Photodynamic diagnosis of shed prostate cancer cells in voided urine treated with 5-aminolevulinic acid
© Nakai et al.; licensee BioMed Central Ltd. 2014
Received: 20 February 2014
Accepted: 22 July 2014
Published: 3 August 2014
Past attempts at detecting prostate cancer (PCa) cells in voided urine by traditional cytology have been impeded by undesirably low sensitivities but high specificities. To improve the sensitivities, we evaluate the feasibility and clinical utility of photodynamic diagnosis (PDD) of prostate cancer by using 5-aminolevulinic acid (5-ALA) to examine shed prostate cancer cells in voided urine samples.
One hundred thirty-eight patients with an abnormal digital rectal exam (DRE) and/or abnormal prostate-specific antigen (PSA) levels were recruited between April 2009 and December 2010. Voided urine specimens were collected before prostate biopsy. Urine specimens were treated with 5-ALA and imaged by fluorescence microscopy and reported as protoporphyrin IX (PPIX) positive (presence of cells demonstrating simultaneous PPIX fluorescence) or PPIX negative (lack of cells demonstrating fluorescence).
Of the 138 patients, PCa was detected on needle biopsy in 81 patients (58.7%); of these 81 patients with PCa, 60 were PPIX-positive (sensitivity: 74.1%). Although 57 patients did not harbor PCa by conventional diagnostic procedures, 17 of these at-risk patients were found to be PPIX-positive (specificity: 70.2%). PPIX–PDD was more sensitive compared with DRE and transrectal ultrasound and more specific compared with PSA and PSA density. The incidence of PPIX–PDD positivity did not increase with increasing total PSA levels, tumor stage or Gleason score.
To our knowledge, this is the first successful demonstration of PPIX in urine sediments treated with 5-ALA used to detect PCa in a noninvasive yet highly sensitive manner. However, further studies are warranted to determine the role of PPIX–PPD for PCa detection.
Keywords5-aminolevulinic acid Prostate cancer Photodynamic diagnosis Urine cytology
Detection of prostate cancer (PCa) primarily relies on an abnormal digital rectal examination (DRE) and/or increased prostate specific antigen (PSA) levels. Despite its widespread use, PSA has marginal specificity, and 65%–70% males with elevated PSA levels within 4–10 ng/ml generally reveal a negative biopsy result . Therefore, PSA is not the ideal screening tool for prostate cancer. To improve PSA specificity, various analyses have been introduced such as PSA density, PSA velocity, and free/total PSA; however, these methods have not appreciably improved the cancer detection rates. In addition, because of sampling errors or sampling inefficiencies associated with transrectal ultrasound-guided biopsy of the prostate, some PCa may be missed (false negative). Therefore, there is a clear need for clinically useful biomarkers that would allow earlier detection of clinically significant PCa. For this reason, there is currently considerable interest in novel noninvasive biomarkers (e.g., urine- or serum-based assays) to assist in the diagnosis of PCa . Nevertheless, to date, no routinely used tumor biomarker has replaced PSA.
Upon prostatic massage, prostate cells (including PCa cells) may be dislodged and displaced into the urethra and urine. However, unlike bladder cancer, wherein voided urinary cytology has proven useful in cancer detection and surveillance, past attempts at detecting PCa cells in voided urine by traditional cytology have been impeded by undesirably low sensitivities but high specificities [3–5]. The low sensitivity was presumably attributed to the scant number of prostate cells present in the voided urine and the difficulty in differentiating the malignant prostatic cells from the other shed cells and debris. Thus, based on recent technological advancements, we revisited the cytology-based approach to PCa detection in voided urine samples and investigated the use of photodynamic diagnosis (PDD). For example, in bladder cancer, several investigators have used photodynamic agents such as 5-aminolevulinic acid (5-ALA) to induce protoporphyrin IX (PPIX) accumulation in malignant tissue, which then enables it to be differentiated from benign tissue [6–9]. In the heme biosynthetic pathway, 5-ALA is a precursor in the heme biosynthesis pathway and is metabolized to fluorescent PPIX before being converted to photoinactive heme. PPIX, which temporarily accumulates in the cancer cells after exogenous application of 5-ALA, is the endogenous photosensitizer needed for PDD . Selective PPIX accumulation in malignant tissue provides an intense color contrast between the red fluorescence of malignant lesions and the nonfluorescence of normal tissue.
Previously, Zaak et al. reported the results of a small feasibility study (n = 18), in which prostate tissue samples were analyzed after exposure to 5-ALA for the presence of prostate tumor(s). Briefly, prostate tissue samples from 16 patients who had undergone 5-ALA fluorescence microscopy revealed selective PPIX accumulation in the PCa cells, and only weak PPIX fluorescence could be detected in the benign epithelial cells. The two patients who were not treated with 5-ALA revealed no PPIX fluorescence within the prostate . In this study, our aim was different from those of previous investigators. We evaluated the feasibility and clinical utility of PDD for PCa by using 5-ALA-induced PPIX to examine shed PCa cells in voided urine samples. The use of a fluorescence biomarker for the diagnosis of cancers is an intriguing and still fledgling concept for improving the detection accuracy of PCa.
Characteristic of the study population
Males with positive biopsy (n = 81)
Males with negative biopsy (n = 57)
Total (n = 138)
Free PSA (ng/ml)
Sample preparations and 5-ALA treatment
Photodynamic detection of PPIX in shed prostate cells
5-ALA-treated (and 5-ALA-untreated) urine cytospin slides were investigated for PPIX fluorescence at 100× magnification by fluorescent microscopy with the appropriate fluorescence filter sets (excitation filter, 380–420 nm; emission filter, 590 nm; Nikon Eclipse 400; Nikon Instruments Inc., Melville, NY, USA). The slides were evaluated for the following: (i) PPIX positivity, presence of cells demonstrating simultaneous fluorescence of PPIX and (ii) PPIX negativity, lack of cells demonstrating fluorescence.
The Mann–Whitney test was used to analyze the differences between the patients with or without PCa. We used the Chi-square test for trend to analyze whether the incidence of PPIX–PDD positivity was associated with cancer, PSA, Gleason score, and tumor stage. Multivariate logistic regression analysis was used to evaluate the demographic features, key clinical features [e.g., DRE, total PSA,%fPSA, PSA density (PSAD), and transrectal ultrasonography (TRUS)], and the PPIX–PDD results in order to determine their ability to independently predict PCa on prostate biopsy. Differences were considered statistically significant at p values <0.05.
In this study, 81/138 (58.7%) patients revealed PCa on needle biopsy (Table 1). The median age of the patients without PCa was 67 (range 55–81) years, and the median age of patients with PCa was 72 (range 51–86) years (p = 0.002). The median PSA of the patients without PCa was 7.4 (range 1.4-24.1) ng/ml, and the median PSA of the patients with PCa was 9.7 (range 3.8-690) ng/ml (p = 0.002). Furthermore, the median free PSA (1.5 vs. 1.2 ng/ml, p = 0.008) and PSAD (0.39 vs. 0.20 ng/ml/cm3, p < 0.001) values differed significantly between the patients with and without PCa, respectively. In addition, prostate volume (34.0 vs. 28.7 ml, p = 0.002) and transition zone volume (16.9 vs. 13.3 ml, p = 0.04) were significantly higher in the patients with PCa than in those without PCa.
Sensitivity and specificity of ALA, DRE, PSA, %sPSA, PSAD and TRUS
PSAD (cut-off 0.15)
%fPSA (cut-off 20%)
PSA (cut-off 4ng/ml)
Diagnostic accuracy of PDD according to total PSA, grade (Gleason) and stage
Males with positive biopsy (n=81)
Males with positive PDD (n=60)
Univariate and multivariate analysis of factors to predict PCa on prostate biopsy
% fPSA (>20%/<20%)
PSA (>20/>10, <20/<10)
In this study, we demonstrated the feasibility of PPIX–PDD and 5- ALA in noninvasive detection of PCa cells extracorporeally in voided urine sediments. The use of voided urine samples in an attempt to diagnosis PCa is gaining widespread attention. For example, investigators are using RT-PCR to identify the prostate cancer antigen 3 (PCA-3)  and glutathione S-transferase P112 gene  in voided urine samples. Particularly, the sensitivity and specificity of PCA-3 have been reported to be 54% and 74%, respectively, for the entire cohort and 53% and 71%, respectively, for PSA within 4–10 ng/ml. In the present study, the sensitivity and specificity of PPIX–PDD were 74.1% and 70.2% in the entire cohort and 70.0% and 67.6% in the gray zone cases, which were superior to those reported for PCA-3. Interestingly, the positivity rate of PPIX–PDD did not increase with increasing total PSA or tumor stage. However, the positivity rate of PPIX–PDD did increase with increasing Gleason score though a non-significant trend (p = 0.11) was noted. These results showed a possibility that PCa detection with PPIX–PDD identified more clinically significant PCa compared with PCa detected by other reported methods. Stummer et al. reported that the fluorescence intensity of glioma by PPIX–PDD was associated with cellular density, tumor proliferation, and tumor angiogenesis ; these results supported our results indicating that PPIX–PDD detected more aggressive clinically relevant cancers.
Although no reports regarding fluorescent cytology of shed PCa cells were found in the literature, several reports from the bladder cancer literature are available [15–17]; in these reports, voided urine after intravesical 5-ALA instillation was evaluated. Inoue et al. reported that regardless of the 5-ALA administration route (intravesical or oral), exposure to 5-ALA was well tolerated with only cystitis symptoms being reported . Hence, in this extracorporeal model, shed prostate cells rather than patients were treated with 5-ALA.
Although the findings in the present study were quite compelling, the study had some limitations. First, we obtained limited pathological data on the patients with positive PPIX–PDD results and positive prostate biopsy results who proceeded to undergo radical prostatectomy. Second, with limited follow-up (mean follow-up, 27 months), we must cautiously interpret the false-positive PPIX–PDD results. It is possible that over time, these prostate biopsies will become positive. In this cohort, only 4 false positive patients had been performed repeated prostate needle biopsies after this study. 2 patients of them were diagnosed as prostate cancer. These false positive patients may need strict PSA follow-up or a saturation biopsy. Third, we didn’t perform RT-PCR for every patient. We demonstrated RT-PCR in the first twenty patients. Then we evaluated the expression of PSA. We could confirm the existence of cells from prostate by that. In addition, these false-positive results may be attributed to autofluorescence. Tauber et al. reported that some biological materials had autofluorescence. To address this issue, the authors used bleaching fluorescence; however, the specificity of bleaching fluorescence was comparatively low . In our study, we were able to subjectively estimate autofluorescence by examining the slide not treated with 5-ALA from each patient. Furthermore, cytological examinations are known to present interobserver variability. In the future, we hope to develop a more objective spectrophotometric method to quantify the intensity of PPIX fluorescence.
To our knowledge, this is the first successful demonstration of PPIX in urine sediments treated with 5-ALA for detection of PCa in a noninvasive yet highly sensitive manner. The incidence of PPIX–PDD positivity increased with increasing Gleason score though a non-siginificant trend (p = 0.11) was noted. Further studies are warranted to determine the role of PPIX–PPD for PCa detection.
Digital rectal exam
Glyceraldehyde 3-phosphate dehydrogenase
Prostate cancer antigen 3
Reverse transcription polymerase chain reaction.
This work was supported by Department of Urology in Nara Medical University.
- Draisma G, Etzioni R, Tsodikov A, Marriot A, Wever E, Gulati R, Feuer E, De Koning H: Lead time and overdiagnosis in prostate-specific antigen screening: importance of methods and context. J Nati Cancer Inst. 2009, 101: 374-383. 10.1093/jnci/djp001.View ArticleGoogle Scholar
- Tanaka N, Fujimoto K, Chihara Y, Torimoto M, Hirao Y, Konishi N, Saito I: Prostatic volume and volume-adjusted prostate-specific antigen as predictive parameters for prostate cancer patients with intermediate PSA levels. Prostate Cancer Prostatic Dis. 2007, 10: 274-278.View ArticlePubMedGoogle Scholar
- Sharifi R, Shaw M, Ray V, Rhee H, Nagubadi S, Guinan P: Evaluation of cytologic techniques for diagnosis of prostate cancer. Urology. 1983, 4: 417-420.View ArticleGoogle Scholar
- Bologna M, Vicentini C, Festuccia C, Muzi P, Napolitano T, Biordi L, Miano L: Early diagnosis of prostatic carcinoma based on in vitro culture of viable tumor cells harvested by prostatic massage. Eur Urol. 1988, 14: 474-476.PubMedGoogle Scholar
- Garret M, Jassie M: Cytologic examination of post prostatic massage specimens as an aid in diagnosis of carcinoma of the prostate. Acta Cytol. 1976, 20: 126-131.PubMedGoogle Scholar
- Kriegmair M, Baumgartner R, Knuchel R, Idvall I, Ingvar C, Rydell R, Jocham D, Diddens H, Bown S, Gregory G, Montán S, Andersson-Engels S, Svanberg S: Detection of early bladder cancer by 5-aminolevulinic acid induced porphyrin fluorescence. J Urol. 1996, 155: 105-109.View ArticlePubMedGoogle Scholar
- Filbeck T, Roessler W, Knuechel R, Straub M, Kiel HJ, Wieland WF: Clinical results of the transurethral resection and evaluation of superficial bladder carcinomas by means of fluorescence diagnosis after intravesical instillation of 5-aminolevulinic acid. J Endourol. 1999, 13: 117-121. 10.1089/end.1999.13.117.View ArticlePubMedGoogle Scholar
- König F, McGovern FJ, Larne R, Enquist H, Schomacker KT, Deutsch TF: Diagnosis of bladder carcinoma using protoporphyrin IX fluorescence induced 5-aminolaevulinic acid. BJU Int. 1999, 83: 129-135.View ArticleGoogle Scholar
- Zaak D, Hungerhuber E, Schneede P, Stepp H, Frimberqer D, Corvin S, Schmeller N, Krieqmair M, Hotstetter A, Knuechel R: Role of 5-aminolevulinic acid in the detection of urothelial premalignant lesions. Cancer. 2002, 95: 1234-1238. 10.1002/cncr.10821.View ArticlePubMedGoogle Scholar
- Steinbach P, Weingandt H, Baumgartner R, Kriegmair M, Hofstädter F, Knüchel R: Cellular fluorescence of the endogenous photosensitizer protoporphyrin IX following exposure to 5-aminolevulinic acid. Photochem Photobiol. 1995, 62: 887-895.View ArticlePubMedGoogle Scholar
- Zaak D, Sroka R, Khoder W, Adam C, Tritschler S, Karl A, Reich O, Knuechel R, Baumqartner R, Tilki D, Popken G, Hofstetter A, Stief CG: Photodynamic diagnosis of prostate cancer using 5-aminolevulinic acid—first clinical experiences. Urology. 2008, 72: 345-348. 10.1016/j.urology.2007.12.086.View ArticlePubMedGoogle Scholar
- Deras IL, Aubin SM, Blase A, Day JR, Koo S, Partin AW, Ellis WJ, Marks LS, Fradet Y, Rittenhouse H, Groskopf J: PCA3: a molecular urine assay for predicting prostate biopsy outcome. J Urol. 2008, 179: 1587-1592. 10.1016/j.juro.2007.11.038.View ArticlePubMedGoogle Scholar
- Vener T, Derecho C, Jonathan B, Wang H, Rajpurohit Y, Skelton J, Mehrotra J, Varde S, Chowdary D, Stallings W, Leibovich B, Robin H, Pelzer A, Schäfer G, Auprich M, Mannweiler S, Amersdorfer P, Mazumder A: Development of a multiplexed urine assay for prostate cancer diagnosis. Clin Chem. 2008, 54: 874-882. 10.1373/clinchem.2007.094912.View ArticlePubMedGoogle Scholar
- Stummer W, Reulen HJ, Novotny A, Stepp H, Tonn JC: Fluorescence-guided resections of malignant gliomas–an overview. Acta Neurochir Suppl. 2003, 88: 9-12.PubMedGoogle Scholar
- Tauber S, Schneede P, Bernhard L, Liesmann F, Zaak D, Hofstetter A: Fluorescence cytology of the urinary bladder. Urology. 2003, 61: 1067-1071. 10.1016/S0090-4295(02)02554-2.View ArticlePubMedGoogle Scholar
- Pytel A, Schmeller N: New aspect of photodynamic of bladder tumors: fluorescence cytology. Urology. 2002, 59: 216-219. 10.1016/S0090-4295(01)01528-X.View ArticlePubMedGoogle Scholar
- Tauber S, Stepp H, Meier R, Bone A, Hofstetter A, Stief C: Integral spectrophotometric analysis of 5-aminolaevulinic acid-induced fluorescence cytology of urinary bladder. BJU Int. 2006, 97: 992-996. 10.1111/j.1464-410X.2006.06094.x.View ArticlePubMedGoogle Scholar
- Inoue K, Fukuhara H, Shimamoto T, Kamada M, Liyama T, Miyamura M, Kurabayashi A, Furihata M, Tanimura M, Watanabe H, Shuin T: 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: 1062-1074. 10.1002/cncr.26378.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2490/14/59/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.