large cell CIS with pleomorphism
- Large cell CIS without pleomorphism
- Small cell CIS *
- Clinging CIS
- Cancerization of normal urothelium
- Pagetoid CIS
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.
Immunoprofiles for CIS, Reactive Atypia, and Normal Urothelium
| ||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. 
(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.
(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
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.
 Murphy and
Miller in a cytology study showed that only 5% (1/19) patients developed bladder cancer in 10 years.
Data from studies by Smith et al ,
Althausen et al , Cheng et
and Mostofi and Sesterhenn  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.
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.  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. 
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
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- 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.
- 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,
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
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