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DNA breaks as measured by the alkaline comet assay in exfoliated cells as compared to voided urine cytology in the diagnosis of bladder cancer: a study of 105 subjects
Mutation Research 564 (2004) 57–64
DNA breaks as measured by the alkaline comet assay in exfoliated cells as compared to voided urine cytology in the diagnosis of bladder cancer: a study of 105 subjects夽 Maria Enrica Fracassoa,∗ , Paola Franceschettia , Denise Doriaa , Giorgio Talaminib , Franco Bonettic a
b
Department of Medicine and Public Health, Section of Pharmacology, Faculty of Medicine, University of Verona, Policlinico Borgo Roma, P.le Scuro, 37134 Verona, Italy Department of Biochemical Science and Surgery, University of Verona, Policlinico Borgo Roma, P.le Scuro, 37134 Verona, Italy c Department of Pathology, University of Verona, Policlinico Borgo Roma, P.le Scuro, 37134 Verona, Italy Received 20 February 2004; received in revised form 19 July 2004; accepted 22 July 2004 Available online 19 September 2004
Abstract In this study we evaluated the clinical usefulness of identifying urothelial cells with increased DNA damage with the alkaline comet assay and compare it with voided urine cytology for the assessment of markers indicative of bladder cancer. The analysis was carried out on 105 subjects having clinical suspicion of bladder cancer, and who had undergone cytology for the first time. Urine cytology and alkaline comet assay were performed on the same fresh urine samples obtained from each patient. The subjects were divided according to negative or positive cytology. The Mann-Whitney U-test showed that the comet parameters (tail moment, tail length, and % of DNA in the tail) and the numbers of comets (cells with an arbitrary cut-off value of head intensity <90% of DNA content) in subjects positive in both tests were significantly higher than in the negative group. Sensitivity, specificity, and positive and negative predictive value of the comet assay were compared with those of cytology, which is regarded as the gold standard. Sensitivity was 71.4%, specificity was 91.8%, positive and negative predictive values were 38.5 and 97.8, respectively. Two subjects negative in the comet assay were positive in cytology. Eight patients were positive in the comet assay and negative for cytology. Interestingly, one of these eight patients was later found positive for cytology. Logistic regression analysis indicates that the tail moment is significantly associated with an increased risk for positive cytology. © 2004 Elsevier B.V. All rights reserved. Keywords: Alkaline comet assay; Exfoliated cells; Urine cytology; Bladder cancer
夽 Preliminary data were presented at the Seventh International Symposium on Molecular Basis of Predictive Oncology Intervention and Strategies, Nice, France, 2004. ∗ Corresponding author. Tel.: +39 045 8027606; fax: +39 045 581111. E-mail address:[email protected] (M.E. Fracasso).
1383-5718/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2004.07.010
1. Introduction Bladder cancer is a predominant genitourinary cancer in men and for this reason intensive surveillance is necessary for early detection. According to the registry
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of tumours in Italy from 1993 to 1998, tumours of the urinary bladder rank fourth among men, with a crude incidence of 65.3/100,000 and seventh among women, with a crude incidence of 15.5/100,000 [1]. In general, patients are routinely monitored by urine cytology or cystoscopy with biopsy. Cystoscopy and urine cytology remain the gold standard in the detection of bladder cancer for diagnosis of both primary and recurrent tumours [2,3]. The endoscopic procedure has been the mainstay for bladder cancer detection with additional information provided by urine cytology, but it is invasive, uncomfortable, costly, and its sensitivity is limited to tumours that can be visualized and is relatively poor in the case of low-grade lesions. The voided urine cytology is still commonly used as diagnostic test and in the assessment and follow-up of patients with bladder cancer, although standardization of this method is still lacking [4]. The evaluation of the results is therefore subjective and the analytical accuracy depends on the skill of the cytopathologist. In addition, several other variables including the number of cells in the sample and the correct preservation of the urine, can cause changes in the exfoliated cells in voided urine, and may interfere with the diagnosis [5,6]. Consequently, a non-invasive urine assay that is sensitive, objective and accurate would be a useful adjunct to cytology in the detection of bladder cancer. In these last years an interesting technique for visualizing DNA single-strand breaks in individual cells, the single-cell gel electrophoresis (SCGE) or alkaline comet assay, has been developed [7–9]. The comet assay is one of the most promising methods in that it allows the detection of DNA damage in virtually any eukaryotic cell population that can be obtained as a single-cell suspension, and can be carried out on cycling as well as on quiescent cells. This assay has been widely used for different kinds of studies [10], including DNA repair [11,12], human biomonitoring [13–15] and genetic toxicology [16,17]. Previously, several studies used the alkaline comet assay to measure DNA strand breaks in cells obtained by washing the human urinary bladder. McKelvey-Martin et al. [18] reported that exfoliated cells extracted from bladder washings from patients with transitional cell carcinoma presented high levels of DNA damage compared with specimens from control patients. Recently, Gontijo et al. used the same procedure in urine collection, with modifications of the standard alkaline protocol, and
demonstrated that patients with a smoking history [19] and patients with a history of previous urothelial carcinoma [20] have elevated levels of DNA damage in cytologically normal urothelial cells. Both authors used urinary bladder washings, obtained by either syringe aspiration through the irrigation channel or by drainage via the main cystoscope channel, and these procedures, besides being inconvenient for the patient, can lead to trauma with consequent contamination with non-urothelial cells (i.e., leukocytes). The aim of this study is to compare the results of the alkaline comet assay with the outcome of cytology in exfoliated cells obtained from voided urine of subjects who underwent cytology for the first time, in order to assess sensitivity, specificity and diagnostic accuracy of the comet assay compared with cytology, and to propose the comet assay as a useful non-invasive test in adjunct to cytology for the early detection of bladder cancer. 2. Material and methods 2.1. Patients A total of 105 consecutive subjects (80 males, 25 females; mean age, 62 ± 14 years) with clinical suspicion of bladder cancer or urological disorders provided voided urine samples for cytology for the first time. Informed consent was obtained from all participating subjects. 2.2. Urine samples and cytology Urine samples from each patient were taken fresh in the morning on three consecutive days: urine cytology and alkaline comet assay were immediately conducted on the same samples. For cytological analysis two 10 ml aliquots of each urine sample were centrifuged, the cell pellets were concentrated with a cytospin onto slides, and stained with the standard hematoxylin/eosin staining procedure. Slides were then observed through a microscope and at least 100 cells for each urine sample were morphologically evaluated. The cytological analysis was defined according to the morphological aspects of the urothelial cells present in the specimen and categorized as: normal, inflammatory, atypical and neoplastic. Cytological analysis and the evaluation of abnormalities
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in nuclei and cytoplasm were performed according to the classical procedure described by Koss [21]. Normal and inflammatory cells were then clustered together as negative, and atypical and neoplastic as positive. Urine cytology was performed by a pathologist who was unaware of the results of the alkaline comet assay. 2.3. Alkaline comet assay One 50 ml aliquot of each freshly voided urine sample was centrifuged and the pellet of exfoliated cells was immediately used for the alkaline comet assay. The cells were counted, with cell numbers varying from 20,000 to 150,000 per sample. The cell viability was determined using the trypan-blue exclusion technique, and the mean value ± S.D. was 87.65 ± 12.95%. The alkaline comet assay was basically performed according to Singh et al. [7] with some modifications. Briefly, the modifications of the standard alkaline protocol included the treatment of the cells, once embedded in agarose and layered on a microscope slide, with proteinase K (1 mg/ml; 70 l/slide) (Sigma-Aldrich, Milan, Italy) for 2 h at room temperature; and subsequent immersion of the slides at 4 ◦ C overnight in freshly prepared lysis solution (2.5 M NaCl, 100 mM Na2 -EDTA, 10 mM Tris, 1% N-lauroylsarcosine, pH 10, supplemented immediately prior use with 10% DMSO and 1% Triton X-100) (all provided by Sigma-Aldrich, Milan, Italy). These modified conditions [22–24] had been identified in prior experiments as giving a suitable lysis of the exfoliated cell membrane, because with the traditional solution, the transitional cells remained intact, even after extended lysis. The following steps, pH > 13 to denature the DNA (for 40 min), electrophoresis (0.66 V/cm, for 20 min), and neutralization were performed as in the standard protocol. All of these steps were conducted under dimmed light to prevent induction of additional DNA damage. Following the electrophoretic run, the slides were neutralized, dipped in cold 100% ethanol (JT Baker, Milan Italy), dried at room temperature and kept in a dry atmosphere for a period of up to 3 months until analysis [25]. 2.4. Comet assay analysis Staining was performed with 45 l ethidium bromide (20 g/ml) (Sigma-Aldrich, Milan, Italy) and the
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slides were examined at 400× magnification using a fluorescence microscope under green light. A total of at least 300 cells were counted randomly for each subject (50 cells each on two replicate slides of three urine samples). Among the parameters provided by the image-analysis system (Comet Assay II, Perceptive Instruments, Suffolk, UK), head intensity (HI), tail DNA content (% DNA), tail length in m (TL) and tail moment (TM), which was defined as the product of the comet length and the amount on DNA in the tail [26], were considered for statistical analysis. A cell was defined as a comet when the head intensity (HI) was <90% of the DNA content, an arbitrary cut-off value. The exfoliated urothelial cells were distinguished from other contaminating cells (e.g., leukocytes) on the basis of their size and the form of their comet head. The urothelial cells appeared with irregular edges and larger size compared with that of leukocytes, typically round and small (about 20 m) according to Gontijo et al. [19]. We have considered as potential apoptotic cells, those comets that clearly showed a morphological distinguishable feature usually depicted as structures with diffuse fan-like tail, very small head, ghost cells or clouds [8,27–29]. The presumed apoptotic cells were recorded only as percentage and not included in the evaluation of comet parameters. The Comet assay was performed by investigators who were unaware of the cytology results. The median of each parameter was used as a representative value for each subject, and the mean of all medians was used for statistical analysis. 2.5. Statistical analysis The statistical analysis was carried out using the Statgraphics program, version 5.0. The correlation between positive or negative cytology responses and positive or negative results in the alkaline comet assay was evaluated with the chi-square test and by use of the Spearman’s correlation coefficient. The MannWhitney U-test was used to test the differences between groups. P-values below 0.05 were considered to correspond with statistical significance. The sensitivity was calculated as true positive results/all patients with positive cytology; specificity was calculated as true negative results/all patients with negative cytology. The role of each possible prognostic factor (univariate analysis) and the joint effect of all these factors
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Table 1 Comparison of tail moment (TM), tail length (TL) and percentage of DNA in the comet tail (%DNA), number of comets and presumed apoptotic cells of subjects divided according to negative or positive urine cytology P-valuea
Cytology Negativeb (n = 98)
Positiveb (n = 7)
Comet parameters TM % DNA TL
0.45 ± 0.43 4.78 ± 2.23 17.26 ± 8.86
4.53 ± 5.66 19.18 ± 19.75 33.87 ± 18.61
0.012 0.027 0.005
No. of cometsc (%)
16.48 ± 16.75
44.57 ± 31.93
0.003
6.91 ± 12.53
14.86 ± 19.14
0.416
No. of presumed apoptotic cellsc (%) a b c
Mann-Whitney U-test. Mean ± S.D.; in brackets number of subjects. 50 Cells scored on each of two replicate slides from three urine samples for each subject (300 randomly selected cells).
(multivariate analysis) were explored using the multivariate logistic regression analysis. The final results of this analysis are Odds ratio (95% – CI). The likelihood ratio statistic was used to test the following hypothesis: Odds ratio = 1.0 and P-value < 0.05 were considered significant.
3. Results Voided urine cytology was performed with samples from 105 patients according to standard procedures and the alkaline comet assay was carried out on the same urine samples. Of these patients, 98 (93.3%) reported negative cytology (normal and inflammatory) and 7 (6.6%) were positive (atypical and neoplastic). The comet parameters in exfoliated urothelial cells divided in negative and positive cytology are reported in Table 1. All parameters characterizing DNA strand breaks showed mean values significantly higher in positive than in negative cytology patients analyzed by Mann-Whitney U-test (TM, P = 0.012; % DNA, P = 0.027; TL, P = 0.005). The mean number of comets, assessed on 300 randomly selected cells (50 cells each on two replicate slides from three urine samples for each subject), markedly increased in the positive cytology compared with the negative cytology group (P = 0.003), whereas the difference in the number of presumed apoptotic cells was not significant. The negative and positive responses in the comet assay compared with the respective results of the cytology, as gold standard, are reported in Table 2. The comet assay on urothelial cells was positive in
5 of 7 patients with positive cytology, a sensitivity of 71.4% (33.0–94.9) and a positive predictive value (PPV) of 38.5; negative responses in the comet assay were seen in 90 of 98 subjects with negative cytology, a specificity of 91.83% (85.1–96.1), with a negative predictive value (NPV) of 97.8. The statistical analysis shows that the results of the comet assay are significantly correlated with the cytological results (chi-square = 24.1, P < 0.0001; Spearman correlation r = 0.48, P < 0.0001). In the present study the comet test was negative in two subjects with positive cytology (false negative, 28.6%) and positive in eight subjects with negative cytology (false positive, 8.2%). Table 3 shows the descriptive parameters of the alkaline Table 2 Frequency of negative and positive responses in the comet assay compared to the respective results in cytologya Cytology Positive Comet positive Sensitivity (%) Range PPVb
5 71.4 33.0–94.9 38.5
Total Negative 8
Comet negative Specificity (%) Range NPVc
2
90 91.83 85.1–96.1 97.8
Total
7
98
13
92
105
Statistical test: χ2 -test = 24.1; P < 0.0001. Spearman correlation: r = 0.48; P < 0.0001. b PPV, positive predictive value. c NPV, negative predictive value. a
5.68 (11.73) 20.75 (13.81), P = 0.0016 4 (5.65) 19.20 (21.43), NSb
Gender Age Cell viability No. of comets No. of presumed apoptotic cells Tail moment
a
b
Mean (S.D.) Mean (S.D.) Negative/positive (2) Positive/positive (5)
Mann-Whitney U-test; negative/negative group vs. positive/negative and negative/negative vs. positive/positive group. NS: not significant vs. negative/negative group.
13.08 (11.82) 54.75 (16.98), P = 0.0001 14 (1.41) 56.80 (29.58), P = 0.0014
Comets (%) TL
16.03 (0.69) 31.15 (15.46), P = 0.0002 14.93 (1.21) 41.44 (16.37), P = 0.0004 4.38 (0.16) 9.29 (2.73), P = 0.0001 4.41 (0.07) 25.09 (20.79), P = 0.0076
% DNA
Mean (S.D.) Mean (S.D.) Negative/negative (90) Positive/negative (8)
TM Statistical dataa Groups (no. of patients)
Table 3 Descriptive parameters [mean (S.D.)] of each group evaluated according to the responses in comet assay/cytology
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Table 4 Result of multivariate logistic regression analysis considering positive and negative subjects for cytology (n = 105)
0.37 (0.18) 1.34 (1.07), P = 0.0001 0.31 (0.09) 6.21 (5.92), P = 0.0007
Presumed apoptotic cells (%)
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Odds ratio
95 % CI
P-value
1.4 1.07 1.03 1.01 1.03
0.29–17.6 0.94–1.23 0.95–1.10 0.92–1.09 0.96–1.10
0.19 0.32 0.38 0.96 0.46
3.12
1.52–6.40
0.002
comet assay among groups of subjects, considering the responses obtained from both the comet and the cytology tests (negative/negative; positive/negative; negative/positive; positive/positive). The statistical analysis by a non-parametric test (Mann-Whitney Utest) showed that the group of patients that was positive in both tests (positive/positive) and the group positive only in the comet assay (positive/negative) presented mean values of TM, % DNA, TL and number of comets significantly higher than the negative/negative patients. The positive/negative group was not statistically different from the positive/positive group, and showed a number of presumed apoptotic cells that was significantly higher than did the negative/negative group. The two subjects negative in comet assay but with positive cytology (negative/positive) displayed mean values of comet parameters, number of comets and presumed apoptotic cells similar to the negative/negative group. A multivariate logistic regression analysis was carried out dividing the patients into positive or negative groups for cytology (Table 4). Considering the gender, age, tail moment (TM), number of comets, number of presumed apoptotic cells, and cellular viability as independent predictors with a method backward, the tail moment was statistically and significantly correlated with positive cytology. The Odds ratio for TM versus positive cytology was 3.12 (95% CI, 1.52–6.40; P = 0.002).
4. Discussion The aim of our study was to compare, for the same group of patients, the results of the alkaline comet assay with the classical voided urine cytology, for the detection of cytological anomalies.
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Urine cytology is still the most commonly used non-invasive test and remains the gold standard for bladder cancer screening [4]. It is the test against which all others are compared when evaluating potential bladder tumour markers. Although the ability of cytology to detect low-grade bladder tumours is limited, urine cytology has excellent specificity with only few false-positive cases [30,31]. Thus, there is a need of a molecular tumour marker assay that may improve the sensitivity for low-grade lesions, and at the same time, easy to perform. Several promising bladder tumour markers have been investigated as potential screening tools, e.g., BTA, bladder tumour antigen or NMP, nuclear matrix protein [32–36]. As no markers have 100% sensitivity and 100% specificity, the selection of markers or a combination of markers may be optimized for clinical use. The alkaline version of the comet assay detects DNA double-, single-strand breaks, alkali-labile adducts, and incomplete repair sites [37]. Some authors using the comet assay have found increased DNA damage in cells in bladder washings from patients with current urothelial carcinoma in comparison with a matched control group [18]. The present study, instead, was aimed to identify by use of the comet assay positive or negative subjects among patients that are only suspected of being positive for bladder cancer, and to compare the results with that of cytology, as gold standard. The new application that we have considered is related to the possibility of an early identification of neoplastic cells, in that some cells may have damaged DNA in spite of a normal morphology. In our study, the alkaline comet assay found eight patients as false-positives, but it is interesting to note that one of these came out positive in cytology after a following check (4 months later) and even in this case the comet response was positive. This is of particular importance, because it is possible that other subjects, that initially resulted as negative in cytology but clearly positive in the comet assay (thus considered false-positive), may become positive for cytology in the subsequent urinary follow-up. In our laboratory a follow-up of these patients is still in progress. It should also be noted that, when the patients were analyzed by evaluating the responses in both tests (comet/cytology), the false-positive group showed values that were significantly higher than those of the negative/negative group. Some authors using the comet assay detected a signif-
icant increase in DNA damage of exfoliated urothelial cells from smokers without urinary bladder neoplasia compared with a control group of non-smokers, and no difference was observed between ex-smokers and current smokers [19]. Considering these findings, we carried out a retrospective analysis to see if the subjects of our false-positive group might be heavy smokers. This group consisted of three female non-smokers (aged 65.33 ± 6.10 years), and five males (aged 66.00 ± 8.60 years), four of whom were smokers (12 ± 7 cigarettes per day). As not all subjects in this group are smokers, it seems logical to consider, therefore, that the smoking habits are only one of the causes leading to a positive response in the comet assay. In this way, our investigations by use of the comet assay on active and passive smoking effects in lymphocytes and urothelial cells gave similar results: even if the lymphocytes from smokers showed considerable DNA damage, the urothelial cells presented basal levels of DNA damage (data not shown). As tail moment, tail length and % of DNA in the tail were closely correlated, only the tail moment was used as independent variable for DNA damage. The multivariate logistic regression analysis, dividing the patients in those with positive or negative cytology, identified the tail moment as a variable associated with positive cytology. In conclusion, our results demonstrate that the alkaline comet assay in exfoliated cells in voided urine could be a useful adjunct to cytology for the detection of bladder cancer. We have shown that cytology may miss a percentage of positive cases, so it is possible that patients with negative cytology but with clearly positive results in the comet assay could be considered subjects at risk for developing cancer.
Acknowledgement This work was partially supported by grant no. 2003063319 (MIUR-COFIN 2003).
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DNA breaks as measured by the alkaline comet assay in exfoliated cells as compared to voided urine cytology in the diagnosis of bladder cancer: a study of 105 subjects夽 Maria Enrica Fracassoa,∗ , Paola Franceschettia , Denise Doriaa , Giorgio Talaminib , Franco Bonettic a
b
Department of Medicine and Public Health, Section of Pharmacology, Faculty of Medicine, University of Verona, Policlinico Borgo Roma, P.le Scuro, 37134 Verona, Italy Department of Biochemical Science and Surgery, University of Verona, Policlinico Borgo Roma, P.le Scuro, 37134 Verona, Italy c Department of Pathology, University of Verona, Policlinico Borgo Roma, P.le Scuro, 37134 Verona, Italy Received 20 February 2004; received in revised form 19 July 2004; accepted 22 July 2004 Available online 19 September 2004
Abstract In this study we evaluated the clinical usefulness of identifying urothelial cells with increased DNA damage with the alkaline comet assay and compare it with voided urine cytology for the assessment of markers indicative of bladder cancer. The analysis was carried out on 105 subjects having clinical suspicion of bladder cancer, and who had undergone cytology for the first time. Urine cytology and alkaline comet assay were performed on the same fresh urine samples obtained from each patient. The subjects were divided according to negative or positive cytology. The Mann-Whitney U-test showed that the comet parameters (tail moment, tail length, and % of DNA in the tail) and the numbers of comets (cells with an arbitrary cut-off value of head intensity <90% of DNA content) in subjects positive in both tests were significantly higher than in the negative group. Sensitivity, specificity, and positive and negative predictive value of the comet assay were compared with those of cytology, which is regarded as the gold standard. Sensitivity was 71.4%, specificity was 91.8%, positive and negative predictive values were 38.5 and 97.8, respectively. Two subjects negative in the comet assay were positive in cytology. Eight patients were positive in the comet assay and negative for cytology. Interestingly, one of these eight patients was later found positive for cytology. Logistic regression analysis indicates that the tail moment is significantly associated with an increased risk for positive cytology. © 2004 Elsevier B.V. All rights reserved. Keywords: Alkaline comet assay; Exfoliated cells; Urine cytology; Bladder cancer
夽 Preliminary data were presented at the Seventh International Symposium on Molecular Basis of Predictive Oncology Intervention and Strategies, Nice, France, 2004. ∗ Corresponding author. Tel.: +39 045 8027606; fax: +39 045 581111. E-mail address:[email protected] (M.E. Fracasso).
1383-5718/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2004.07.010
1. Introduction Bladder cancer is a predominant genitourinary cancer in men and for this reason intensive surveillance is necessary for early detection. According to the registry
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of tumours in Italy from 1993 to 1998, tumours of the urinary bladder rank fourth among men, with a crude incidence of 65.3/100,000 and seventh among women, with a crude incidence of 15.5/100,000 [1]. In general, patients are routinely monitored by urine cytology or cystoscopy with biopsy. Cystoscopy and urine cytology remain the gold standard in the detection of bladder cancer for diagnosis of both primary and recurrent tumours [2,3]. The endoscopic procedure has been the mainstay for bladder cancer detection with additional information provided by urine cytology, but it is invasive, uncomfortable, costly, and its sensitivity is limited to tumours that can be visualized and is relatively poor in the case of low-grade lesions. The voided urine cytology is still commonly used as diagnostic test and in the assessment and follow-up of patients with bladder cancer, although standardization of this method is still lacking [4]. The evaluation of the results is therefore subjective and the analytical accuracy depends on the skill of the cytopathologist. In addition, several other variables including the number of cells in the sample and the correct preservation of the urine, can cause changes in the exfoliated cells in voided urine, and may interfere with the diagnosis [5,6]. Consequently, a non-invasive urine assay that is sensitive, objective and accurate would be a useful adjunct to cytology in the detection of bladder cancer. In these last years an interesting technique for visualizing DNA single-strand breaks in individual cells, the single-cell gel electrophoresis (SCGE) or alkaline comet assay, has been developed [7–9]. The comet assay is one of the most promising methods in that it allows the detection of DNA damage in virtually any eukaryotic cell population that can be obtained as a single-cell suspension, and can be carried out on cycling as well as on quiescent cells. This assay has been widely used for different kinds of studies [10], including DNA repair [11,12], human biomonitoring [13–15] and genetic toxicology [16,17]. Previously, several studies used the alkaline comet assay to measure DNA strand breaks in cells obtained by washing the human urinary bladder. McKelvey-Martin et al. [18] reported that exfoliated cells extracted from bladder washings from patients with transitional cell carcinoma presented high levels of DNA damage compared with specimens from control patients. Recently, Gontijo et al. used the same procedure in urine collection, with modifications of the standard alkaline protocol, and
demonstrated that patients with a smoking history [19] and patients with a history of previous urothelial carcinoma [20] have elevated levels of DNA damage in cytologically normal urothelial cells. Both authors used urinary bladder washings, obtained by either syringe aspiration through the irrigation channel or by drainage via the main cystoscope channel, and these procedures, besides being inconvenient for the patient, can lead to trauma with consequent contamination with non-urothelial cells (i.e., leukocytes). The aim of this study is to compare the results of the alkaline comet assay with the outcome of cytology in exfoliated cells obtained from voided urine of subjects who underwent cytology for the first time, in order to assess sensitivity, specificity and diagnostic accuracy of the comet assay compared with cytology, and to propose the comet assay as a useful non-invasive test in adjunct to cytology for the early detection of bladder cancer. 2. Material and methods 2.1. Patients A total of 105 consecutive subjects (80 males, 25 females; mean age, 62 ± 14 years) with clinical suspicion of bladder cancer or urological disorders provided voided urine samples for cytology for the first time. Informed consent was obtained from all participating subjects. 2.2. Urine samples and cytology Urine samples from each patient were taken fresh in the morning on three consecutive days: urine cytology and alkaline comet assay were immediately conducted on the same samples. For cytological analysis two 10 ml aliquots of each urine sample were centrifuged, the cell pellets were concentrated with a cytospin onto slides, and stained with the standard hematoxylin/eosin staining procedure. Slides were then observed through a microscope and at least 100 cells for each urine sample were morphologically evaluated. The cytological analysis was defined according to the morphological aspects of the urothelial cells present in the specimen and categorized as: normal, inflammatory, atypical and neoplastic. Cytological analysis and the evaluation of abnormalities
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in nuclei and cytoplasm were performed according to the classical procedure described by Koss [21]. Normal and inflammatory cells were then clustered together as negative, and atypical and neoplastic as positive. Urine cytology was performed by a pathologist who was unaware of the results of the alkaline comet assay. 2.3. Alkaline comet assay One 50 ml aliquot of each freshly voided urine sample was centrifuged and the pellet of exfoliated cells was immediately used for the alkaline comet assay. The cells were counted, with cell numbers varying from 20,000 to 150,000 per sample. The cell viability was determined using the trypan-blue exclusion technique, and the mean value ± S.D. was 87.65 ± 12.95%. The alkaline comet assay was basically performed according to Singh et al. [7] with some modifications. Briefly, the modifications of the standard alkaline protocol included the treatment of the cells, once embedded in agarose and layered on a microscope slide, with proteinase K (1 mg/ml; 70 l/slide) (Sigma-Aldrich, Milan, Italy) for 2 h at room temperature; and subsequent immersion of the slides at 4 ◦ C overnight in freshly prepared lysis solution (2.5 M NaCl, 100 mM Na2 -EDTA, 10 mM Tris, 1% N-lauroylsarcosine, pH 10, supplemented immediately prior use with 10% DMSO and 1% Triton X-100) (all provided by Sigma-Aldrich, Milan, Italy). These modified conditions [22–24] had been identified in prior experiments as giving a suitable lysis of the exfoliated cell membrane, because with the traditional solution, the transitional cells remained intact, even after extended lysis. The following steps, pH > 13 to denature the DNA (for 40 min), electrophoresis (0.66 V/cm, for 20 min), and neutralization were performed as in the standard protocol. All of these steps were conducted under dimmed light to prevent induction of additional DNA damage. Following the electrophoretic run, the slides were neutralized, dipped in cold 100% ethanol (JT Baker, Milan Italy), dried at room temperature and kept in a dry atmosphere for a period of up to 3 months until analysis [25]. 2.4. Comet assay analysis Staining was performed with 45 l ethidium bromide (20 g/ml) (Sigma-Aldrich, Milan, Italy) and the
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slides were examined at 400× magnification using a fluorescence microscope under green light. A total of at least 300 cells were counted randomly for each subject (50 cells each on two replicate slides of three urine samples). Among the parameters provided by the image-analysis system (Comet Assay II, Perceptive Instruments, Suffolk, UK), head intensity (HI), tail DNA content (% DNA), tail length in m (TL) and tail moment (TM), which was defined as the product of the comet length and the amount on DNA in the tail [26], were considered for statistical analysis. A cell was defined as a comet when the head intensity (HI) was <90% of the DNA content, an arbitrary cut-off value. The exfoliated urothelial cells were distinguished from other contaminating cells (e.g., leukocytes) on the basis of their size and the form of their comet head. The urothelial cells appeared with irregular edges and larger size compared with that of leukocytes, typically round and small (about 20 m) according to Gontijo et al. [19]. We have considered as potential apoptotic cells, those comets that clearly showed a morphological distinguishable feature usually depicted as structures with diffuse fan-like tail, very small head, ghost cells or clouds [8,27–29]. The presumed apoptotic cells were recorded only as percentage and not included in the evaluation of comet parameters. The Comet assay was performed by investigators who were unaware of the cytology results. The median of each parameter was used as a representative value for each subject, and the mean of all medians was used for statistical analysis. 2.5. Statistical analysis The statistical analysis was carried out using the Statgraphics program, version 5.0. The correlation between positive or negative cytology responses and positive or negative results in the alkaline comet assay was evaluated with the chi-square test and by use of the Spearman’s correlation coefficient. The MannWhitney U-test was used to test the differences between groups. P-values below 0.05 were considered to correspond with statistical significance. The sensitivity was calculated as true positive results/all patients with positive cytology; specificity was calculated as true negative results/all patients with negative cytology. The role of each possible prognostic factor (univariate analysis) and the joint effect of all these factors
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Table 1 Comparison of tail moment (TM), tail length (TL) and percentage of DNA in the comet tail (%DNA), number of comets and presumed apoptotic cells of subjects divided according to negative or positive urine cytology P-valuea
Cytology Negativeb (n = 98)
Positiveb (n = 7)
Comet parameters TM % DNA TL
0.45 ± 0.43 4.78 ± 2.23 17.26 ± 8.86
4.53 ± 5.66 19.18 ± 19.75 33.87 ± 18.61
0.012 0.027 0.005
No. of cometsc (%)
16.48 ± 16.75
44.57 ± 31.93
0.003
6.91 ± 12.53
14.86 ± 19.14
0.416
No. of presumed apoptotic cellsc (%) a b c
Mann-Whitney U-test. Mean ± S.D.; in brackets number of subjects. 50 Cells scored on each of two replicate slides from three urine samples for each subject (300 randomly selected cells).
(multivariate analysis) were explored using the multivariate logistic regression analysis. The final results of this analysis are Odds ratio (95% – CI). The likelihood ratio statistic was used to test the following hypothesis: Odds ratio = 1.0 and P-value < 0.05 were considered significant.
3. Results Voided urine cytology was performed with samples from 105 patients according to standard procedures and the alkaline comet assay was carried out on the same urine samples. Of these patients, 98 (93.3%) reported negative cytology (normal and inflammatory) and 7 (6.6%) were positive (atypical and neoplastic). The comet parameters in exfoliated urothelial cells divided in negative and positive cytology are reported in Table 1. All parameters characterizing DNA strand breaks showed mean values significantly higher in positive than in negative cytology patients analyzed by Mann-Whitney U-test (TM, P = 0.012; % DNA, P = 0.027; TL, P = 0.005). The mean number of comets, assessed on 300 randomly selected cells (50 cells each on two replicate slides from three urine samples for each subject), markedly increased in the positive cytology compared with the negative cytology group (P = 0.003), whereas the difference in the number of presumed apoptotic cells was not significant. The negative and positive responses in the comet assay compared with the respective results of the cytology, as gold standard, are reported in Table 2. The comet assay on urothelial cells was positive in
5 of 7 patients with positive cytology, a sensitivity of 71.4% (33.0–94.9) and a positive predictive value (PPV) of 38.5; negative responses in the comet assay were seen in 90 of 98 subjects with negative cytology, a specificity of 91.83% (85.1–96.1), with a negative predictive value (NPV) of 97.8. The statistical analysis shows that the results of the comet assay are significantly correlated with the cytological results (chi-square = 24.1, P < 0.0001; Spearman correlation r = 0.48, P < 0.0001). In the present study the comet test was negative in two subjects with positive cytology (false negative, 28.6%) and positive in eight subjects with negative cytology (false positive, 8.2%). Table 3 shows the descriptive parameters of the alkaline Table 2 Frequency of negative and positive responses in the comet assay compared to the respective results in cytologya Cytology Positive Comet positive Sensitivity (%) Range PPVb
5 71.4 33.0–94.9 38.5
Total Negative 8
Comet negative Specificity (%) Range NPVc
2
90 91.83 85.1–96.1 97.8
Total
7
98
13
92
105
Statistical test: χ2 -test = 24.1; P < 0.0001. Spearman correlation: r = 0.48; P < 0.0001. b PPV, positive predictive value. c NPV, negative predictive value. a
5.68 (11.73) 20.75 (13.81), P = 0.0016 4 (5.65) 19.20 (21.43), NSb
Gender Age Cell viability No. of comets No. of presumed apoptotic cells Tail moment
a
b
Mean (S.D.) Mean (S.D.) Negative/positive (2) Positive/positive (5)
Mann-Whitney U-test; negative/negative group vs. positive/negative and negative/negative vs. positive/positive group. NS: not significant vs. negative/negative group.
13.08 (11.82) 54.75 (16.98), P = 0.0001 14 (1.41) 56.80 (29.58), P = 0.0014
Comets (%) TL
16.03 (0.69) 31.15 (15.46), P = 0.0002 14.93 (1.21) 41.44 (16.37), P = 0.0004 4.38 (0.16) 9.29 (2.73), P = 0.0001 4.41 (0.07) 25.09 (20.79), P = 0.0076
% DNA
Mean (S.D.) Mean (S.D.) Negative/negative (90) Positive/negative (8)
TM Statistical dataa Groups (no. of patients)
Table 3 Descriptive parameters [mean (S.D.)] of each group evaluated according to the responses in comet assay/cytology
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Table 4 Result of multivariate logistic regression analysis considering positive and negative subjects for cytology (n = 105)
0.37 (0.18) 1.34 (1.07), P = 0.0001 0.31 (0.09) 6.21 (5.92), P = 0.0007
Presumed apoptotic cells (%)
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Odds ratio
95 % CI
P-value
1.4 1.07 1.03 1.01 1.03
0.29–17.6 0.94–1.23 0.95–1.10 0.92–1.09 0.96–1.10
0.19 0.32 0.38 0.96 0.46
3.12
1.52–6.40
0.002
comet assay among groups of subjects, considering the responses obtained from both the comet and the cytology tests (negative/negative; positive/negative; negative/positive; positive/positive). The statistical analysis by a non-parametric test (Mann-Whitney Utest) showed that the group of patients that was positive in both tests (positive/positive) and the group positive only in the comet assay (positive/negative) presented mean values of TM, % DNA, TL and number of comets significantly higher than the negative/negative patients. The positive/negative group was not statistically different from the positive/positive group, and showed a number of presumed apoptotic cells that was significantly higher than did the negative/negative group. The two subjects negative in comet assay but with positive cytology (negative/positive) displayed mean values of comet parameters, number of comets and presumed apoptotic cells similar to the negative/negative group. A multivariate logistic regression analysis was carried out dividing the patients into positive or negative groups for cytology (Table 4). Considering the gender, age, tail moment (TM), number of comets, number of presumed apoptotic cells, and cellular viability as independent predictors with a method backward, the tail moment was statistically and significantly correlated with positive cytology. The Odds ratio for TM versus positive cytology was 3.12 (95% CI, 1.52–6.40; P = 0.002).
4. Discussion The aim of our study was to compare, for the same group of patients, the results of the alkaline comet assay with the classical voided urine cytology, for the detection of cytological anomalies.
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Urine cytology is still the most commonly used non-invasive test and remains the gold standard for bladder cancer screening [4]. It is the test against which all others are compared when evaluating potential bladder tumour markers. Although the ability of cytology to detect low-grade bladder tumours is limited, urine cytology has excellent specificity with only few false-positive cases [30,31]. Thus, there is a need of a molecular tumour marker assay that may improve the sensitivity for low-grade lesions, and at the same time, easy to perform. Several promising bladder tumour markers have been investigated as potential screening tools, e.g., BTA, bladder tumour antigen or NMP, nuclear matrix protein [32–36]. As no markers have 100% sensitivity and 100% specificity, the selection of markers or a combination of markers may be optimized for clinical use. The alkaline version of the comet assay detects DNA double-, single-strand breaks, alkali-labile adducts, and incomplete repair sites [37]. Some authors using the comet assay have found increased DNA damage in cells in bladder washings from patients with current urothelial carcinoma in comparison with a matched control group [18]. The present study, instead, was aimed to identify by use of the comet assay positive or negative subjects among patients that are only suspected of being positive for bladder cancer, and to compare the results with that of cytology, as gold standard. The new application that we have considered is related to the possibility of an early identification of neoplastic cells, in that some cells may have damaged DNA in spite of a normal morphology. In our study, the alkaline comet assay found eight patients as false-positives, but it is interesting to note that one of these came out positive in cytology after a following check (4 months later) and even in this case the comet response was positive. This is of particular importance, because it is possible that other subjects, that initially resulted as negative in cytology but clearly positive in the comet assay (thus considered false-positive), may become positive for cytology in the subsequent urinary follow-up. In our laboratory a follow-up of these patients is still in progress. It should also be noted that, when the patients were analyzed by evaluating the responses in both tests (comet/cytology), the false-positive group showed values that were significantly higher than those of the negative/negative group. Some authors using the comet assay detected a signif-
icant increase in DNA damage of exfoliated urothelial cells from smokers without urinary bladder neoplasia compared with a control group of non-smokers, and no difference was observed between ex-smokers and current smokers [19]. Considering these findings, we carried out a retrospective analysis to see if the subjects of our false-positive group might be heavy smokers. This group consisted of three female non-smokers (aged 65.33 ± 6.10 years), and five males (aged 66.00 ± 8.60 years), four of whom were smokers (12 ± 7 cigarettes per day). As not all subjects in this group are smokers, it seems logical to consider, therefore, that the smoking habits are only one of the causes leading to a positive response in the comet assay. In this way, our investigations by use of the comet assay on active and passive smoking effects in lymphocytes and urothelial cells gave similar results: even if the lymphocytes from smokers showed considerable DNA damage, the urothelial cells presented basal levels of DNA damage (data not shown). As tail moment, tail length and % of DNA in the tail were closely correlated, only the tail moment was used as independent variable for DNA damage. The multivariate logistic regression analysis, dividing the patients in those with positive or negative cytology, identified the tail moment as a variable associated with positive cytology. In conclusion, our results demonstrate that the alkaline comet assay in exfoliated cells in voided urine could be a useful adjunct to cytology for the detection of bladder cancer. We have shown that cytology may miss a percentage of positive cases, so it is possible that patients with negative cytology but with clearly positive results in the comet assay could be considered subjects at risk for developing cancer.
Acknowledgement This work was partially supported by grant no. 2003063319 (MIUR-COFIN 2003).
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