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From Vysis, Inc.,
*
Downers Grove, Illinois; and the Department of Laboratory Medicine and Pathology,
Mayo Clinic and Foundation, Rochester, Minnesota
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our understanding of the genetic changes that accompany urothelial tumor carcinoma initiation and progression are increasingly being elucidated.12, 13, 14 Homozygous deletions of the P16 gene at 9p21 are one of the most common alterations in UC and occur early in the development of both papillary UC and UC in situ.15, 16, 17, 18, 19, 20, 21 UC progression is accompanied by increased chromosomal instability and aneuploidy.12, 22, 23, 24, 25 Cytogenetic studies reveal frequent alterations of a variety of chromosomes including chromosomes 9, 17, 7, 11, 1, and others.12 These chromosomal alterations can be detected with fluorescence in situ hybridization (FISH).26, 27, 28, 29, 30, 31, 32 FISH utilizes fluorescently labeled DNA probes to chromosomal centromeres or unique loci to detect cells with numerical or structural abnormalities indicative of malignancy.
Previous investigators have demonstrated that FISH can be used to detect UC in voided urine or bladder washing specimens.26, 27, 28, 29, 30, 31, 32 However, despite its apparent promise for this use, FISH has not been used clinically to detect UC. Possible explanations for the slow introduction of FISH for UC detection include (i) previous studies have generally used single FISH probes for their analyses, which may have limited the sensitivity and specificity of the assay, and (ii) the FISH procedures that have been used to evaluate urine specimens for evidence of UC have been too time-consuming to be clinically practical. The goal of this study was to develop a FISH assay for the detection of UC in urine specimens that can be used in clinical laboratories to monitor patients with superficial UC and possibly to screen high risk patients (eg, smokers) for UC. In this paper we have determined (i) which FISH probes (of a set of eight) have the highest sensitivity for UC detection, (ii) the sensitivity and specificity of a multicolor, multitarget FISH probe set containing the four most sensitive of these eight probes for UC in prospectively obtained urine specimens from patients under evaluation for bladder cancer, (iii) the methodology that should be used to evaluate slides for malignant cells, and (iv) the diagnostic criteria that should be used to consider a case positive for UC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Urine Processing
Specimens for FISH were processed within 24 hours. Cells from
voided urine were sedimented at 1200 x g for 8
minutes. The cell pellet was resuspended in 15 ml hypotonic solution
(0.75 mol/L KCl) for 10 minutes. The cells were then sedimented again
at 1200 x g for 8 minutes and resuspended in 10 ml of
a 3:1 solution of methanol:glacial acetic acid. This was
repeated two more times, and the final cell pellet was generally
resuspended in 50 to 200 µl (depending on cell pellet size) of
residual 3:1 methanol:acetic acid.
Slide Preparation
For the probe selection portion of the study, slides were prepared
by dropping a portion of the cell pellet suspension onto standard glass
slides with a Pasteur pipette and visualizing the cellularity of the
slide with a phase contrast microscope. Additional cell suspension was
dropped onto the slide, if necessary, until an appropriate cellularity
had been reached. We subsequently found that the cells could be
confined to a smaller area of the slide with the use of 12-well, 0.6-cm
Shandon-Lipshaw slides (Shandon Inc., Pittsburgh, PA). By confining the
cells to a smaller area of the slide, we decreased the amount of time
that was required to analyze the cells by microscopy after FISH
hybridization had been performed. Thus, for the larger study of 179
patients, slides were prepared by placing 3 µl, 10 µl, and 30 µl
of cell suspension into three separate wells. The use of 3 µl, 10
µl, and 30 µl of cell suspension ensured that most samples had at
least one circle with the appropriate density of cells. If the density
of the cells was too high or too low, additional dilution or
concentration of the cell suspension was performed and applied to a
fourth well.
Probe Sets Used to Determine Optimal Probe Set
Probes to the pericentromeric regions of chromosomes 3, 7, 8, 9,
11, 15, 17, 18, and Y and to the 9p21 band were examined for their
relative sensitivities at detecting UC in urine specimens. The choice
of these probes was based on a review of the literature that revealed
that numerical alterations of these chromosomes or chromosomal loci are
among the most frequent chromosomal alterations in
UC.12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25
Directly labeled fluorescent probes to the
pericentromeric regions of chromosomes (CEP probes) and the probe to
the 9p21 band (LSI 9p21) were purchased from Vysis (Downers Grove, IL).
These labeled probes were then combined into one of three probe sets.
The probes contained in each of these probe sets and the fluorophore
used to label each probe are shown in Table 1
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Enumeration of FISH Signals
The criteria used to enumerate FISH signals were as previously
described.33
Overlapping cells and cells with indistinct
or blurry signals were not scored. Care was taken not to interpret
split signals as two signals.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. This was done by enumerating the FISH signals for each of the probes
in probe sets A, B, and C in 100 consecutive cells of voided urine
specimens from 9 normal donors. The CEP15 and CEP Y probe signal
intensities were weak, and we were unable to collect data for these two
probes. Monosomy rates for the centromeric probes were relatively high
and ranged from 7.8 to 14.4% (Table 2)
. Trisomy and tetrasomy rates
for the centromeric probes were lower and ranged from 1.0 to 3.0% and
0.4 to 1.5%, respectively. The frequency of hemizygous and homozygous
9p21 loss was 10.3 and 4.5%, respectively. The presence of normal
multinucleated umbrella cells did not impact the observed polysomy
rates because the individual nuclei of the umbrella cells were disomic
and distinct from one another by 1-4, phenylenediamine (DAPI) stain.
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. On the other hand, tetrasomy or greater was a
frequent finding in cells from UC patients (Table 3)
. This suggested that the finding of tetrasomic or greater cells in
urine specimens might be more indicative of UC than the finding of
trisomic cells. For this reason, we used tetrasomy or greater as our
criterion for evidence of FISH abnormality with individual probes in
the patients with UC. A specimen was considered positive for cancer if
the percentage of cells with tetrasomy or greater for a centromeric
probe was greater than or equal to the mean number of cells + 3
SD with tetrasomy or greater observed in normal donors. The percentage
of urine specimens from the 21 patients with UC that were FISH-positive
by this criterion for each centromeric probe is shown at the bottom of
Table 3
. The sensitivity of tetrasomy of the centromeric probes for UC
ranged from 42.1% for CEP18 to 76.2% for CEP 7.
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and because it has been reported to
be a frequent alteration in early stage UC.15, 16, 17, 18, 19, 20, 21
The
sensitivity of homozygous 9p21 deletion for UC in the 21 UC cases was
28.6% (Table 3)
. Additionally, we also determined the sensitivity of
chromosome 9 monosomy (ie, single copy of CEP 9 probe) for UC in the 21
UC cases (data not shown in Table 3
), since this has also been shown to
be a frequent event in UC.15, 16, 17, 18, 19, 20, 21
The sensitivity of
chromosome 9 monosomy in the 21 UC cases, using a cutoff of 30.8%
(mean percentage in normals + 3 SD), was 1/21 (4.8%).
Complementation Analysis to Determine the Combination of Probes
with the Greatest Sensitivity for UC
Complementation analysis was performed to determine the
combination of probes that provides the greatest overall sensitivity
for UC detection (Table 3)
. Comple-mentation analysis is
important because a probe might have relatively low sensitivity for UC
when used alone but might nonetheless enhance the overall sensitivity
of the assay by complementing other probes. As noted above, the CEP7
probe was the most sensitive, having detected 16 of the 21 patients
(darkly shaded cells in Table 3
). The CEP3 and CEP17 probes
complemented the CEP7 probe by detecting abnormalities in two and one
additional patients, respectively, who did not show abnormalities with
the CEP7 probe (lightly shaded cells in Table 3
). Additionally, the
9p21 locus-specific probe detected two more UC cases (patients 223 and
239) that were not detected with any of the centromeric probes (lightly
shaded cells in Table 3
). Thus, with this group of UC patients, the
highest sensitivity of UC detection was achieved with probes to the
centromeres of chromosomes 3, 7, and 17 and a probe to the 9p21 band.
Together these probes detected 20 of the 21 (95.2%) UC cases. For
these same 21 UC cases, urine cytology was positive, equivocal, and
negative for 13, 3, and 5 patients, respectively. If equivocal cytology
results are scored as positive, the sensitivity of cytology for this
set of patients was 76%.
Based on the results of the probe selection study we concluded that a probe set containing the CEP7, CEP3, CEP17, and the LSI probe 9p21 would have the greatest sensitivity for UC detection. The CEP7, CEP3, CEP17, and LSI 9p21 probes were labeled with Spectrum Green, Spectrum Red, Spectrum Aqua, and Spectrum Gold, respectively, and combined into a single probe set. This probe set was then used to prospectively analyze 181 urine samples from 179 patients with a history of UC or other genitourinary signs and symptoms. The goals were further refinement of the criteria that should be used to determine whether a case is positive or negative for cancer and determination of the sensitivity and specificity of FISH based on these criteria.
Development of Scanning Method for Evaluation of FISH Slides for UC
Our experience with FISH from patients with biopsy-proven UC and
positive urine cytology findings has revealed that cells with FISH
abnormalities suggestive of malignancy (eg, gains of multiple
chromosomes) generally exhibit nuclear abnormalities. An example of the
nuclear abnormalities observed in cells that are also abnormal by FISH
is shown in Figure 1
. The nuclear abnormalities observed include nuclear enlargement,
irregular nuclear borders, and patchy (ie, nonhomogeneous) DAPI
staining. We have also noted that polysomy (ie, gains of two or more
different chromosomes in the same cell) is a more specific indicator
that a cell is malignant than the gain of a single chromosome. Gains of
a single chromosome appear more often to be artifactual, perhaps
resulting from factors such as signal splitting or cross-hybridization.
Based on these observations, we concluded that the most efficient way
to evaluate slidesfor cells with FISH abnormalities is to scan
for cells with nuclear abnormalities suggestive of malignancy and to
determine the CEP and LSI signal copy numbers in the suspicious cells.
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For the portion of the study that used scanning, a cell was considered
abnormal if it was polysomic (ie, showed gains of multiple
chromosomes). Representative examples of normal and polysomic cells are
shown in Figure 2
. For the purpose of data collection and standardization of the scanning
technique, the slide was scanned for 20 morphologically abnormal cells
and the FISH signal pattern in those cells recorded.
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4 of the 20
cells (the mean percentage + 3 SD of cells with polysomy observed in
specimens from normal donors) demonstrated polysomy. The specificity of
the two approaches was comparable: 93.2% for counting method
versus 88.4% for the scanning method
(P = 0.46). However, the sensitivity of scanning
method was significantly better than for the counting method (86.2%
versus 62.0%, respectively; P = 0.04).
Further Refinement of the Criteria for Considering a Case Positive
for UC
We then used the CEP3, CEP7, CEP17, LSI 9p21 probe set and the
scanning method to evaluate prospectively 181 urine specimens from 179
patients and 43 normal donors. Patient samples were evaluated without
knowledge of the patient’s history, clinical or biopsy findings.
Ninety-three patients had a previous history of bladder cancer and 86
did not. Biopsies or surgical resections were performed on 74 of the
179 patients. The surgical pathology results for these 74 patients were
classified as positive for UC in 46 patients. Among these 46 tumors
were 22 pTa tumors, 12 pTis tumors, and 12 pT1-pT4 tumors. The tumor
grade for the 46 tumors was grade 1 for 8 tumors, grade 2 for 14
tumors, and grade 3 for 22 tumors. Although rare polysomic cells could
be found in the urine of normal donors, the number of cells with
polysomy never exceeded 5 cells for any of the 43 normal donors.
Additionally, only 8.2% of the patients without evidence of UC
(63 of the 179 patients) but with other genitourinary symptoms and
signs had more than 5 cells with polysomy. Of the patients with
biopsy-proven UC (n = 46), 84.2% had five or
more cells with polysomy.
A receiver-operator characteristic curve was generated to determine the
effect of various cutoffs on the sensitivity and specificity of the
assay (Figure 3)
. The sensitivity and specificity of the assay for UC was 89.5% and
87.8%, respectively, if 4 or more cells with polysomy was used as a
cutoff for positivity and 84.2% and 91.8%, respectively, using a
cutoff of 5 or more cells with polysomy for positivity (Figure 3)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our findings clearly show that the use of more than one probe improves the sensitivity of UC detection. With the CEP7 probe alone, the sensitivity of the assay for UC would have been only 76% (16/21 patients). However, the addition of CEP3, CEP17, and the LSI 9p21 probes to the set increased the sensitivity to 95% (20/21 patients). These findings are consistent with those of Sauter et al, who have shown that the fraction of paraffin-embedded UCs demonstrating chromosomal abnormalities with FISH increases when the number of probes used is increased from one to four.37 They did not find any further increase in the fraction of tumors detected when 4 to 6 probes were applied. Although the overall sensitivity of the LSI 9p21 probe was low (29%; 6/21 patients), it enhanced the sensitivity of the assay by detecting tumors that were not detected with the CEP probes. The CEP probes and LSI 9p21 probe are complementary because the CEP probes detect hyperdiploidy, a common feature of carcinoma in situ and invasive UC, but not non-invasive papillary UC, whereas the LSI 9p21 probe detects deletions of the 9p21 band, a common feature of non-invasive papillary UC.15, 16, 17, 18, 19, 20, 21
The CEP9 probe was, surprisingly, found to be one of the least
sensitive probes (Table 3)
. This may seem surprising since loss of all
or part of chromosome 9 has been reported as the most frequent
chromosomal abnormality in UC. An explanation for this paradox may be
that if chromosome 9 loss occurs before tetraploidization (a common
event in solid tumors), then the copy number of chromosome 9 would go
from monosomy to disomy.22, 23, 38
Thus, the copy number of
chromosome 9 would appear normal despite the fact that the cell is
hyperdiploid.
Our experience with FISH has led us to realize that the finding of polysomy (ie, gains of two or more different chromosomes) in a cell is a more specific indicator that a cell is likely to be malignant than the gain of a single chromosome. For example, our calculations show that the specificity of our assay using the criteria 5 cells with polysomy (gains of multiple chromosomes) was 91.8% (45/49 patients). If we use as a criterion for cancer positivity 5 cells with gains of a single chromosome (for example, CEP3), then the specificity of the assay drops to 81.6% (40/49 patients). This fact can be easily explained statistically, because it is less likely that the artifacts (such as a split signal) occur simultaneously on two independent chromosomes in the same cell.
We also found that a scanning method was more sensitive and faster than counting for the analysis of FISH slides. The higher sensitivity of the scanning method is probably due to the fact that it allows the observer to examine a much larger population of cells (up to thousands) for evidence of malignancy. This is not possible with counting, due to the large amount of time required to count cells. Counting may miss rare abnormal cells if the percentage of abnormal cells is low. We found this to be especially true of cases that had FISH abnormal cells hidden among inflammatory or normal epithelial cells.
The scanning technique can generally be performed in 1 to 10 minutes by an experienced observer. Specimens with high percentages of FISH abnormal cells can be categorized as positive for malignancy in approximately 1 minute; specimens with relatively few FISH abnormal cells may require up to 10 minutes to evaluate. Counting 100 cells, on the other hand, generally takes 30 to 60 minutes per case.
A receiver operator characteristic curve (Figure 3)
revealed that a
cutoff of 5 or more cells with polysomy as the criteria for evidence of
UC gave a sensitivity and specificity of 84.2% and 91.8%,
respectively. A slightly higher sensitivity (89.5%) but lower
specificity (87.8%) were obtained with a cutoff of 4 or more cells. To
maintain a high specificity, we recommend that the cutoff of 5 or more
cells with gains of 2 or more different chromosomes be used to classify
cases as malignant.
In summary, this study reveals that a FISH probe set containing probes to the centromeres of chromosomes 3, 7, and 17 and to the 9p21 band has high sensitivity and specificity for the detection of UC. Our studies suggest that the best way to assess slides is to scan the slide for cells with morphologically abnormal features and then determine whether the cell has chromosomal aberrations suggestive of malignancy by viewing the FISH signals. Our studies also suggest that the sensitivity of this FISH assay for UC is superior to urine cytology and yet maintains the high specificity of cytology. It is possible that regular surveillance of UC patients for tumor recurrence with FISH could reduce UC mortality, because false negative urine cytology results place superficial UC patients at risk of undetected progression to less curable muscle-invasive UC.
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Footnotes |
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Supported by a grant from Vysis, Inc. (Amoco 608-910-9900).
Accepted for publication June 15, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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