15
Urinary Tract
cytometry has been applied not only for the diagnosis but for
the classification of urothelial tumors.69,70 Patients with diploid
tumors rarely experience progression or recurrence, whereas
in aneuploid tumors, the progression rate is 10% for tetra-
ploid lesions and 50% for those with nontetraploid aneuploid
lesions.71 Because most low-grade tumors are diploid and a
high histologic grade is generally associated with aneuploidy,
DNA studies in these two groups of cases offer no significant
additional prognostic information. Grade II tumors, however,
can be subclassified as diploid or tetraploid (biologically less
aggressive) or aneuploid (biologically aggressive), with signifi-
cant prognostic information.40,72
Sensitivity and specificity varies from 45 to 95% and 83 to
100%, respectively.7 Dual color FC using monoclonal antibodies
for cytokeratin 18 along with DNA content can further increase
the sensitivity of the assay.
immunocytochemistry
Immunohistochemical stains can be performed on urine speci-
mens including Papanicolaou-stained smears after removal of
the coverslips. They can also be performed on cytospins, Thin-
Preps, and cell blocks. The clinical utility of immunohistochem-
istry in the diagnosis of tumors of the urinary bladder is well
documented in the literature. More recently several biomarkers
have emerged as important adjuncts to morphologic evaluation
of tumors especially for poorly differentiated urothelial carci-
nomas, adenocarcinomas, and mesenchymal lesions. Establish-
ing the urothelial origin of a metastatic tumor is an additional
application of the method. A number of biomarkers may prove
to be important indicators of prognosis or response to chemo-
therapy.73
Immunoperoxidase stains are also useful for identifying
viruses
(e.g.
polyomavirus)
and for classifying malignant
lymphomas as B or T types.
For additional information see Chapter 35, Immunocyto-
chemistry.
Morphometry
Morphometry using a microscope equipped with a television
camera attachment and a computer can generate accurate data
about the nuclear perimeter, nuclear area, and the nucleocyto-
plasmic ratio. These measurements permit classification of the
various groups of urothelial cells, and the nuclear measurements
correlate closely with the grade of tumor. Boon and co-workers
were unable to distinguish normal urothelial cells from cells
that exfoliated from grade I tumors because of similar cellular
and nuclear dimensions.30 They did demonstrate that when
compared with grade II tumors, the nucleocytoplasmic ratio of
grade I tumor cells is smaller. They found grade II tumor cells to
be larger and associated with anisocytosis.
Blood Group Antigen Predictors
The deletion of A, B, and H blood group isoantigens is associ-
ated with biochemical and structural changes of the cell surface
in neoplastic transformation and in particular with alterations
of glycolipid and glycoprotein components. The A, B, and H
blood group substances are decreased in high-grade and aggres-
sive tumors. This deletion is accompanied by the appearance
of precursors or cryptic antigens. Positive staining of tumor
cells for the isoantigens correlates with a low rate of recurrence,
whereas negative staining predicts a low survival rate. Chichara
et al. suggests that loss of ABO gene and/or its promoter hyper-
methylation is a specific marker for urothelial carcinoma.74
When evaluated with DNA ploidy, lack of positive staining for
BGi-A correlates with DNA aneuploidy, whereas the lack of posi-
tive staining for BGi-H appears to correlate with DNA diploidy.75
Although of interest, these studies are not yet widely used.
BTA stat and BTA TRAK
Cellular proliferation in bladder cancer is associated with the
presence of urinary tumoral antigens, which are not produced
by normal cells. The first-generation BTA test consisted of a
latex agglutination test carried out in several steps to detect
basement membrane antigen complexes in the urine. The BTA
stat (Bard Diagnostic Sciences, Redmond, WA) is a second-
generation test which can detect a bladder tumor antigen, a
high-molecular-weight protein related to the H factor of human
complement (hcfHrp), through an immunochromatographic
assay and can be performed with only five drops of recently
voided urine. The results are available in 5 minutes. The sensi-
tivity of this test varies from 57 to 78% and the specificity from
52 to 93%.76-80
The BTA Trak (Bard Diagnostic Sciences) is a quantitative
enzyme immunoassay which requires two different monoclonal
antibodies to bind the same antigen as the BTA stat. The extent
of the enzyme reaction is determined by measuring the absorb-
ance at 405 nm. The absorbance obtained is proportional to
the concentration of antigen in the test sample in the range of
0-100 U/mL and is compared with the absorbance obtained in
controls. The TRAK normal range is defined as 0-14 U/mL. The
sensitivity of the test varies from 62 to 76% and the specificity
from 51 to 98%.81
Nuclear Matrix Protein (NMP22)
The NMP22 (Matritech, Inc., Newton, MA) assay targets com-
plexed and fragmented forms of the nuclear mitotic apparatus
protein in voided urine, a specific protein of the non-chromatin
internal framework of the nucleus involved in DNA replication,
RNA transcription, and gene expression. The intracellular nuclear
mitotic apparatus protein concentration is about 25-fold higher
in bladder cancer cell lines when compared to the mean level
in urothelial cells of normal bladder. This test is a quantitative
enzyme immunoassay which utilizes two monoclonal antibod-
ies (mAb302-18, mAb 302-22). The manufacturer recommends
a reference cut-off level of 10 U/mL but lower cut-off levels have
been used with higher degree of accuracy. The sensitivity of this
test varies from 48 to 100% and the specificity varies from 70 to
91%.76,82-86
Fluorescence in Situ Hybridization (FiSH)
and immunocyt test
Fluorescence in situ is a hybridization technique with fluores-
cent-labeled DNA probes to detect cells with numerical or struc-
tural abnormalities. The probe mixture consists of fluorescent
probes targeted to the pericentromeric regions of chromosomes
3, 7, and 17 (CEP3, CEP7, CEP17) and to band 9p21 locus
(LSI 9p21). Samples are scanned using a fluorescence micro-
scope with a DAPI filter. The identification of five or more cells
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