Photodiagnosis and Photodynamic Therapy (2015) 12, 282—288
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Protoporphyrin IX induced by 5-aminolevulinic acid in bladder cancer cells in voided urine can be extracorporeally quantified using a spectrophotometer Yasushi Nakai, Satoshi Anai, Sayuri Onishi, Kuwada Masaomi, Yoshihiro Tatsumi, Makito Miyake, Yoshitomo Chihara, Nobumichi Tanaka, Yoshihiko Hirao, Kiyohide Fujimoto MD, PhD ∗ Department of Urology, Nara Medical University, 840 Shijo-cho, Kashihara-shi, Nara 634-8522, Japan Received 10 August 2014; received in revised form 25 December 2014; accepted 26 December 2014 Available online 13 January 2015
KEYWORDS 5-Aminolevulinic acid; Photodynamic detection; Urinary cytology; Spectrophotometer
∗
Summary Background: We evaluated the feasibility of photodynamic diagnosis of bladder cancer by spectrophotometric analysis of voided urine samples after extracorporeal treatment with 5aminolevulinic acid (ALA). Methods: Sixty-one patients with bladder cancer, confirmed histologically after the transurethral resection of a bladder tumor, were recruited as the bladder cancer group, and 50 outpatients without history of urothelial carcinoma or cancer-related findings were recruited as the control group. Half of the voided urine sample was incubated with ALA (ALA-treated sample), and the rest was incubated without treatment (ALA-untreated sample). For detecting cellular protoporphyrin IX levels, intensity of the samples at the excitation wavelength of 405 nm was measured using a spectrophotometer. The difference between the intensity of the ALA-treated and ALA-untreated samples at 635 nm was calculated. Results: The differences in the bladder cancer group were significantly greater than those in the control group (p < 0.001). These differences were also significantly greater in patients with highgrade tumors than in those with low-grade tumors (p = 0.004), and also in patients with invasive bladder cancer than in those with noninvasive bladder cancer (p = 0.007). The area under the curve was 0.84. Sensitivity and specificity of the method were 82% and 80%, respectively.
Corresponding author. Tel.: +81 744 22 3051; fax: +81 744240345. E-mail address: [email protected] (K. Fujimoto).
http://dx.doi.org/10.1016/j.pdpdt.2014.12.010 1572-1000/© 2015 Elsevier B.V. All rights reserved.
Protoporphyrin IX induced by 5-aminolevulinic acid in bladder cancer cells in voided
283
Conclusions: We demonstrated that protoporphyrin IX levels in urinary cells treated with ALA could be quantitatively detected by spectrophotometer in patients with bladder cancer. Therefore, this cancer detection system has a potential for clinical use. © 2015 Elsevier B.V. All rights reserved.
Introduction
Materials and methods
Although some researchers have attempted to establish less-invasive new diagnostic methods, voided urinary cytology remains the most established noninvasive method for detecting bladder cancer. Voided urinary cytology has high specificity but low sensitivity and large interobserver variability [1—3]. To solve these problems, two reports about voided urinary cytology using 5-aminolevulinic acid (ALA) have been published [4,5]. ALA is a precursor in heme biosynthesis and causes fluorescent endogenous porphyrins, mainly protoporphyrin IX, to accumulate significantly more in tumor cells than in healthy cells because of factors, such as decreased ferrochelatase activity. Under excitation at 405 nm, protoporphyrin IX emits red fluorescence which has a peak at the wavelength 635 nm [6,7]. In these cytological studies using ALA, the researchers obtained urine samples after the instillation of ALA into the bladder and analyze the urinary cells using fluorescence microscopy. Although the sensitivity was 86% and 98% in these studies [6,7], the specificity was 75% [7]. The limitations of these reports are their subjective methodology. Autofluorescence makes it difficult to distinguish cancer cells from normal cells with cellular fluorochromes. Tauber et al. [8] reported on integral spectrophotometric analysis of ALA-induced fluorescence cytology of urinary bladder using the technique of photobleaching. They identified protoporphyrin IX fluorescence, obtained by subtracting the spectra measured after exposure of light from the first spectrum. The reported sensitivity and specificity were 96% and 50%, respectively, with the specificity being low. In addition, all reports about ALA-induced urine cytology have used voided urine samples after intravesical ALA instillation followed by in situ incubation. Thus, they were unable to compare samples obtained from the same patient, one treated with ALA with a control sample without such treatment. This may be the cause of the low specificity. Another limitation is that this method needs catheterization and cannot avoid adverse effects of ALA, even though they are not severe [9]. As a noninvasive, more precise, and more objective method, we proposed the extracorporeal exposure to ALA of voided urine and the detection of fluorescence-positive cells in urine using a spectrophotometer. This extracorporeal method enables us to compare two samples simultaneously collected from the same patient, with a test sample being treated with ALA and a control sample without treatment. The purpose of this work was to evaluate the feasibility and clinical utility of photodynamic diagnosis in bladder cancer using the spectrophotometric analysis of the ALA-induced protoporphyrin IX levels to detect bladder tumor cells exfoliated in voided urine samples.
Cell culture The T24 cell line, which originates from advanced high-grade bladder cancer, was purchased from ATCC (Manassas, VA, USA). The cell line was maintained in the RPMI-1640 culture medium (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (ICN Biomedicals, Aurora, OH, USA), 100U/mL penicillin, and 100-g/mL streptomycin (Gibco, Grand Island, NY, USA) in a standard humidified incubator at 37 ◦ C in an atmosphere containing 5% CO2 .
Patients After we received approval of the Nara Medical University Hospital Institutional Review Board (authorization no. 362), 61 patients with histologically confirmed bladder cancer after the transurethral resection of a bladder tumor were recruited between August 2012 and August 2013 as the bladder cancer group. Fifty outpatients who did not have a history of urothelial carcinoma or cancer-related findings were recruited as the control group during the same period. All patients enrolled in this prospective feasibility study after the provided written informed consent. Hematuria was defined as ≥5 red blood cells in a high-power field and pyuria was defined as ≥5 white blood cells per high-power field.
Collection of urine samples More than 150 mL of voided urine of bladder cancer patients was collected before the transurethral resection of bladder tumors. Fifty milliliter of voided urine was used for voided urinary cytology and 100 mL for ALA-induced fluorescence cytology. Voided urine samples were stored at 4 ◦ C until processing, which was generally performed within 2 h of collection (Fig. 1). Urine samples were centrifuged at 1500 rpm for 5 min. The supernatant was decanted, and the pellets were resuspended in phosphate-buffered saline, from which two samples were collected. One sample was treated with 1mM ALA (Sigma-Aldrich, Saint Louis, MO, USA), diluted with phosphate-buffered saline, and then incubated at 37 ◦ C for 2 h. The other sample was diluted with phosphatebuffered saline and incubated without ALA. The solution was centrifuged at 1500 rpm for 5 min, the supernatant was decanted, and 40 L of phosphate-buffered saline was added to resuspend the pellet. Next, one half of the suspension was pipetted into the measuring chamber of a 384-well plate (Greiner UV-Star 384-well plates; SigmaAldrich; Fig. 1).
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Y. Nakai et al. subtracted from the difference between the intensity of ALA-treated and ALA-untreated samples at 635 nm (Fig. 3a and b). After that, the adjusted difference was divided by the intensity of the ALA-untreated sample at 635 nm. This adjusted change ratio was used for the diagnosis of bladder cancer (the method of ALA-induced fluorescence cytology).
Statistical analysis All calculations were performed in SPSS for Windows (version 20.0; IBM, Armonk, NY, USA). The Mann—Whitney U test was used for the comparison of continuous variables. The chi-squared test was used for the comparison of categorical variables. Receiver operating characteristic analysis was performed and the area under the curve was assessed. The optimal cutoff value that maximizes the sum of sensitivity and specificity was determined as the point closest to the upper left-hand corner. ROCKIT [10] was used to evaluate the difference between two receiver operating characteristic curves. Differences were considered statistically significant if p values were 103 cells. Subtle peaks were observed in samples containing 5 × 102 ALA-positive cells. The adjusted change ratios of the samples that included 104 , 5 × 103 , 103 , 5 × 102 , and 102 positive cells were 6.38, 3.19, 0.5, 0.28, and 0.001, respectively.
Background of bladder cancer and control groups There were no significant differences in age, gender, and the presence of hematuria or pyuria between the bladder cancer and control groups (Table 1). Of 61 bladder cancer patients, 24 had stage Ta disease, 13 had T1, 13 had carcinoma in situ, and 10 had cancer at stage T ≥ 2. The tumor cell grading was high in 33 patients and low in 28.
Analysis of adjusted change ratios The adjusted change ratio of the bladder cancer group was significantly higher than that of the control group (p < 0.001; Fig. 4). The adjusted change ratio for high-grade tumors was significantly higher than that for low-grade tumors
Protoporphyrin IX induced by 5-aminolevulinic acid in bladder cancer cells in voided
285
Fig. 2 Quantitative detection of protoporphyrin IX using a fluorescence spectrophotometer. Fluorescence spectra of representative cases are shown. Black curves are spectra of samples treated with 5-aminolevulinic acid, whereas gray curves are spectra of a control not treated with 5-aminolevulinic acid. (a) A urothelial carcinoma case (high-grade pT1) in which the black spectra showed a peak at 635 nm. (b) A control case in which the black spectra did not show a peak and the two spectra were almost the same. ALA, aminolevulinic acid; PPIX, protoporphyrin IX.
Table 1
Characteristics of the study population.
Median age (range, years) Male:female Hematuria Pyuria Pathological stage Tis Ta T1 T≥2 Grade High Low a b
Bladder cancer (%)(n = 61)
Control (%) (n = 50)
p-Value
73 (34—91) 49:12 23 (38) 19 (31)
74 (25—92) 43:7 10 (20) 8 (16)
0.19a 0.41b 0.06b 0.08b
13 (21) 24 (39) 13 (21) 10 (16)
— — — —
33 (54) 28 (46)
— —
Mann—Whitney U test. Chi-square test.
(p = 0.004; Fig. 4a). In muscle-invasive bladder cancer, the adjusted change ratio was higher than in non-muscleinvasive bladder cancer (p = 0.007; Fig. 4b). A receiver operating characteristic curve is shown in Fig. 5; the area under the curve was 0.84. Accuracy was improved after the adjustments (p = 0.04; Fig. 5). Sensitivity of the conventional cytology and ALA-induced fluorescence cytology was 49% and 82%, respectively, whereas the specificity was 100% and 80%, respectively (Table 2).
Table 2
Comparison of results of the conventional cytology and ALA-induced fluorescence cytology The overall sensitivity values for the conventional cytology and ALA-induced fluorescence cytology were 49% and 82%, respectively. The results of the conventional cytology and ALA-induced fluorescence cytology were compared using contingency tables (Table 2). The sensitivity of ALA-induced
Comparison of positive rates between conventional cytology and 5-ALA induced fluorescent cytology. Bladder cancer
Control
No. patients (n = ) Method of cytology
Low High Ta 28 33 24 No. positive patient (%)
CIS 13
T1 14
T2≤ 10
Total 61
50
Conventional cytology 5-ALA induced fluorescent cytology
5(18) 21(75)
10(77) 10(77)
7(50) 13(93)
9(90) 10(100)
30(49) 50(82)
0(0) 10(20)
Low, low grade tumor; high, high grade tumor.
25(76) 29(88)
4(17) 17(71)
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Y. Nakai et al.
Fig. 3 The method for adjustment of the change ratio. Black curves are spectra of samples treated with 5-aminolevulinic acid, and gray curves are spectra of a control case not treated with 5-aminolevulinic acid. (a) A bladder cancer case with the difference between samples treated and not treated with 5-aminolevulinic acid; between 550 nm and 600 nm. The difference at 600 nm was subtracted from the intensity at 635 nm (↓). A peak at 635 nm was detected even after adjustment for the difference at 600 nm. (b) A control case without a peak at 635 nm and with a difference between samples treated and not treated with 5-aminolevulinic acid; from 550 nm to 640 nm. The difference was subtracted from the intensity with 5-aminolevulinic acid (↓). The black spectra show spectra adjusted for the difference in intensity between the samples treated and not treated with 5-aminolevulinic acid at 600 nm. ALA, aminolevulinic acid.
Fig. 4 (a) Comparison of the adjusted change ratios among the control, low-grade, and high-grade groups. (b) Comparison of the adjusted change ratios among the control, non-muscle-invasive tumor, and muscle-invasive tumor groups. Statistically significant differences between two factors were determined using the Mann—Whitney U test; * p < 0.05, ** p < 0.01.
Protoporphyrin IX induced by 5-aminolevulinic acid in bladder cancer cells in voided
Fig. 5 A receiver operating characteristic plot of the adjusted change ratio (black curve) and the change ratio (gray curve). The area under the curve of the adjusted change ratio and the change ratio were 0.84 and 0.74, respectively.
fluorescence cytology was higher than that of the conventional cytology in low-grade tumors and non-muscle-invasive bladder tumors.
Discussion Although several new urine-based tests for the detection of bladder cancer have been reported, voided urinary cytology remains the most established noninvasive method for detecting bladder cancer. However, this method has several drawbacks including low sensitivity, low cost effectiveness, a lack of interobserver variability, and technical instability [11,12]. To solve these problems, here we demonstrate feasibility of spectrophotometric detection using a method that involves the extracorporeal treatment of bladder cancer cells from voided urine sediments with ALA. This method seems to be noninvasive, objective, cost effective, and easy, with a sensitivity and specificity of 84% and 80%, respectively (area under the curve: 0.84). Thus, this method shows moderate accuracy, particularly with high sensitivity in lowgrade or low-stage tumors. Previous studies on ALA-induced cytology used urine obtained after ALA instillation into the bladder [4,5,8]. This approach requires catheterization and allows the adverse effects of administered ALA, even though they are not severe [9]. Our extracorporeal method is less invasive than those previous techniques. Two reports demonstrated the principle of extracorporeal fluorescence cytology. Olivo et al. reported ex vivo fluorescence cytology using a novel photosensitizer, hypericin, using a fluorescence microscope [13]. This detection method lacks objectivity. Cunderlíková et al. demonstrated another approach to fluorescence cytology: using extracorporeal treatment with hexaminolevulinate and flow cytometry. Unfortunately, the method showed low sensitivity (46%) and specificity (50%), and the study population was small [14]. Our study results show that ALA-induced fluorescence cytology is more effective for the detection of highgrade tumors and muscle-invasive tumors than low-grade and noninvasive tumors. Olivo et al. reported that after transurethral resection of higher-grade and higher-stage bladder tumors can produce and accumulate higher levels of protoporphyrin IX after ALA instillation [15]. Their findings support our results. Miyake and colleagues showed that low expression or molecular defects of ferrochelatase
287
in urothelial cancer correlate with intracellular protoporphyrin IX accumulation [16]. Hagiya and coworkers reported that expression levels of peptide transporter 1 and human ATP-binding cassette transporter 2 play key roles in ALA-induced protoporphyrin IX accumulation [17]. Ogino et al. reported the stimulation of ALA-induced protoporphyrin IX accumulation in T24 cells by a ferrochelatase inhibitor and a human ATP-binding cassette transporter 2 inhibitor [18]. Several factors seem to be involved in the mechanisms underlying the increased accumulation of protoporphyrin IX in high-grade tumors. Which factor plays the predominant role is a subject of future research. In our study, false-positive results were detected in 10 control group patients. Bacteria may have to be taken into account because they are an important cause of low specificity of such tests. Bacteria accumulate photoactive porphyrins when ALA is supplied exogenously [19,20]. Those authors [19,20] demonstrated increased levels of photoactive porphyrins using 5-aminolevurinic acid and incubation for 4 h with gram-positive and gram-negative bacteria, including Escherichia coli and Staphylococcus aureus. The amount of photoactive porphyrin accumulation varies by bacterial species and even by strains within a species; Enterococcus faecalis does not accumulate photoactive porphyrin. In the present study, four of the nine patients with bacteriuria (E. coli 7 cases, E. faecalis 1 case, and methicillin-resistant S. aureus 1 case) in our control group showed positive results. All four of these test-positive patients had an E. coli infection. In all E. coli cases both the sample with and without ALA showed a peak at approximately 635 nm. Nevertheless, in test-positive cases, the intensity at 635 nm of the sample treated with ALA is higher than that without ALA. This difference may be caused by photoactive porphyrin of bacteria and may change with bacterial species, strains, incubation time, bacterial numbers, or ALA levels. These questions should be addressed in the future. Fontinos and coworkers reported that bacteria do not produce photoactive porphyrin when hexyl ester is used [20]. Therefore, the use of this reagent may increase specificity. Possible causes other than bacterial contamination of false positive results are technical errors or underlying urinary tract malignant tumors. This detection method should be assessed in another cohort, which includes more patients with chronic kidney diseases, chronic urinary infections, lower urinary tract symptoms, or asymptomatic hematuria. A limitation of our study is a lack of follow-up of patients in the control group who received a positive diagnosis using ALA-induced fluorescence cytology. Some of those patients may be diagnosed as having urothelial carcinoma later. Another limitation is the single-center design and a small study population. This detection method should be assessed in another cohort that includes a larger study population with a variety of diseases.
Conflict of interest statement The authors have no conflicts of interest.
Acknowledgments This study was supported by the Ministry of Education, Culture, Sports, Sciences and Technology of Japan, Grantsin-Aid for Science Research (B) JSPS KAKENHI Grant Number
288 22791490 (SA) and (C) JSPS KAKENHI Grant Number 21592057 (KF and YH), and Nara Cancer Society, Grants-in-Aid for Cancer Research Grant Number H240401 (KF).
References [1] Lokeshwar VB, Habuchi T, Grossman HB, et al. Bladder tumor markers beyond cytology: International Consensus Panel on bladder tumor markers. Urology 2005;66:35—63. [2] Badalament RA, Hermansen DK, Kimmel M, et al. The sensitivity of bladder wash flow cytometry, bladder wash cytology, and voided cytology in the detection of bladder carcinoma. Cancer 1987;60:1423—7. [3] Brown FM, Urine cytology. Is It still the gold standard for screening? Urol Clin North Am 2000;27:25—37. [4] Tauber S, Schneede P, Liedl B, et al. Fluorescence cytology of the urinary bladder. Urology 2003;61:1067—71. [5] Pytel A, Schmeller N. New aspect of photodynamic diagnosis of bladder tumors: fluorescence cytology. Urology 2002;59:216—9. [6] Steinbach P, Kriegmair M, Baumgartner R, et al. Intravesical instillation of 5-aminolevulinic acid (ALA): the fluorescent metabolite is limited to urothelial cells. Urology 1994;44:676—81. [7] Datta S, Loh C, MacRobert A, et al. Quantitative studies of the kinetics of 5-aminolaevulinic acid-induced fluorescence in bladder transitional cell carcinoma. Br J Cancer 1998;78:1113—8. [8] Tauber S, Stepp H, Meier R, et al. Integral spectrophotometric analysis of 5-aminolaevulinic acid-induced fluorescence cytology of the urinary bladder. BJU Int 2006;97: 992—6. [9] Inoue K, Fukuhara H, Shimamoto T, et al. Comparison between intravesical and oral administration of 5-aminolevulinic acid in the clinical benefit of photodynamic diagnosis for nonmuscle invasive bladder cancer. Cancer 2012;118:1062—74.
Y. Nakai et al. [10] Metz CE, Herman BA, Roe CA. Statistical comparison of two ROC-curve estimates obtained from partially-paired datasets. Med Decis Making 1998;18:110—21. [11] Xylinas E, Kluth LA, Rieken M, et al. Urine markers for detection and surveillance of bladder cancer. Urol Oncol 2014;32:222—9, http://dx.doi.org/10.1016/j.urolonc.2013.06.001. [12] Goodison S, Rosser CJ, Urquidi V. Bladder cancer detection and monitoring: assessment of urine- and blood-based marker tests. Mol Diagn Ther 2013;17:71—84. [13] Olivo M, Lau W, Manivasager V, et al. Novel photodynamic diagnosis of bladder cancer: ex vivo fluorescence cytology using hypericin. Clin Int J Oncol 2003;23:1501—4. [14] Cunderlíková B, Wahlqvist R, Berner A, et al. Detection of urinary bladder cancer with flow cytometry and hexaminolevulinate in urine samples. Cytopathology 2007;18:87—95. [15] Olivo M, Lau W, Manivasager V, Hoon TP, et al. Fluorescence confocal microscopy and image analysis of bladder cancer using 5-aminolevulinic acid. Int J Oncol 2003;22:523—8. [16] Miyake M, Ishii M, Kawashima K, et al. siRNA-mediated knockdown of the heme synthesis and degradation pathways: modulation of treatment effect of 5-aminolevulinic acid-based photodynamic therapy in urothelial cancer cell lines. Photochem Photobiol 2009;85:1020—7. [17] Hagiya Y, Fukuhara H, Matsumoto K, et al. Expression levels of PEPT1 and ABCG2 play key roles in 5-aminolevulinic acid (ALA)induced tumor-specific protoporphyrin IX (PpIX) accumulation in bladder cancer. Photodiagn Photodyn Ther 2013;10:288—95. [18] Ogino T, Kobuchi H, Munetomo K. Serum-dependent export of protoporphyrin IX by ATP-binding cassette transporter G2 in T24 cells. Mol Cell Biochem 2011;358:297—307. [19] Nitzan Y, Salmon-Divon M, Shporen E, et al. ALA induced photodynamic effects on gram positive and negative bacteria. Photochem Photobiol Sci 2004;3:430—5. [20] Fotion N, Convert N, Piffaretti JC, et al. Effects on gram-negative and gram-positive bacteria mediated by 5aminolevulinic acid and 5-aminolevulinic acid derivatives. Antimicrob Agents Chemother 2008;52:1366—73.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/pdpdt
Protoporphyrin IX induced by 5-aminolevulinic acid in bladder cancer cells in voided urine can be extracorporeally quantified using a spectrophotometer Yasushi Nakai, Satoshi Anai, Sayuri Onishi, Kuwada Masaomi, Yoshihiro Tatsumi, Makito Miyake, Yoshitomo Chihara, Nobumichi Tanaka, Yoshihiko Hirao, Kiyohide Fujimoto MD, PhD ∗ Department of Urology, Nara Medical University, 840 Shijo-cho, Kashihara-shi, Nara 634-8522, Japan Received 10 August 2014; received in revised form 25 December 2014; accepted 26 December 2014 Available online 13 January 2015
KEYWORDS 5-Aminolevulinic acid; Photodynamic detection; Urinary cytology; Spectrophotometer
∗
Summary Background: We evaluated the feasibility of photodynamic diagnosis of bladder cancer by spectrophotometric analysis of voided urine samples after extracorporeal treatment with 5aminolevulinic acid (ALA). Methods: Sixty-one patients with bladder cancer, confirmed histologically after the transurethral resection of a bladder tumor, were recruited as the bladder cancer group, and 50 outpatients without history of urothelial carcinoma or cancer-related findings were recruited as the control group. Half of the voided urine sample was incubated with ALA (ALA-treated sample), and the rest was incubated without treatment (ALA-untreated sample). For detecting cellular protoporphyrin IX levels, intensity of the samples at the excitation wavelength of 405 nm was measured using a spectrophotometer. The difference between the intensity of the ALA-treated and ALA-untreated samples at 635 nm was calculated. Results: The differences in the bladder cancer group were significantly greater than those in the control group (p < 0.001). These differences were also significantly greater in patients with highgrade tumors than in those with low-grade tumors (p = 0.004), and also in patients with invasive bladder cancer than in those with noninvasive bladder cancer (p = 0.007). The area under the curve was 0.84. Sensitivity and specificity of the method were 82% and 80%, respectively.
Corresponding author. Tel.: +81 744 22 3051; fax: +81 744240345. E-mail address: [email protected] (K. Fujimoto).
http://dx.doi.org/10.1016/j.pdpdt.2014.12.010 1572-1000/© 2015 Elsevier B.V. All rights reserved.
Protoporphyrin IX induced by 5-aminolevulinic acid in bladder cancer cells in voided
283
Conclusions: We demonstrated that protoporphyrin IX levels in urinary cells treated with ALA could be quantitatively detected by spectrophotometer in patients with bladder cancer. Therefore, this cancer detection system has a potential for clinical use. © 2015 Elsevier B.V. All rights reserved.
Introduction
Materials and methods
Although some researchers have attempted to establish less-invasive new diagnostic methods, voided urinary cytology remains the most established noninvasive method for detecting bladder cancer. Voided urinary cytology has high specificity but low sensitivity and large interobserver variability [1—3]. To solve these problems, two reports about voided urinary cytology using 5-aminolevulinic acid (ALA) have been published [4,5]. ALA is a precursor in heme biosynthesis and causes fluorescent endogenous porphyrins, mainly protoporphyrin IX, to accumulate significantly more in tumor cells than in healthy cells because of factors, such as decreased ferrochelatase activity. Under excitation at 405 nm, protoporphyrin IX emits red fluorescence which has a peak at the wavelength 635 nm [6,7]. In these cytological studies using ALA, the researchers obtained urine samples after the instillation of ALA into the bladder and analyze the urinary cells using fluorescence microscopy. Although the sensitivity was 86% and 98% in these studies [6,7], the specificity was 75% [7]. The limitations of these reports are their subjective methodology. Autofluorescence makes it difficult to distinguish cancer cells from normal cells with cellular fluorochromes. Tauber et al. [8] reported on integral spectrophotometric analysis of ALA-induced fluorescence cytology of urinary bladder using the technique of photobleaching. They identified protoporphyrin IX fluorescence, obtained by subtracting the spectra measured after exposure of light from the first spectrum. The reported sensitivity and specificity were 96% and 50%, respectively, with the specificity being low. In addition, all reports about ALA-induced urine cytology have used voided urine samples after intravesical ALA instillation followed by in situ incubation. Thus, they were unable to compare samples obtained from the same patient, one treated with ALA with a control sample without such treatment. This may be the cause of the low specificity. Another limitation is that this method needs catheterization and cannot avoid adverse effects of ALA, even though they are not severe [9]. As a noninvasive, more precise, and more objective method, we proposed the extracorporeal exposure to ALA of voided urine and the detection of fluorescence-positive cells in urine using a spectrophotometer. This extracorporeal method enables us to compare two samples simultaneously collected from the same patient, with a test sample being treated with ALA and a control sample without treatment. The purpose of this work was to evaluate the feasibility and clinical utility of photodynamic diagnosis in bladder cancer using the spectrophotometric analysis of the ALA-induced protoporphyrin IX levels to detect bladder tumor cells exfoliated in voided urine samples.
Cell culture The T24 cell line, which originates from advanced high-grade bladder cancer, was purchased from ATCC (Manassas, VA, USA). The cell line was maintained in the RPMI-1640 culture medium (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (ICN Biomedicals, Aurora, OH, USA), 100U/mL penicillin, and 100-g/mL streptomycin (Gibco, Grand Island, NY, USA) in a standard humidified incubator at 37 ◦ C in an atmosphere containing 5% CO2 .
Patients After we received approval of the Nara Medical University Hospital Institutional Review Board (authorization no. 362), 61 patients with histologically confirmed bladder cancer after the transurethral resection of a bladder tumor were recruited between August 2012 and August 2013 as the bladder cancer group. Fifty outpatients who did not have a history of urothelial carcinoma or cancer-related findings were recruited as the control group during the same period. All patients enrolled in this prospective feasibility study after the provided written informed consent. Hematuria was defined as ≥5 red blood cells in a high-power field and pyuria was defined as ≥5 white blood cells per high-power field.
Collection of urine samples More than 150 mL of voided urine of bladder cancer patients was collected before the transurethral resection of bladder tumors. Fifty milliliter of voided urine was used for voided urinary cytology and 100 mL for ALA-induced fluorescence cytology. Voided urine samples were stored at 4 ◦ C until processing, which was generally performed within 2 h of collection (Fig. 1). Urine samples were centrifuged at 1500 rpm for 5 min. The supernatant was decanted, and the pellets were resuspended in phosphate-buffered saline, from which two samples were collected. One sample was treated with 1mM ALA (Sigma-Aldrich, Saint Louis, MO, USA), diluted with phosphate-buffered saline, and then incubated at 37 ◦ C for 2 h. The other sample was diluted with phosphatebuffered saline and incubated without ALA. The solution was centrifuged at 1500 rpm for 5 min, the supernatant was decanted, and 40 L of phosphate-buffered saline was added to resuspend the pellet. Next, one half of the suspension was pipetted into the measuring chamber of a 384-well plate (Greiner UV-Star 384-well plates; SigmaAldrich; Fig. 1).
284
Y. Nakai et al. subtracted from the difference between the intensity of ALA-treated and ALA-untreated samples at 635 nm (Fig. 3a and b). After that, the adjusted difference was divided by the intensity of the ALA-untreated sample at 635 nm. This adjusted change ratio was used for the diagnosis of bladder cancer (the method of ALA-induced fluorescence cytology).
Statistical analysis All calculations were performed in SPSS for Windows (version 20.0; IBM, Armonk, NY, USA). The Mann—Whitney U test was used for the comparison of continuous variables. The chi-squared test was used for the comparison of categorical variables. Receiver operating characteristic analysis was performed and the area under the curve was assessed. The optimal cutoff value that maximizes the sum of sensitivity and specificity was determined as the point closest to the upper left-hand corner. ROCKIT [10] was used to evaluate the difference between two receiver operating characteristic curves. Differences were considered statistically significant if p values were 103 cells. Subtle peaks were observed in samples containing 5 × 102 ALA-positive cells. The adjusted change ratios of the samples that included 104 , 5 × 103 , 103 , 5 × 102 , and 102 positive cells were 6.38, 3.19, 0.5, 0.28, and 0.001, respectively.
Background of bladder cancer and control groups There were no significant differences in age, gender, and the presence of hematuria or pyuria between the bladder cancer and control groups (Table 1). Of 61 bladder cancer patients, 24 had stage Ta disease, 13 had T1, 13 had carcinoma in situ, and 10 had cancer at stage T ≥ 2. The tumor cell grading was high in 33 patients and low in 28.
Analysis of adjusted change ratios The adjusted change ratio of the bladder cancer group was significantly higher than that of the control group (p < 0.001; Fig. 4). The adjusted change ratio for high-grade tumors was significantly higher than that for low-grade tumors
Protoporphyrin IX induced by 5-aminolevulinic acid in bladder cancer cells in voided
285
Fig. 2 Quantitative detection of protoporphyrin IX using a fluorescence spectrophotometer. Fluorescence spectra of representative cases are shown. Black curves are spectra of samples treated with 5-aminolevulinic acid, whereas gray curves are spectra of a control not treated with 5-aminolevulinic acid. (a) A urothelial carcinoma case (high-grade pT1) in which the black spectra showed a peak at 635 nm. (b) A control case in which the black spectra did not show a peak and the two spectra were almost the same. ALA, aminolevulinic acid; PPIX, protoporphyrin IX.
Table 1
Characteristics of the study population.
Median age (range, years) Male:female Hematuria Pyuria Pathological stage Tis Ta T1 T≥2 Grade High Low a b
Bladder cancer (%)(n = 61)
Control (%) (n = 50)
p-Value
73 (34—91) 49:12 23 (38) 19 (31)
74 (25—92) 43:7 10 (20) 8 (16)
0.19a 0.41b 0.06b 0.08b
13 (21) 24 (39) 13 (21) 10 (16)
— — — —
33 (54) 28 (46)
— —
Mann—Whitney U test. Chi-square test.
(p = 0.004; Fig. 4a). In muscle-invasive bladder cancer, the adjusted change ratio was higher than in non-muscleinvasive bladder cancer (p = 0.007; Fig. 4b). A receiver operating characteristic curve is shown in Fig. 5; the area under the curve was 0.84. Accuracy was improved after the adjustments (p = 0.04; Fig. 5). Sensitivity of the conventional cytology and ALA-induced fluorescence cytology was 49% and 82%, respectively, whereas the specificity was 100% and 80%, respectively (Table 2).
Table 2
Comparison of results of the conventional cytology and ALA-induced fluorescence cytology The overall sensitivity values for the conventional cytology and ALA-induced fluorescence cytology were 49% and 82%, respectively. The results of the conventional cytology and ALA-induced fluorescence cytology were compared using contingency tables (Table 2). The sensitivity of ALA-induced
Comparison of positive rates between conventional cytology and 5-ALA induced fluorescent cytology. Bladder cancer
Control
No. patients (n = ) Method of cytology
Low High Ta 28 33 24 No. positive patient (%)
CIS 13
T1 14
T2≤ 10
Total 61
50
Conventional cytology 5-ALA induced fluorescent cytology
5(18) 21(75)
10(77) 10(77)
7(50) 13(93)
9(90) 10(100)
30(49) 50(82)
0(0) 10(20)
Low, low grade tumor; high, high grade tumor.
25(76) 29(88)
4(17) 17(71)
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Fig. 3 The method for adjustment of the change ratio. Black curves are spectra of samples treated with 5-aminolevulinic acid, and gray curves are spectra of a control case not treated with 5-aminolevulinic acid. (a) A bladder cancer case with the difference between samples treated and not treated with 5-aminolevulinic acid; between 550 nm and 600 nm. The difference at 600 nm was subtracted from the intensity at 635 nm (↓). A peak at 635 nm was detected even after adjustment for the difference at 600 nm. (b) A control case without a peak at 635 nm and with a difference between samples treated and not treated with 5-aminolevulinic acid; from 550 nm to 640 nm. The difference was subtracted from the intensity with 5-aminolevulinic acid (↓). The black spectra show spectra adjusted for the difference in intensity between the samples treated and not treated with 5-aminolevulinic acid at 600 nm. ALA, aminolevulinic acid.
Fig. 4 (a) Comparison of the adjusted change ratios among the control, low-grade, and high-grade groups. (b) Comparison of the adjusted change ratios among the control, non-muscle-invasive tumor, and muscle-invasive tumor groups. Statistically significant differences between two factors were determined using the Mann—Whitney U test; * p < 0.05, ** p < 0.01.
Protoporphyrin IX induced by 5-aminolevulinic acid in bladder cancer cells in voided
Fig. 5 A receiver operating characteristic plot of the adjusted change ratio (black curve) and the change ratio (gray curve). The area under the curve of the adjusted change ratio and the change ratio were 0.84 and 0.74, respectively.
fluorescence cytology was higher than that of the conventional cytology in low-grade tumors and non-muscle-invasive bladder tumors.
Discussion Although several new urine-based tests for the detection of bladder cancer have been reported, voided urinary cytology remains the most established noninvasive method for detecting bladder cancer. However, this method has several drawbacks including low sensitivity, low cost effectiveness, a lack of interobserver variability, and technical instability [11,12]. To solve these problems, here we demonstrate feasibility of spectrophotometric detection using a method that involves the extracorporeal treatment of bladder cancer cells from voided urine sediments with ALA. This method seems to be noninvasive, objective, cost effective, and easy, with a sensitivity and specificity of 84% and 80%, respectively (area under the curve: 0.84). Thus, this method shows moderate accuracy, particularly with high sensitivity in lowgrade or low-stage tumors. Previous studies on ALA-induced cytology used urine obtained after ALA instillation into the bladder [4,5,8]. This approach requires catheterization and allows the adverse effects of administered ALA, even though they are not severe [9]. Our extracorporeal method is less invasive than those previous techniques. Two reports demonstrated the principle of extracorporeal fluorescence cytology. Olivo et al. reported ex vivo fluorescence cytology using a novel photosensitizer, hypericin, using a fluorescence microscope [13]. This detection method lacks objectivity. Cunderlíková et al. demonstrated another approach to fluorescence cytology: using extracorporeal treatment with hexaminolevulinate and flow cytometry. Unfortunately, the method showed low sensitivity (46%) and specificity (50%), and the study population was small [14]. Our study results show that ALA-induced fluorescence cytology is more effective for the detection of highgrade tumors and muscle-invasive tumors than low-grade and noninvasive tumors. Olivo et al. reported that after transurethral resection of higher-grade and higher-stage bladder tumors can produce and accumulate higher levels of protoporphyrin IX after ALA instillation [15]. Their findings support our results. Miyake and colleagues showed that low expression or molecular defects of ferrochelatase
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in urothelial cancer correlate with intracellular protoporphyrin IX accumulation [16]. Hagiya and coworkers reported that expression levels of peptide transporter 1 and human ATP-binding cassette transporter 2 play key roles in ALA-induced protoporphyrin IX accumulation [17]. Ogino et al. reported the stimulation of ALA-induced protoporphyrin IX accumulation in T24 cells by a ferrochelatase inhibitor and a human ATP-binding cassette transporter 2 inhibitor [18]. Several factors seem to be involved in the mechanisms underlying the increased accumulation of protoporphyrin IX in high-grade tumors. Which factor plays the predominant role is a subject of future research. In our study, false-positive results were detected in 10 control group patients. Bacteria may have to be taken into account because they are an important cause of low specificity of such tests. Bacteria accumulate photoactive porphyrins when ALA is supplied exogenously [19,20]. Those authors [19,20] demonstrated increased levels of photoactive porphyrins using 5-aminolevurinic acid and incubation for 4 h with gram-positive and gram-negative bacteria, including Escherichia coli and Staphylococcus aureus. The amount of photoactive porphyrin accumulation varies by bacterial species and even by strains within a species; Enterococcus faecalis does not accumulate photoactive porphyrin. In the present study, four of the nine patients with bacteriuria (E. coli 7 cases, E. faecalis 1 case, and methicillin-resistant S. aureus 1 case) in our control group showed positive results. All four of these test-positive patients had an E. coli infection. In all E. coli cases both the sample with and without ALA showed a peak at approximately 635 nm. Nevertheless, in test-positive cases, the intensity at 635 nm of the sample treated with ALA is higher than that without ALA. This difference may be caused by photoactive porphyrin of bacteria and may change with bacterial species, strains, incubation time, bacterial numbers, or ALA levels. These questions should be addressed in the future. Fontinos and coworkers reported that bacteria do not produce photoactive porphyrin when hexyl ester is used [20]. Therefore, the use of this reagent may increase specificity. Possible causes other than bacterial contamination of false positive results are technical errors or underlying urinary tract malignant tumors. This detection method should be assessed in another cohort, which includes more patients with chronic kidney diseases, chronic urinary infections, lower urinary tract symptoms, or asymptomatic hematuria. A limitation of our study is a lack of follow-up of patients in the control group who received a positive diagnosis using ALA-induced fluorescence cytology. Some of those patients may be diagnosed as having urothelial carcinoma later. Another limitation is the single-center design and a small study population. This detection method should be assessed in another cohort that includes a larger study population with a variety of diseases.
Conflict of interest statement The authors have no conflicts of interest.
Acknowledgments This study was supported by the Ministry of Education, Culture, Sports, Sciences and Technology of Japan, Grantsin-Aid for Science Research (B) JSPS KAKENHI Grant Number
288 22791490 (SA) and (C) JSPS KAKENHI Grant Number 21592057 (KF and YH), and Nara Cancer Society, Grants-in-Aid for Cancer Research Grant Number H240401 (KF).
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