From the Pathology and Laboratory Medicine Service,
Veterans Affairs Maryland Health Care System, Baltimore, Maryland [1];
Department of Pathology, University of Maryland Medical System,
Baltimore, Maryland [2]; and
Department of Pathology, The Johns Hopkins Medical Institutions,
Baltimore, Maryland [3].
Berman JJ, Seidman JD, Yetter RA, Moore GW.
Clear cell dysplasia of the bladder:
Report of a case with flow cytometric analysis.
Anal Quant Cytol Histol. 1991 Dec;13(6):391-394.
PMID: 1807281.
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ABSTRACT.
Clear cell dysplasia of bladder is a described morphologic entity
that has been found in association with transitional cell carcinoma
of bladder. Its biological role in bladder tumorigenesis is unknown,
and no instances of its ploidy analysis have been reported. The authors
describe a case of clear cell dysplasia of bladder found in association
with a primary adenocarcinoma of bladder. Flow cytometric analysis
of bladder tissue involved by clear cell dysplasia, adenocarcinoma,
and cystitis cystica (all from the same bladder) demonstrated no DNA
aneuploid populations. Cells from the area of clear cell dysplasia
had an S+G2+M fraction of 7%, indicating that it is a proliferative lesion.
Cells from the adenocarcinoma had an S+G2+M phase of 18%, and cells
from an area of cystitis cystica had an S+G2+M phase of 4%.
INTRODUCTION.
Clear cell dysplasia of bladder is a described morphologic entity
characterized by the focal replacement of normal transitional mucosa
by cells having abundant clear cytoplasm, nuclear enlargement,
and a granular chromatin pattern.
[18]
Further features of nuclear atypia
generally associated with urothelial dysplasia can also be found. Sekine
has observed that clear cell foci with either mild or marked nuclear
dysplasia are frequently associated with high grade transitional cell
carcinoma.
[17]
Clear cell change has been associated with several
urothelial neoplasms and proliferative lesions.
[4,
20,
9]
Because these lesions are uncommonly observed, little is known
of their biologic potential. Clear cell dysplasia has been observed
in bladders containing invasive transitional cell carcinoma, but there
is no study showing that clear cell dysplasia can itself progress to cancer.
Flow cytometry permits analysis of large numbers of cells
from lesions as well as sampling of many lesions collected from
a paraffin archive. A wide variety of dysplastic (preneoplastic) lesions
have been examined by flow cytometry to determine whether abnormalities
in nuclear DNA content are present. Aneuploid populations have been found
in precancerous lesions of the cervix,
[12]
skin,
[14]
respiratory,
[2,
15]
and gastrointestinal tracts.
[8,
13,
16]
Clear cell dysplasia is a lesion whose ploidy has, until now, escaped examination. Bladder biopsies obtained at cystoscopy are usually too small for flow analysis. Bladders obtained at autopsy usually show autolysis, making preparation of adequate flow specimens difficult. Bladders obtained at surgery, although ideal specimens for flow cytometry, are removed for reasons other than the presence of clear cell dysplasia and usually do not contain a sampling of this uncommonly reported lesion. Recently, our laboratory received a bladder removed for primary adenocarcinoma. Microscopic examination of the bladder uninvolved by tumor showed widespread clear cell dysplasia. Flow cytometry was performed.
CASE HISTORY.
The patient was a 64 year old man admitted for urethral stricture.
At surgery, the urethral stricture was opened and a small isolated lesion
was found at the base of the bladder near the left ureteral orifice.
The lesion was resected and was found to be a mass measuring
0.5 x 0.5 x 0.3 cm, diagnosed as moderately differentiated adenocarcinoma
(Fig. 1).
Prostate-specific antigen and prostate-specific acid phosphatase immunostaining (DAKO) was negative in tumor cells. Colonoscopic evaluation was negative for a colon primary. The tumor was diagnosed as a primary adenocarcinoma of bladder, and has been reviewed with concurrence at the Armed Forces Institute of Pathology. Radical prostatocystectomy was subsequently performed. No residual tumor was present in the bladder and pelvic lymph nodes, prostate, and seminal vesicles were without tumor. Multiple sections of bladder were examined and most of the bladder mucosa was replaced by Brunn's nest hyperplasia, cystitis cystica, and multifocal clear cell dysplasia
(Fig. 2).
MATERIALS AND METHODS.
Buffered formalin-fixed, paraffin-embedded tissues were prepared for flow cytometric analysis as described by Hedley et al] , as modified by Hitchcock et al] .
[10,
11]
Briefly, two to five 70-micron sections were deparaffinized with HistoSolv (Biochemical Sciences, Inc., Bridgeport, NJ), rehydrated with Flex (Richard Allan Medical Industries, Richland, MI), and digested for two hours with 0.2% pepsin (Sigma Chemical Co., St. Louis, MO). The pepsin, ribonuclease A, propidium iodide, and phosphate-buttered saline (PBS) solutions included 3% polyethylene glycol-8000 (Sigma), and the 0.1% Triton X-100 PBS solution contained 1 mg/ml bovine serum albumin (Sigma). Isolated nuclei were examined on an Epics Profile Flow Cytometer (Coulter Electronics, Hialeah, FL). Forward scatter versus log side scatter plots were examined. Histograms were prepared using Epics Profile software, version 2.0. The flow cytometer was checked (as recommended by Givan
[7]
) to insure a channel number of zero for unstained particles. No correction was required.
Data were downloaded into the File Manager (FileMan) database management system of the Department of Veterans Affairs for analysis and into a spreadsheet for display. The diploid mean (i.e., G0/G1 channel mean, 2n) was initially estimated as the mean of the entire histogram. The tetraploid mean (i.e., G2/M channel mean, 4n) was taken as two times the diploid value. The mean height of channels between 2.5n and 3.5n was taken as the mean S-phase channel height. Mean S-phase height was then subtracted from the histogram between channels 2n and 4n. In the remaining histogram, all channels above 3n (i.e., channels mostly in the tetraploid range) were assigned half their actual channel number. A new diploid mean was calculated from this reduced histogram. The procedure was repeated on the original histogram until two consecutive diploid means were identical. In our experience, this procedure converges to the same diploid mean for a given histogram, regardless of the initial diploid value.
RESULTS.
Figure 3
shows a histogram of 16,475 cells prepared from an area of bladder with cystitis cystica. There were 96% of cells in G0/G1 phase, and 4% of cells in S/G2/M phase.
Figure 4
shows a histogram of 23,828 cells prepared from an area of bladder with clear cell dysplasia. There were 93% of cells in G0/G1 phase, and 7% of cells in S/G2/M phase. Figure 5 shows a histogram of 18,877 cells prepared from an area of bladder with adenocarcinoma. There were 82% of cells in G0/G1 phase, and 18% of cells in S/G2/M phase. None of the tissue areas in the case showed an aneuploid population.
DISCUSSION.
The morphologic identification of dysplastic lesions
has great clinical significance, as it permits curative excision
of lesions that might otherwise progress to cancer, and identifies
patients at risk for cancer at a specific tissue site. Moreover,
it permits study of cell populations that may have acquired
some of the genetic lesions leading to the malignant phenotype,
thus leading to further classification of the carcinogenic process
in a specific tissue.
The morphologic entity of clear cell dysplasia of bladder has been associated with transitional cell carcinoma of bladder, but its potential to progress to carcinoma is as yet undetermined. In the present report, clear cell dysplasia of bladder was found associated with a primary bladder adenocarcionma. Flow cytometry of uninvolved bladder mucosa, clear cell dysplasia and, in this instance, adenocarcinoma, are shown in
Figure 3,
Figure 4,
and
Figure 5,
In each case, a single G0/G1 peak was detected with no second (aneuploid)
peak noted and G0/G1 peak CV's within an expected range for single population
curve (<12%). The mean peak channel for the adenocarcinoma (channel 76) was very close to the mean peak channel for cystitis cystica (channel 77) and clear cell dysplasia (channel 74). The adenocarcinoma specimen was removed prior to cystectomy and fixed separately. Variations in formalin fixation time can vary the G0/G1 mean channel location with differences up to 13 channels.
[5]
The differences in mean channel peak in the present study presumably results from fixation artefact and cannot be interpreted to represent a difference in nuclear DNA content in the samples.
These findings are similar to a recently reported experience
with clear cell (cribriform) hyperplasia of prostate,
[6]
where clear cell atypia was found to be diploid. The absence
of an aneuploid peak, of course, does not rule out the presence
of neoplastic genetic alterations, as the majority
of low-grade transitional cell tumors of bladder are DNA diploid.
[1,
3]
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ILLUSTRATIONS.
Fig. 1. Bladder adenocarcinoma underlying urothelium,
with Brunn's nest hyperplasia and cystitis cystica.
Carcinoma invades deeply into bladder wall. Hematoxylin and Eosin x100.
Fig. 2. Clear cell dysplasia of bladder. Hematoxylin and Eosin x300.
Fig. 3. Histogram of cells prepared from an area of bladder
with cystitis cystica.
Fig. 4. Histogram of cells prepared from an area of bladder
with clear cell dysplasia.
Fig. 5. Histogram of cells prepared from an area of bladder
with adenocarcinoma.
Last updated: 1/26/2008, by
G. William Moore, MD, PhD.