DNA Ploidy Analysis by Flow Cytometry or Digital Image Analysis
Flow cytometry and digital image analysis (DIA) can be used to detect aneuploid cells in the urine that are consistent with a diagnosis of UC.16,17 The sensitivity of flow cytometry is limited by the fact that it will fail to detect UC cells if the aneuploid cells represent only a small proportion of all the cells in the urine. Rare UC cells cannot be distinguished from normal cells in S phase in the "aneuploid" regions of the DNA ploidy histogram. DIA is a technique in which the cells on the slide are stained with a Feulgen stain, which stains the DNA in a stoichiometric fashion, followed by image analysis in an image cytometer to assess for aneuploidy. The advantage that DIA has over flow cytometry is that one can select the cells that are assessed for ploidy status. Cells that are clearly nonneoplastic can be ignored. This makes it possible to identify smaller populations of aneuploid cells in a background of diploid cells. Several studies suggest that the sensitivity and specificity of DIA ploidy analysis for the detection of UC are relatively high.16,17 For example, Cajulis et al. showed that the sensitivity of DIA, flow cytometry, and conventional cytology for UC were 91%, 72%, and 61%, respectively, while the specificity of DIA, flow cytometry, and conventional cytology were 83%, 80%, and 100%, respectively. The main disadvantage of DIA is that it is relatively labor-intensive.
Fluorescence in situ hybridization is a technique that utilizes fluorescently labeled DNA probes to detect chromosomal abnormalities. There are two general types of FISH probes: chromosome enumeration probes (CEP) and locus-specific indicator (LSI) probes. CEP provide useful information on whether the cells exhibit significant aneu-somy and CIN. Aneusomy is defined as an abnormal copy number for a specific chromosome. LSI probes hybridize to specific loci and can be used to detect alterations of the TP53, HER2/NEU, MYC, and other cancer-related genes.
Most UC are characterized by numerical and structural chromosomal abnormalities and a marked degree of CIN with variation in the chromosomal abnormalities found from cell to cell. The finding of aneusomy and CIN in a population of cells by FISH is virtually pathognomonic of malignancy.
A FISH assay for the detection of UC in urine has been developed. This assay utilizes a multiprobe mixture that contains CEP3, CEP7, CEP17, and LSI 9p21 probes labeled with red, green, aqua, and yellow fluorophores, respec-tively.3,18 This probe set, now known as UroVysion (Abbott
Figure 25-2. Representative examples of nonneoplastic urothelial cells (panel a) and UC cells (panels b and c) with FISH. Nonneoplastic cells generally have two signals for each of the four probes, though occasional nonneoplastic cells show only one signal for one or more of the probes due to random overlap of signals or imperfect hybridization efficiency. UC cells generally exhibit gains for two or more of the probes (i.e., polysomy) of the UroVysion probe set. The finding ofjust a few cells with polysomy is virtually pathognomic of malignancy. Red signals, CEP3; green signals, CEP7; aqua signals, CEP17; yellow signals, LSI 9p21.
Laboratories, Waukegan, IL), received FDA approval in August 2001 for monitoring UC patients for tumor recurrence. Representative examples of patients with FISH-positive and -negative findings are shown in Figure 25-2.
FISH using the UroVysion probe set is significantly more sensitive than urine cytology for the detection of recurrent UC.3,16,19-21 (Note that the author receives industry funding from Abbott Laboratories and royalties from the sale of the UroVysion probe set). The sensitivity of UroVysion for the detection of CIS, invasive UC, and high-grade papillary tumors is greater than 95%.3 The sensitivity of UroVysion is lower for low-grade papillary tumors than other UC but is still significantly better than cytology for low-grade tumors. Though more studies need to be performed, it is possible that the low-grade tumors that FISH fails to detect are less dangerous and the intervals between cystoscopy could be extended.
UroVysion not only has good sensitivity for UC in patients with biopsy-proven UC but also can detect recurrent UC before it is clinically evident by cystoscopy.3,19-21 In the trial that led to FDA approval,21 Sarosdy et al. reported that there were 36 patients with a negative cystoscopic examination but a positive FISH result. With continued follow-up, 15 (41.7%) of these cases were found to have biopsy-proven tumor recurrence with time to tumor diagnosis of 3 to 16 months (mean 6.0 months). Conversely, among 68 patients who had a negative cystoscopy and a negative FISH result, only 13 (19.1%) had a biopsy-proven recurrence at 3 to 19 months (mean 11.2 months). The time to recurrence was significantly less (p = 0.014) for the patients with a positive FISH result but a negative cystoscopy than for patients with a negative FISH result and a negative cystoscopy.21
One of the greatest advantages of urine cytology over other assays for UC is its high specificity. New assays that improve on the sensitivity of UC detection but have worse specificity than urine cytology are not particularly useful since the urologist has to perform cystoscopy and perhaps other studies to determine whether the result is a true- or false-positive result. A number of the antigen-based assays such as the FDA-approved BTA stat test have been shown to have higher sensitivity than cytology but significantly worse specificity. For example, the specificity of the BTA stat test is typically about 75%.22 By contrast, FISH with UroVysion, like cytology, has high specificity for UC detection, typically exceeding 95%.3,16,19-21
The primary disadvantage of the FISH assay is that it requires more effort than conventional cytology or point-of-care assays such as the BTA stat test. Typical turnaround times for the FISH assay are 1 to 2 days, though the test can be performed in a single day. Automated "dot counters" are under development, and these may increase the ease of FISH test performance, reduce the cost of testing, and increase the throughput and sophistication of the data that can be obtained. Another shortcoming of the FISH test is its inability to detect some low-grade papillary tumors. An assay for UC cells that harbor FGFR3 mutations (see FGFR3 section below) may complement FISH and allow for the detection of virtually all UC.
Sidransky and coworkers have described an assay that utilizes MA to detect recurrent UC.23,24 The principles underlying this test are that UC exhibit frequent AI of certain loci (e.g., 9p21 and 17p13) and the detection of AI in urinary cells provides evidence that the patient has UC. The test also is able to identify tumor if there is evidence of MSI in the DNA extracted from the cells of the urine. In practice, however, the majority of cases found to be positive by MA are positive because there is evidence of AI.12
A reference normal specimen, most commonly peripheral blood or buccal mouthwash, is required to perform this test. DNA is isolated from urinary cells and the normal tissue specimen from the same patient. Polymerase chain reaction (PCR) of 20 or more polymorphic microsatellite markers is performed separately for both the normal and tumor tissue, and the PCR products are analyzed by poly-acrylamide gel or capillary electrophoresis. The markers used include loci that are frequently altered in UC, such as loci on 3p, 8p, 9p, 11p, 17p, and 18q. Differences in the pattern of PCR products are used to identify LOH and/or MSI in the tumor cells. Representative examples of AI and MSI can be seen in Figure 23-1 in chapter 23.
An initial study, in a relatively small population of patients (20 patients with biopsy-proven bladder cancer), revealed that the sensitivity of MA was 95% compared to 50% for urine cytology.23 Most cases were detected due to 9p21 loss, with only a few cases being detected due to the presence of MSI.
Several studies have demonstrated the utility of MA for the detection of UC in urine specimens.25-28 In a phase II trial, MA was compared to the BTA stat test and cytology.25 The sensitivity of MA, the BTA stat test, and cytology were 74%, 56%, and 22%, respectively, while the specificity of MA, the BTA stat test, and cytology were 82%, 79%, and 95%, respectively.
The MA method is promising but currently has several limitations, which include labor-intensiveness, poorly defined criteria for a positive result, and poor analytical sensitivity. The criteria used to decide whether a case is positive or negative for LOH, MSI, or both have not been well defined. Studies are needed to define these criteria and determine how varying these criteria affects the sensitivity and specificity of the test. As for other tests, less-stringent criteria for a positive case would increase sensitivity and decrease specificity, while more-stringent criteria would decrease sensitivity but increase specificity. Most studies suggest that MA has high sensitivity and specificity. However, a study by Christensen et al. suggests that MA analysis lacks specificity.29 They found that patients with benign prostatic hyperplasia and cystitis showed evidence of MSI and LOH.
The analytical sensitivity (and consequently diagnostic sensitivity) of MA is limited by the fact that as the percentage of neoplastic cells in the urine becomes smaller, detection of LOH or MSI becomes more difficult. The presence of urinary leukocytes compromises the diagnostic sensitivity of MA.30 For this reason, MA would likely not be useful for detecting recurrent tumor in superficial UC patients receiving Bacillus Calmette-Guerin (BCG) therapy, since the these patients frequently have numerous leukocytes in their urine. The sensitivity of MA also would be expected to be low for urine samples with high percentages of benign squamous cells, a common finding in nonclean catch urine specimens.
Telomerase is an enzyme that normally maintains the length of the telomeres in stem cells. Interestingly, telomerase is upregulated in most malignant cells and is thought to be necessary for the maintenance of the "immortal" phenotype that typifies malignant cells. Because telomerase is over-expressed in almost all malignancies but not in most normal tissues, it has been suggested that telomerase may be a good marker for the presence of malignant cells in various cyto-logic specimens, including urine and oral washings.
The utility of telomerase for detecting UC in urine specimens has been evaluated.31-35 Most of the early studies assessed for the presence of telomerase activity with the telomere repeat amplification protocol (TRAP) assay.36 Representative examples of patients with TRAP-positive and -negative results are shown in Figure 25-3. The sensitivity of the TRAP assay for UC has varied widely. Three studies have shown that the sensitivity of the TRAP assay for UC is higher for bladder wash specimens than for voided urine specimens.33,37 Most of the studies demonstrate that the TRAP assay has high specificity for UC. One possible reason for the relatively poor sensitivity of the TRAP assay observed in some studies is that the telomerase enzyme is labile. For example, an initial study by Ramakumar et al. had a much higher sensitivity of telomerase than a follow-up study by the same group.31,38
Figure 25-3. Representative examples of telomerase positive and negative cases. Lane 1,thyroid cancer cell line (positive control); lanes 2 and 3, positive telomerase results for urinary sediments from patients with UC; lane 4, negative telomerase result for urinary sediment from patient without UC. The positive cases demonstrate the ladder typically seen when telomerase activity is present in the specimen.
The most likely explanation is that in the original study, the urine specimens were placed on ice and processed quickly for telomerase activity, while in the follow-up study, the urine specimens were collected at ambient temperature and processed several hours after collection.
Nonfastidious assays for telomerase are needed for practical reasons. Several investigators have attempted to overcome telomerase lability by assaying for the RNA component of telomerase using a reverse transcription-polymerase chain reaction (RT-PCR) assay rather than for telomerase activity.34,35 These studies suggest that the RT-PCR assay is more sensitive but less specific than the TRAP assay. For example, Neves et al. reported a sensitivity and specificity of 75% and 69%, respectively, for RT-PCR detection of telomerase.32 It should be possible to define cutoffs for the RT-PCR assay that improve the specificity of the assay.
The detection of cells in the urine that harbor FGFR3 mutations is a promising way to detect the low-grade papillary tumors that are not discovered with assays such as FISH or MA.14,30,39 FGFR3 is a tyrosine kinase receptor. Germline point mutations in various domains of FGFR3 are associated with human skeletal disorders such as hypochon-droplasia and achondroplasia, and somatic mutations of FGFR3 have been identified in bladder cancer and myeloma. Interestingly, two groups have demonstrated a high frequency of somatic FGFR3 point mutations in low-grade papillary UC and urothelial papilloma but not in high-grade papillary UC, CIS, or invasive UC.14,39 Billerey et al. found that the frequency of FGFR3 mutations by stage was pTa 74%, pTis 0%, pTl 21%, and pT2 to pT4 16%. Grade 1 showed 84%, grade 2 showed 55%, and grade 3 showed 7%. The most commonly identified FGFR3 mutation was an S249C mutation (33 of 48 tumors; 69%), but R248C, G372C, Y375C, and K652E mutations also were identified. The difference in the frequency of FGFR3 mutations between low-grade and high-grade tumors was highly significant (P < 0.0001) and is consistent with the current model of bladder tumor progression in which the most common precursor of invasive UC is CIS (Figure 251).
TP53 mutations are common in UC, especially in highgrade UC.40 Assays that assess for TP53 status could potentially be used to assess prognosis and detect tumor recurrence. Some studies have shown that TP53 overexpression detected by immunohistochemical analysis of paraffin-embedded tumors is associated with worse prognosis and higher risk of muscle invasion,41,42 while others have not.43 Immunohistochemical analysis of bladder tumors for TP53 expression has not been widely utilized by urologists or pathologists. An assay that detects urinary cells with TP53 mutations could be used to detect recurrent tumors.44 However, the heterogeneity of TP53 mutations present in different bladder cancers makes it difficult to design an assay for clinical use.
A few studies have shown that the antiapoptotic protein survivin may be a sensitive and specific marker for the detection of recurrent UC,45 but blinded prospective studies are needed to further evaluate the clinical utility of this assay. Alterations in certain genes such as glutathione S-transferase M1 and N-acetyltransferase,that encode proteins that metabolize carcinogens, may increase an individual's risk of developing bladder cancer especially among smokers, but assays for these alterations also have not been used clinically.46
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