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Keywords:

  • 5-aminolaevulinic acid;
  • protoporphyrin IX;
  • fluorescence;
  • diagnosis;
  • bladder neoplasm

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Objective

To report the results of a clinical study investigating the diagnosis of malignant and dysplastic bladder lesions by protoporphyrin IX (PPIX) fluorescence and to compare them with those from earlier studies.

Patients and methods

The study included 55 patients with suspected bladder carcinoma (at initial diagnosis or at tumour follow-up visits); 130 bladder biopsies from 49 patients were classified by pathological analysis. All patients received an intravesical instillation of 50 mL of a 3% 5-aminolaevulinic acid (ALA) solution a mean of 135 min before cystoscopy, which was then performed under white and blue light. Malignant/dysplastic lesions showing red fluorescence under blue-light excitation were noted and the increase in detection rate calculated.

Results

There were 63 benign and 67 malignant/dysplastic areas biopsied; 10 malignant/dysplastic lesions (four transitional cell carcinoma, two carcinoma in situ, four dysplasia) were not detected during routine white-light cystoscopy but were identified under blue light. Fluorescence cystoscopy improved the overall diagnosis of malignant/dysplastic bladder lesions by 18% over standard white-light cystoscopy. The improvement was greater for dysplastic lesions and carcinoma in situ (50%). However, the improvement over standard cystoscopy was less than that found by other groups.

Conclusion

The ALA-based fluorescence detection system significantly enhanced the diagnosis of malignant/dysplastic bladder lesions. However, determining the optimum drug exposure time requires further investigation using well-characterized instrumentation and study protocols, which would then allow comparison of the results from different groups.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

At least two other groups are currently using 5-aminolaevulinic acid (ALA) to enhance the diagnosis of bladder carcinoma [1,2]. The results of these clinical studies indicate a sensitivity of up to 97% for ALA-based photodetection (PD) of malignant bladder lesions [1]. The PD of malignancies is based on the fluorescence of dyes (fluorophores) that localize preferentially in neoplastic tissue.

Porphyrins and other tetrapyroles are the most exploited group of dyes for PD and photodynamic therapy (PDT). The use of haematoporphyrin (Hp) as a fluorophore and photosensitizer was described as early as 1913 [3]. Photofrin® , a purified form of haematoporphyrin derivative (HpD), is approved for PDT in several countries and currently the only approved PDT drug in the USA. In principle, Photofrin® could be used for PD, but it has the disadvantage of causing skin photosensitization, which can last for several weeks. In contrast, the intravesical instillation of ALA, first described in 1993 by Baumgartner et al. [4] shows no systemic side-effects and leads to an increase of protoporphyrin IX (PPIX) in neoplastic bladder lesions. ALA is a precursor of PPIX, which is in turn the immediate precursor of haeme in the biosynthetic pathway for haeme. All mammalian nucleated cells have the capacity to synthesize haeme, as haeme-containing enzymes are required for aerobic energy metabolism. Therefore, PPIX is an endogenous photosensitizer, which fluoresces red under excitation with blue light [5]. We report the use of ALA for diagnosing bladder cancer and compare our results with those of earlier studies.

Patients and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The study included 55 patients (11 women and 44 men, mean age 66 years, range 31–87) with suspected bladder carcinoma (at initial diagnosis or at tumour follow-up visits). An intravesical instillation of 50 mL of a 3% ALA-solution (Levulan® , DUSA Pharmaceuticals Inc., Tarrytown, NY, USA) was administered to all these patients before cystoscopy. The solution was freshly prepared immediately before instillation by mixing crystalline ALA hydrochloride (molecular weight 167.6) with a bicarbonate buffer (first 19 patients) or for better stability, with a potassium phosphate buffer (the last 36 patients). The mixture was then filter-sterilized by passage through a sterile filter (0.22 μm pore size). Sterility and pyrogenicity were tested and the safety of the final ALA solution confirmed. The interval between intravesical instillation of the drug and cystoscopic procedure was 51–710 min (mean 135); the duration of drug exposure, defined as the time between intravesical instillation and drainage of the bladder (voiding or start of cystoscopy), was 30–470 min (mean 126).

The detection system used to capture and display PPIX-fluorescence images was described previously [5]; briefly, it consists of a standard cystoscopy xenon light source with an added internal filter assembly which passes primarily blue light (200–400 mW at 425 nm with a full width at half maximum intensity of ≈50 nm). A foot pedal allows the surgeon to conveniently switch between standard white light and blue light, used for illumination or fluorescence excitation of the bladder surface, respectively. To visualize the red fluorescence light, a yellow filter in the cystoscope was used to block most of the blue excitation light. The bladder was studied primarily with the naked eye. To capture bladder images, a colour charged-coupled device (CCD) camera with integrating capability was used. Images from the CCD camera were displayed on a standard colour monitor and recorded on videotape.

Fluorescence cystoscopy was always carried out with two surgeons in attendance. Initially, using white and blue light, the bladder surface was examined thoroughly and the results documented. Unaware of the results of this initial investigation, the second surgeon (one of six senior urologists participating in the study) performed a standard cystoscopic procedure under white light, including clinically necessary mucosal biopsies and/or TURB. After this procedure, a second blue-light examination was performed to take biopsies from all remaining fluorescent areas. Finally, if possible, all fluorescent areas were resected. All biopsied and resected samples were fixed in formalin and submitted for routine pathological analysis.

The sensitivity, specificity, positive and negative predictive values (PPV, NPV) of the ALA-induced PPIX-fluorescence method were determined using standard statistical methods and compared with values obtained using standard cystoscopy.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Table 1 shows the histological classification of the 130 biopsies obtained from the 49 patients analysed; there were 63 benign areas and 67 malignant/dysplastic lesions. Table 2 shows the analysis of lesions that were missed during standard white-light cystoscopy but detected under blue light (Fig. 1). In all, 10 additional malignant/dysplastic lesions were found; thus the number of diagnosed lesions was increased by 18%. However, considering only dysplasia and carcinoma in situ (Cis), the lesions most readily missed by white-light cystoscopy, the increase was substantially greater (six blue light only/12 white light). Table 3 compares the conditions and results of the present study with those of earlier studies.

Table 1.  The correlation between histopathological diagnosis and PPIX fluorescence Thumbnail image of
Table 2.  Histopathological diagnosis of 10 lesions not detected during white-light cystoscopy but detected under blue light, together with the total number of lesions of each type Thumbnail image of
image

Figure 1. White light and corresponding blue light image of a TaG2 tumour located near the air bubble and missed during standard white light cystoscopy.

Download figure to PowerPoint

Table 3.  Comparison of three clinical studies of ALA for the detection of bladder carcinoma Thumbnail image of

Analysing the data from all 130 lesions, the sensitivity, specificity, PPV and NPV of this method for the diagnosis of malignant/dysplastic bladder lesions were 87%, 59%, 69% and 80%, respectively. False-positive results were obtained mainly from inflammatory lesions (Table 1). In the present study, white-light cystoscopy had a sensitivity of 84% and 89% for detecting malignant/dysplastic and malignant lesions, respectively. As most of the lesions that were not detected under blue light had a papillary structure or were classified as suspicious under white light, the overall sensitivity (combined white- and blue-light cystoscopy) was 99%. One mild dysplastic lesion (1%) was detected solely by random biopsies. There were no differences in sensitivity or specificity when using the two buffer solutions noted above.

None of the patients showed signs of systemic side-effects of PD (e.g. phototoxicity of the skin). One patient reported transient (<24 h) dysuria and one patient developed an urinary tract infection (nonhaemolytic streptococci), which was treated with antibiotics.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

TCC of the bladder is known to have a particularly high recurrence rate, up to 60% [6]. One of the most likely explanations for this phenomenon is that during the initial cystoscopy, early malignant and dysplastic lesions are overlooked because their appearance is similar to that of inflamed or normal tissue.

In the present study, and in two earlier studies (Table 3) the ALA-induced PPIX fluorescence detection method improved the diagnosis of bladder cancer by detecting more malignant/dysplastic lesions than did the standard white-light procedure. However, in the present study, the improvement of 18% was relatively low compared with the results of Kriegmair et al. (38%) and Jichlinski et al. (76%) [1,2]. If the improvement in the detection of only dysplasia and Cis is considered, there was a 50% increase in the present study; from the results of Kriegmair et al. [1] we calculate a 140% increase for these lesions. In the study by Jichlinski et al. [2] all the dysplastic lesions and Cis (total 43) found under blue light were defined as undetectable under white light, as their appearance was similar to unspecific inflammatory changes of the urothelium. In contrast, we considered every lesion which was not chosen randomly for biopsy as detectable, even if it was not obviously malignant (papillary, sessile, etc.). This could be one explanation for the lower increase in detection rate of malignant/dysplastic lesions found in the present study. Otherwise, the reason for the discrepancy in the results of the three clinical studies remains unclear. At least three more possibilities must be considered: (i) differences in the amount of PPIX formed, possibly arising from different ALA dosing schedules; (ii) differences in the sensitivity of white-light cystoscopy in different hands; and (iii) differences in the efficiency of generation and detection of PPIX-fluorescence.

For (i), in the study of Jichlinski et al. [2] the interval between the instillation of the drug and the start of fluorescence cystoscopy and the duration of drug exposure were substantially longer than in the present study (Table 3). Despite the considerably longer intervals, Jichlinski et al. did not report a significantly higher rate of false-positive results (43%) than in the present study (41%). Comparing these two studies might suggest that longer intervals lead to more detected neoplastic lesions per patient (Table 3) during fluorescence cystoscopy. However, recent animal studies by Heil et al. [7], using a rat tumour model and 60 min of exposure to ALA, indicate that optimum tumour:normal contrast is obtained 60 min after beginning the instillation and maximum tumour fluorescence occurs at about 150 min after beginning the instillation. While the conditions of the animal and human studies differ, the results suggest that the optimum ALA dosing conditions for humans have yet to be determined. Given the differences in results among the three studies cited, meaningful results will probably require that the different time regimens be studied by one group while keeping all other conditions constant.

For (ii), the different design of the present study (the surgeon was unaware of the results of the ALA-method) from that in earlier studies could also partly explain the relatively few additional malignant/dysplastic lesions detected under blue light. Knowing that the results will be assessed, the urologist could be biased to consider more regions suspicious than usual and perform more biopsies/resections than under routine conditions. Different levels of experience and skill could influence the number of malignant/dysplastic lesions detected during white-light cystoscopy. Although the present study found less enhancement in detection than previous reports, the participants were senior urologists in a teaching hospital, as a ‘gold standard’. Greater improvement would be expected when white-light cystoscopy is performed by less experienced urologists.

For (iii), while the calculated sensitivity and specificity of the ALA-method in this study were comparable with the results of Jichlinski et al. [2], Kriegmair et al. [1], reported higher values (Table 3). These differences should be interpreted cautiously as the true sensitivity and specificity of any new diagnostic method can be determined only by including many random biopsies from each bladder which, in clinical studies, is difficult because there are ethical problems. To obtain sufficient data, fluorescence studies on cystectomy specimens from patients who preoperatively received an ALA-solution intravesically are currently underway at our laboratory. When considering sensitivity, specificity and enhanced detection of malignant/dysplastic lesions, it should be noted that the instrumentation and measurement systems have not yet been standardized. Only minor differences in the spectral characteristics of the filter used in the cystoscope to occlude most of the blue light could result in a loss of contrast and lead to a lower sensitivity of the system for the detection of small amounts of PPIX in the target tissue. Furthermore, the use of CCD cameras with a higher sensitivity and integrating capabilities than that available here would allow the improved detection of less fluorescent malignant/dysplastic lesions and could explain discrepancies in the results of the three studies. Indeed, the camera system used in the present study cannot exceed the sensitivity of the human eye at acceptable integration times (≈0.25 s) and therefore was used only if pictures were needed for documentation or image analysis.

The mechanism of the induction of PPIX fluorescence after intravesical instillation of ALA is of interest for both the potential improvement of PD and for the application of ALA to PDT. The possibility that cellular characteristics predispose to selective PPIX accumulation was investigated by Steinbach et al. [8], who created multicellular spheroids from co-cultured RT4 cells (a cell line derived from a papillary bladder carcinoma) and N1 cells (skin fibroblast cell line). The PPIX accumulation characteristics (as ALA-induced fluorescence) of RT4 cells were examined and compared with those of a cell line (J82) derived from a poorly differentiated invasive urothelial carcinoma and a cell line (HCV29) derived from normal urothelium. The N1 skin fibroblast line and human umbilical cord endothelial cell (HUVEC) were also used as controls. An ALA concentration of 30 μg/mL and incubation time of 4 h were standard for all studies. Sub-confluent N1 monolayers exhibited fluorescence equivalent to RT4 and J82 cells, all much higher than untransformed HCV29 cells. However, in the form of multicellular spheroids, the fluorescence of RT4 cells was at least twice that of any other cell line. RT4 cells also had the greatest increase in fluorescence as a function of increasing ALA concentration, with HCV29 cells showing the least increase in fluorescence regardless of the presence or absence of serum in the medium. Fluorescence was concentrated in the perinuclear area, but did not appear specifically associated with mitochondrial bodies. After 48 h of continuous exposure to ALA, fluorescence was primarily concentrated in the cell membrane. The authors interpreted these data as suggesting that differences inherent in cell type determine their ability to accumulate PPIX, although the degree of fluorescence did not correlate with the degree of differentiation of the tumour from which the cell lines were derived.

In addition to possible differences in the level of cellular metabolism, there are structural characteristics of diseased urothelium which could lead to increased ALA transport and account for the increased PPIX fluorescence. Normal transitional epithelium of the human bladder (urothelium) acts as a barrier to water and sodium [9]. That ALA exposure times of up to 7.8 h in the present study did not lead to any loss of tumour:normal fluorescence contrast, as often is observed in animal models [10], and is consistent with normal urothelium acting as a diffusion barrier for ALA. Scanning electron microscopy studies have shown two distinct components of the urothelial permeability barrier; tight junctions (zonulae occludentes), which are apically located areas of membrane union at the lateral surfaces of superficial urothelial cells (umbrella cells) and which provide an intercellular barrier to diffusion; and unique plaques of thickened asymmetrical unit membrane (AUM) that are embedded in and surrounded by interplaque membrane. In TCC of the bladder in humans and in similar tumours in rats, the ultrastructure of the luminal membrane is altered [11]. While in normal urothelium tight junctions consist of a network of three to five intramembrane strands, tight junctions in tumours are focally attenuated to a single strand. In higher-grade tumours, intramembrane strands are often discontinuous. Fellows et al. [9] have shown that the presence of well differentiated lesions did not alter the permeability, whereas urinary infection and undifferentiated tumours show an increased permeability of the urothelium. The results of the present study seem to be consistent with these findings, and we hypothesize that increasing urothelial permeability for ALA occurs with inflammation and increasing degrees of dedifferentiation. While 43% of mild dysplastic lesions showed no detectable PPIX-fluorescence, 43% of inflammatory lesions and >90% of the more dedifferentiated lesions (moderate and severe dysplasia, Cis, G2 and G3 tumours) showed detectable PPIX-fluorescence. In further support of this hypothesis, Kriegmair et al. [12] found, among the group of fluorescent lesions, a higher intensity of PPIX-fluorescence for malignant/dysplastic than for inflammatory lesions. However, 88% of well differentiated G1 tumours and 67% of mild dysplastic lesions in the present study showed PPIX fluorescence, which either contradicts the results of Fellows et al. [9] or indicates that in addition to diffusion that there is an active mechanism for ALA transport into the cell, suggesting that there are indeed differences in the ALA/PPIX-metabolism between normal and dedifferentiated cells. The existence of an active transport mechanism is supported by animal studies, which have shown that substances with biochemical properties similar to ALA (molecular weight <200, water soluble and yet sufficiently lipophilic for binding with plasma membranes) can be absorbed by the urothelium [13[14]–15]. After instillation of amino acids and glucose, a concentration gradient from the mucosa to serosal surfaces has been detected [13,14]. However, these were animal studies and to our knowledge there is still no evidence for an active ALA-transport into human urothelium in vivo.

Finally, the increased PPIX fluorescence in malignant/dysplastic lesions could partly arise because these tissue changes are often characterized by hyperproliferation of the urothelium. More fluorescent urothelial layers in malignant/dysplastic areas than in normal epithelium may cause higher fluorescence intensity at the bladder surface.

In summary, the mechanism for increased PPIX fluorescence in malignant/dysplastic and inflammatory bladder lesions after the intravesical instillation of ALA is not fully understood and should be studied further.

During fluorescence cystoscopy, the intensity of red fluorescence decreased under prolonged illumination (5–10 min) with blue light. This phenomenon is probably photobleaching, which has been observed in cells using blue light and in mouse skin using red light [16,17]. Further clinical studies are needed to determine the dose for 50% photobleaching of PPIX in bladder tissue using blue light and to establish guidelines for blue-light cystoscopy.

To understand the differences between the results of earlier studies, future work should quantify as many of the technical variables in fluorescence excitation and detection as possible. The output of blue-light can vary with time, suggesting periodic measurements of lamp output using a power meter. The use of a fluorescent phantom of PPIX in solution to check what concentration of fluorophore can be detected by eye or by camera at a specified distance from the end of the cystoscope would also allow comparison of the sensitivities of different systems.

The results of the present and earlier studies [1,2] suggest caution in the use of ALA for PDT of bladder carcinoma. Despite a recent initial clinical study providing encouraging results, animal experiments in rats have shown that tumour necrosis was never complete in muscle-invasive tumours after PDT [18,19]. In addition, the sensitivity of PPIX fluorescence cystoscopy indicates that 3–13% of malignant and dysplastic lesions show no detectable PPIX fluorescence after intravesical instillation of a 3% ALA solution. The fluorescence within malignant/dysplastic lesions is not homogenous but shows areas of little or no fluorescence. Furthermore, the proportion of normal-appearing malignant/dysplastic lesions that show no PPIX fluorescence may be even higher than reported here, as only a few biopsies was taken from non-fluorescent (34) and random (12) areas of the bladder. However, the 3% ALA solution used for the diagnosis of bladder carcinoma is not representative of PDT dosing, where higher doses are usually applied; in the initial clinical study [18], 17% ALA solution was used. Moreover, the rat animal model may not be representative of the situation in humans. These considerations suggest that trials of intravesical ALA for PDT of bladder cancer be preceded or accompanied by biodistribution studies of PPIX in resected bladder biopsies. Comparison of oral and intravesical ALA administration may also be warranted. Finally, the evaluation of molecular biomarkers associated with bladder cancer, such as nuclear matrix proteins [20], microsatellite DNA markers [21] or proliferation markers (KI-67 [22], proliferating cell nuclear antigen [23]) may detect early neoplastic lesions among the group of ‘false-positive’ PPIX-fluorescent lesions. Indeed, a recent study has shown that 58% of ‘false-positive’ PPIX-fluorescent hyperplastic urothelium showed genetic changes similar to those of papillary carcinomas found in the same patient [24]. While the hyperplastic urothelium was classified as benign by the pathologist, these lesions are characterized by genetic abnormalities, which may indicate malignant proliferation in the future. Therefore, there may be fewer ‘false-positives’ than defined by standard pathological classification.

The only two reported adverse experiences in this study were one case of transient dysuria and one case of postoperative urinary tract infection. While dysuria is a common symptom after any transurethral manipulation, the second patient had a history of UTI, which required treatment with antibiotics preoperatively.

We conclude that the ALA-based fluorescence detection system is a safe and simple procedure which enhances the detection of malignant/dysplastic bladder lesions. The importance of the duration of drug exposure for optimum fluorescence detection requires further investigation using well-characterized instrumentation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

S.W. McDougal, S.P. Dretler, N. Heney, A.F. Althausen, H. Young and F.J. McGovern are the six senior urologists and J. Grocella, S. Batter, D. Stampfer, C. Kelly, D. Rudnick and B. Spencer are the clinical residents who actively participated in the study. We also thank Storz for loaning the light source and camera system, and P. Jichlinski, R. Baumgartner and S.L. Marcus for helpful discussions. Supported by grants from the Department of Energy (DOE #DE-FG02–91-ER61228), the Deutsche Forschungsgemeinschaft (DFG #Ko1664/1–1) and DUSA Pharmaceuticals.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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