Ruth Knüchel

Detection of Early Bladder Cancer by 5-Aminolevulinic Acid Induced Porphyrin Fluorescence

PURPOSE We determined whether the sensitivity of detecting dysplasia or early bladder cancer can be improved by 5-aminolevulinic acid induced porphyrin fluorescence. MATERIALS AND METHODS A 3% 5-aminolevulinic acid solution was instilled intravesically before cystoscopy in 104 patients. The 5-aminolevulinic acid induced porphyrin fluorescence was excited by violet light from a krypton ion laser (wavelength 406.7 nm.). RESULTS The sensitivity of the fluorescence cystoscopy (96.9%) was significantly (p < 0.0001) greater than that of white light cystoscopy (72.7%). There was no impact on specificity. CONCLUSIONS Due to the high sensitivity of the procedure fluorescence guided biopsies are recommended instead of random biopsies.

Endoscopic detection of transitional cell carcinoma with 5-aminolevulinic acid: results of 1012 fluorescence endoscopies

OBJECTIVES The initial encouraging results using 5-aminolevulinic acid (5-ALA) induced fluorescence endoscopy (AFE) have promised a procedure with an outstanding sensitivity for the detection of early stage bladder cancer. Summarized here is our clinical experience and data comprising 1012 fluorescence endoscopies. METHODS Two hours, 30 minutes before endoscopy, 1.5 g 5-ALA dissolved in 50 mL of 5.7% sodium monohydrogen phosphate was instilled in patients intravesically. Before AFE, all patients underwent white light endoscopy, and a bladder washing cytologic specimen was obtained. A special light source provided blue light (375 to 440 nm) for fluorescence excitation. Suspicious sites were identified by their red fluorescence contrasting against backscattered blue light when observed through the long pass filter (445 nm) integrated into the telescope eyepiece. RESULTS Two thousand four hundred seventy-five specimens were obtained (2.4 biopsies per AFE). In 552 AFEs (54.5%), neoplastic urothelial lesions were detected, in 34.2% only because of their positive fluorescence; 38.7% of these additionally detected neoplastic foci had poorly differentiated histologic features. CONCLUSIONS AFE has proved to be a clinically feasible procedure with an outstanding detection rate for flat, urothelial, high-risk lesions.

CELLULAR FLUORESCENCE OF THE ENDOGENOUS PHOTOSENSITIZER PROTOPORPHYRIN IX FOLLOWING EXPOSURE TO 5-AMINOLEVULINIC ACID

Abstract— Supplying 5‐aminolevulinic acid (ALA), a precursor in the biosynthetic pathway to heme from an external source leads to an accumulation of the endogenous fluorescent photosensitizer protoporphyrin IX (PPIX). Following instillation of ALA in the urinary bladder neoplastic tissue can be discerned by fluorescence cystoscopy or treated by illumination with light of an appropriate wavelength. In order to provide a biological rationale for the clinical findings, we have analyzed the capacity of three different cell lines to accumulate PPIX by flow cytometry. Three different urothelial cell lines, normal fibroblasts and endothelial cells were exposed to ALA under varying conditions. Urothelial cell lines J82 and RT4, derived from malignancies of the bladder displayed fluorescence intensities 9‐ and 16‐fold, respectively, above the fluorescence level of the normal urothelial cell line HCV29. Human umbilical cord endothelial cells fluoresced moderately while the fibroblast cell line Nl exhibited a fluorescence level comparable to those of the cancer cells. Fluoresence increased with increasing cell density and was also dependent on the growth of cells as monolayers or multicellular spheroids. Increasing ALA concentrations led to saturation of fluorescence after 4 h of incubation at cell type‐specific fluorescence levels obtained at different ALA concentrations. Continuous incubation in medium containing serum resulted in a linear rise of fluorescence during the first 4 h, which was followed by a saturation period (8–24 h) and a renewed rise. In the case of serum depletion, fluorescence intensities were significantly higher and increased linearly during the entire 48 h incubation period. By replacing serum with albumin, it could be shown that the emission of PPIX into the medium in the presence of serum is mainly caused by this protein. The ALA‐induced fluorescence was predominantly perinuclear after 4 h of incubation and relocated toward the cell membrane after prolonged incubation. This study demonstrated the complexity of factors influencing the ALA‐induced fluorescence and should stimulate further research in this field.

Fluorescence endoscopy for the detection of low and high grade dysplasia in ulcerative colitis using systemic or local 5-aminolaevulinic acid sensitisation

Background and aims: Longstanding ulcerative colitis (UC), especially in the presence of epithelial dysplasia, is associated with an increased risk of developing cancer. As dysplasia is not visible during routine endoscopy, at least 40–50 random biopsies in four quadrants every 10 cm are recommended. Fluorescence endoscopy after sensitisation with 5-aminolaevulinic acid (5-ALA) was assessed for the detection of dysplasia in ulcerative colitis by taking optical guided biopsies. 5-ALA is converted intracellularly into the sensitiser protoporphyrin IX which accumulates selectively in neoplastic tissue allowing the detection of dysplasia by typical red fluorescence after illumination with blue light. Methods: In 37 patients with UC, 54 examinations were performed with fluorescence endoscopy after oral (20 mg/kg) or local (either with an enema or by spraying the mucosa with a catheter) sensitisation with 5-ALA. A total of 481 biopsies of red fluorescent (n=218) and non-fluorescent (n=263) areas of the colonic mucosa were taken. Results: Forty two biopsies in 12 patients revealed either low grade (n=40) or high grade (n=2) dysplasia. Sensitivity of fluorescence for dysplastic lesions was excellent and ranged from 87% (95% confidence interval (CI) 0.73–1.00) to 100% (95% CI 1.00–1.00) after local sensitisation, in contrast with only 43% (95% CI 0.17–0.69) after systemic administration. Specificity did not differ for both forms of local sensitisation (enema 51% (95% CI 0.44–0.57) and spray catheter 62% (95% CI 0.51–0.73)); after systemic sensitisation specificity was 73% (95% CI 0.69–0.83). Negative predictive values of non-fluorescent mucosa for exclusion of dysplasia were very high; 89% after systemic sensitisation and 98–100% after local sensitisation. Positive predictive values were 13% and 14% after local sensitisation with enema and spray catheter, and 21% after oral sensitisation with 20 mg/kg ALA. The overall number of biopsies per examination was less than five from fluorescent positive areas. Conclusion: Fluorescence endoscopy after 5-ALA sensitisation is a possible tool to visualise dysplastic lesions in ulcerative colitis using 5-ALA sensitisation. Local sensitisation is a promising alternative approach compared with systemic administration of 5-ALA. A randomised controlled study is now indicated to compare the efficacy of endoscopic fluorescence detection with the standard technique of four quadrant random biopsies.

Endoscopic fluorescence detection of dysplasia in patients with Barrett's esophagus, ulcerative colitis, or adenomatous polyps after 5-aminolevulinic acid–induced protoporphyrin IX sensitization

BACKGROUND Surveillance of patients with Barretts esophagus or ulcerative colitis for dysplasia is confined to biopsy specimens taken randomly during endoscopy because dysplasia remains undetectable by visual inspection. We attempted to visualize dysplastic tissue during endoscopy after sensitization with 5-aminolevulinic acid (5-ALA) leading to accumulation and formation of protoporphyrin IX and induction of characteristic red fluorescence of the latter substance using blue light illumination. METHODS Six patients with histologically proven low- or high-grade dysplasia (Barretts esophagus 2, ulcerative colitis 1, Billroth-II stomach 1, rectal polyps 2) were treated with oral administration of different concentrations of 5-ALA (10 to 20 mg/kg) or by local instillation of 3 gm 5-ALA in the rectum. Endoscopic fluorescence detection was performed 1 to 6 hours after sensitization using a blue light source and compared with conventional white light endoscopy. Biopsies of fluorescent and nonfluorescent areas were compared with histologic findings. RESULTS Normal duodenal mucosa and squamous epithelium showed more intense 5-ALA-induced background red fluorescence compared with normal mucosa in the stomach or Barretts mucosa. Histologically, dysplasia was exclusively found in areas with red fluorescence. False-positive fluorescence was associated with microscopic inflammation of the mucosa or feces in the colon. CONCLUSIONS 5-ALA-induced protoporphyrin IX fluorescence may be useful in the detection of dysplasia in the gastrointestinal tract by enhancement of endoscopic surveillance of patients at a high risk for dysplasia.

Photodynamic Therapy by Means of 5-ALA Induced PPIX in Human Prostate Cancer – Preliminary Results

Summary Radical retropubic prostatectomy (RRP), external beam radiation and brachytherapy are considered to be the main treatment modalities in localized prostate cancer (PCA). However, with respect to the incidence of complications, efforts have been made to establish alternative treatment modalities of PCA, such as photodynamic therapy (PDT). In a clinical study, the localisation of 5-ALA induced PPIX in PCA was investigated. Patients (n = 14) with histologically proven prostate cancer underwent RRP. Prior to anaesthesia they received a solution of 5-ALA of 20mg/kg body weight (by oral application). Frozen sections of the removed tissue were examined by fluorescence microscopy. Only in carcinoma cells fluorescence was observed, while the epithelial cells and the stromal tissue showed no fluorescence. In control-prostates the tissue was completely fluorescence-negative. Following this experience 5 patients received interstitial PDT, transurethrally (n = 3) or transperineally (n = 2). Light of a diode laser (λ = 633 nm) was coupled into a fibre with a 1 cm cylindrical diffuser tip which was introduced into the tissue. An irradiation of 250 J/cm of laser light (irradiance: 0.5 W/cm, irradiation time: 500 s) was applied. 6 weeks after interstitial PDT the PSA values were reduced by 20% up to 70%. With regard to the side effects no patient complained about incontinence or dysuria after PDT. The presented study demonstrates that interstitial PDT of prostate cancer by means of 5-ALA induced PPIX is a safe and simple procedure. Further investigations concerning light delivery and dosimetry are warranted, to achieve the goal of a complete and curative treatment of prostate cancer.

Intravesical instillation of 5-aminolevulinic acid: the fluorescent metabolite 1s limited to urothelial cells*

OBJECTIVES For photodynamic therapy 5-aminolevulinic acid is an attractive compound, since it is a physiologic endogenous substance, and its application in excess results in the accumulation of the metabolite protoporphyrin IX, a very effective photosensitizer. The topical application of 5-aminolevulinic acid in the urinary bladder led to pronounced fluorescence in neoplasias when excited with violet laser light during cystoscopy. The aim of this study was the determination of the transmural distribution of protoporphyrin IX in order to estimate potential efficacy and side effects of a therapeutic application of 5-aminolevulinic acid. METHODS 5-Aminolevulinic acid was instilled prior to cystoscopy and biopsies were taken of lesions that were either fluorescing or nonfluorescing. Fluorescence distribution was analyzed by fluorescence microscopy on cryostat sections prepared from 72 biopsy specimens. In addition, multicellular spheroids grown from tumor cells and fibroblasts were exposed to 5-aminolevulinic acid and analyzed accordingly. RESULTS Biopsy preparations showed that the fluorescence of protoporphyrin IX was limited to normal and neoplastic urothelial cells. Clinical findings were supported by the in vitro data, which showed negative protoporphyrin IX fluorescence in fibroblasts. CONCLUSIONS Thus, 5-aminolevulinic acid might be superior in selective accumulation to conventional sensitizers known to localize also in endothelial cells of the tumor stroma. The data appear to hold great promise for 5-aminolevulinic acid in photodynamic diagnosis and therapy in bladder cancer, as phototoxicity will be limited to mucosal lesions. Bladder shrinkage due to photodamage of subepithelial structures even in case of high light doses is not expected.

QUANTIFICATION OF 5-AMINOLEVULINIC ACID INDUCED FLUORESCENCE IMPROVES THE SPECIFICITY OF BLADDER CANCER DETECTION

PURPOSE 5-Aminolevulinic acid induced fluorescence endoscopy has outstanding sensitivity for detecting early stage bladder cancer. Nevertheless, a third of the lesions that show specific fluorescence are histologically benign. We decreased the false-positive rate of 5-aminolevulinic acid induced fluorescence endoscopy by incorporating protoporphyrin IX fluorescence quantification into the standard cystoscopy procedure. MATERIALS AND METHODS In 25 cases (53 biopsies) of a history of or suspicion for bladder cancer 5-aminolevulinic acid induced fluorescence endoscopy and fluorescence image quantification were performed. For fluorescence image quantification images obtained with a target integrating color charge-coupled device camera were digitized and stored in a personal computer. Red-to-blue ratios were calculated from fluorescence positive lesions and results were correlated with hematoxylin and eosin histology. RESULTS Malignant fluorescence positive lesions showed significantly stronger fluorescence intensity than fluorescing lesions with benign histology. A threshold was established that decreased the false-positive rate by 30% without affecting sensitivity. CONCLUSIONS Fluorescence image quantification is a new endoscopic method for objectively selecting multicolor fluorescence bladder lesion images for biopsy. It has the potential of eliminating human error by different surgeons with variable experience in fluorescence endoscopy.

Cell proliferation assessment in oncology

A review of the current knowledge on cell cycle control and the techniques used to assess proliferation of normal and neoplastic cells was the focus of a workshop in Regensburg, Germany, held under the joint auspices of the Graduiertenkolleg: Therapieforschung Onkologie and the Committee on AgNOR Quantification. An overview of the recently discovered group of cyclins and their specific kinases, and of other proliferation-associated antigens, such as Ki67, PCNA and topoiseromase II alpha, was given. The topics continued with a reappraisal of modern imaging and flow-cytometric techniques. An update of the relation of AgNORs to cellular proliferation and differentiation was the link to presentations on clinical data, problems and strategies for standardization, as well as guidelines to establish the prognostic value of marker molecules. These lectures were supported by posters. Bringing together researchers from life sciences, technically oriented workers, pathologists, and clinicians resulted in a lively and constructive disussion, which is briefly summarized in the Concluding remarks.

Fluorescence cystoscopy following intravesical instillation of 5-aminolevulinic acid: a new procedure with high sensitivity for detection of hardly visible urothelial neoplasias.

Methods have been sought for the in vivo marking of tiny papillary tumors of the bladder and flat urothelial lesions such as dysplasia or carcinoma in situ, which can easily be missed during conventional endoscopy under white light. A new procedure is reported for the fluorescence detection of urothelial dysplasia and early bladder cancer. The method is based on intravesical application of 5-aminolevulinic acid (ALA). ALA if applied exogenously induces accumulation of protoporphyrin IX (PPIX) in the urothelium of the bladder. PPIX is an intensively red fluorescing agent. The mean ratio of fluorescence intensity between urothelial cancer and normal epithelium was found to be 17:1. Fluorescence excitation was achieved by violet light from a krypton ion laser (lambda = 406.7 nm) or from a xenon arc lamp with a bandpass filter system (lambda = 375-440 nm). Both light sources proved to be of equal suitability for fluorescence excitation. Fluorescence microscopy revealed that the PPIX fluorescence is strictly limited to the urothelium. It could not be detected from the submucosa or muscle of the bladder. Bladder wall biopsies were taken from 90 patients with suspicion of bladder cancer under fluorescence view. The fluorescence detection proved to be of high sensitivity (98%). No serious side effects which would preclude further clinical testing, especially no cutaneous photoreaction, were observed. Tumor-associated fluorescence induced by topical ALA application offers new perspectives in the diagnosis and treatment of bladder cancer. In case of suspicious or positive urine cytologic findings, ALA fluorescence cystoscopy may be useful for detecting the precise site of the malignancy. The procedure might be helpful in complete resection or coagulation of urothelial neoplasms. Due to this, diminishing recurrence rates are expected. However, this hypothesis has to be studied in prospective clinical trials.