Search

What's known on the subject? and What does the study add? Fluorescence cystoscopy with hexylaminolevulinate (h-ALA, Hexvix®) is known to improve tumour detection in non-muscle-invasive bladder cancer. However, specificity is relatively low and the intensity of the observed fluorescence signal decreases over time due to protoporphyrin IX (PpIX) efflux. This study evaluates in an in vivo model the use of a dendritic 5-aminolevulinic acid compound for fluorescence diagnosis. Fluorescence ratios between tumour and urothelium as well as muscle were significantly better as compared with h-ALA. Sustained synthesis of PpIX accounts for preservation of fluorescence for >24 h. OBJECTIVE • To overcome the relative lack of tumour selectivity of fluorescence-guided cystoscopy using 5-aminolevulinic acid (ALA) or its ester derivative (e.g. hexylaminolevulinate, h-ALA; Hexvix®), we evaluated the use of dendrimers bearing different ALA loads in rats bearing orthotopic bladder tumours. MATERIALS AND METHODS • Rat bladders were instilled with h-ALA or ALA dendrimers and fluorescence ratio between tumour and normal urothelium, as well as tumour and muscle and depth of fluorescence were determined with Image J software. • Quantification of ALA and/or esters systemic reabsorption was evaluated by high-performance liquid chromatography. RESULTS • Slow hydrolysis of ALA from dendrimers as observed in vitro implies a higher initial ALA load and longer resting times in vivo. Sustained synthesis of protoporphyrin IX (PpIX) explains persistence of fluorescence for >24 h. • There were significantly better fluorescence ratios with dendrimers, as well as higher penetration depths and absence of systemic reabsorption. CONCLUSION • The prolonged and sustained PpIX synthesis, the improved tumour selectivity with a deeper penetration and the absence of systemic reabsorption are primary indicators that ALA dendrimers could be an alternative to h-ALA in fluorescence-guided cystoscopy.

International audience; Abstract: Background. Collagen matrices as substitution for connective tissue are known to promote wound healing. Photodynamic therapy has been anecdotally associated with improved wound healing and reduced scarring. The present study investigates the impact of collagen based scaffolding material, embedded with a liposomal formulation of meta-tetra (hydroxyphenyl) chlorin (mTHPC, FoslipÒ) and photodynamic therapy on wound healing in mice. Methods. After incision in the neck region, two different types of collagen material, previously incubated with Foslip® at different concentrations, were implanted followed by illumination at 652 nm (10 J/cm², 100 mW/cm²). Mice were imaged daily up to two weeks, whereafter excision was performed and pathological analysis. Results. Scab detachment was observed at day seven for controls whereas it occurred as early as three days for PDT at the lowest concentrations. In the latter conditions, final matrix remodelling could be observed as evidenced by elastin neosynthesis. Conclusions. Topical application of low dose Foslip® in a collagen matrix followed by illumination considerably accelerates wound healing.

Photodynamic Therapy is a complex treatment modality where a large array of factors can influence therapeutic outcome. Vascularization, vessel permeability, oxygenation and light distribution in the tissue as well as immune response play a key role in the photodynamic process. Each of these factors can be influenced by the choice of the animal model. It is therefore of the utmost importance to chose an appropriate model for pre clinical oncologic PDT studies. Heterotopic models are easy to reproduce and monitor tumor growth and response to treatment and can be useful to answer specific questions. However, since many factors such as vascularization and microenvironment are different from the native organ, they can not be used as a representative model for PDT in the clinic. Orthotopic tumor models present the closest resemblance to the clinical situation with regard to all the elements involved in PDT. Even then, some existing models have to be adapted in order to exhibit the same features as observed during clinical PDT. We present here a brief organ specific overview of the different orthotopic animal models that can be used for in vivo photodynamic therapy studies.

Photodynamic therapy (PDT) is an approved anticancer treatment modality that eliminates unwanted cells by the photochemical generation of reactive oxygen species following absorption of visible light by a photosensitizer, which is selectively taken up by tumor cells. Present study reports the modalities of cell death after photosensitization of human adenocarcinoma HT29 monolayer and spheroid cells with a second generation photosensitizer Foscan ® . Kinetics of apoptosis and necrosis after Foscan ® -PDT in monolayer cells determined by flow cytometry using labeling of cleaved poly(ADP-ribose) polymerase (PARP) and staining with propidium iodide (PI) demonstrated that Foscan ® was not a strong inducer of apoptosis and necrosis was a prevailing mode of cell death. Cytochrome c release (cyt c ) and mitochondrial membrane potential (Δ ψ m ) addressed by flow cytometry technique at different time points post-Foscan ® -PDT demonstrated that cell photoinactivation was governed by these mitochondrial events. Foscan ® -loaded HT29 multicell spheroids, subjected to irradiation with different fluence rates and equivalent light doses, displayed much better tumoricidal activity at the lowest fluence rate used. Apoptosis, measured by caspase-3 activation was evidenced only in spheroids irradiated with the lowest fluence rate and moderate fluence inducing 65% of cell death. Application of higher fluence rates for the same level of photocytotoxicity did not result in caspase-3 activation. The observation of the fluence rate-dependent modulation of caspase-3 activity in spheroids offers the possibility of regulating the mechanism of direct cell photodamage and could be of great potential in the clinical context.

Purpose: To optimize photodynamic therapy (PDT) we investigated the kinetics and biodistribution of hexylester 5-aminolevulinate (hALA) induced protoporphyrin IX (PpIX) and the therapeutic efficacy of PDT at different drug and light doses in an orthotopic rat bladder tumor model. Materials and Methods: Healthy and tumor bearing rats were instilled intravesically with hALA (4, 8 and 16 mM) for 1 hour. Fluorescence was recorded spectroscopically in situ. PpIX fluorescence distribution and quantification across the bladders was visualized with fluorescence microscopy. PDT efficacy at different fluences (15 to 80 J/cm 2 ) was histologically assessed 48 hours and 1 week after treatment. Results: Spectroscopic analysis in normal or tumor bearing rats showed the highest tumor-to-normal ratios 2 or 3 hours after the end of the 8 or 16 mM hALA instillation (5.4 and 5.7, respectively). Within the same tumor bearing animal the same fluorescence levels were observed in normal epithelium and transitional cell carcinoma, whereas the tumor-to-muscle ratio was 3. Tumor necrosis with an intact normal bladder wall was observed with a fluence of 20 J/cm 2 for 8 mM hALA, while 15 J/cm 2 was ineffective and 25 J/cm 2 induced total wall necrosis. Although it induced comparable PpIX fluorescence, 16 mM hALA did not result in tumor eradication at any fluence. Conclusions: An optimal PDT effect was obtained with 8 mM hALA and a fluence of 20 J/cm 2 . While different hALA concentrations induce identical PpIX fluorescence intensities, the PDT outcome was considerably different. Thus, fluorescence does not necessarily predict the therapeutic efficacy of PDT.

Whole-bladder wall photodynamic therapy (PDT) is a promising treatment for carcinoma in situ (CIS) and diffuse premalignant changes of the bladder. After the results of our clinical studies showing that intravesical hypericin selectively accumulates in superficial bladder tumors, we investigated the hypericin-PDT efficacy in an AY-27 orthotopic transitional cell carcinoma rat bladder tumor model. After the instillation of hypericin (30 μM, 2 hr) in the bladder, tumors were irradiated (25–50 mW/cm 6–48 J/cm2) using 595 nm laser light. Data demonstrate that light doses of 12–48 J/cm2 resulted in selective PDT-induced urothelial tumor damage without damaging detrusor musculature. Histological assessment of bladder sections 2 days after PDT showed tumor destruction, with tumor cells shrinking and detaching from the bladder wall. There were tumor regrowth 1–3 weeks after treatment. The in vivo/in vitro clonogenic assay results revealed up to 98% of tumor cell kill by hypericin PDT. In conclusion, hypericin PDT can be used to safely induce a selective urothelial tumor damage without damaging detrusor musculature, when optimum hypericin concentration and light fluences are used. A small percentage (2–5%) of tumor cells that survive the photodynamic treatment resulting in tumor regrowth after a prolonged period of time is likely due to oxygen depletion during light irradiation. © 2003 Wiley-Liss, Inc.

An effective therapeutic outcome in the treatment of bladder cancer is largely defined by its early detection. In this context, big expectations have been placed on the fluorescence-guided diagnosis of bladder cancer. This paper reviews the applications of endo- and exogenous fluorescence for early diagnosis of in situ carcinoma of the bladder. Despite certain advantages of autofluorescence, exogenous fluorescence, based on the intravesical instillation of fluorophores with the following visible light excitation, has been shown to be more effective in terms of sensitivity and specificity for detecting carcinoma in situ. The equipment consists of a slightly modified light source in order to choose between white (conventional endoscopy) or blue light (fluorescence endoscopy) excitation, and specific lenses, in order to enhance maximally the contrast between normal (blue) autofluorescence and red fluorescence from malignancies. Among exogenous fluorophores, a particular emphasis will be put on the 5-aminolevulinic acid (ALA), its ester derivative (h-ALA) and hypericin. These dyes demonstrated an excellent sensitivity above 90% and specificity ranging from 70% to 90%.

Objective To determine the use of hypericin instillation for the fluorescent detection of papillary bladder cancer and carcinoma in situ. Patients and methods Eighty-seven patients with papillary bladder cancer and/or carcinoma in situ received instillations with 40 mL of an 8 µmol/L hypericin solution for at least 2 h. Fluorescent excitation with blue light was effective for up to 16 h, and biopsies were examined by fluorescence microscopy. Results There were no side-effects reported, no photobleaching and all papillary lesions fluoresced red. The sensitivity and specificity for detecting carcinoma in situ was 94% and 95%, respectively. An interval of 4 months is recommended after BCG instillations before using this test. Fluorescence microscopy showed that hypericin was selectively localized in the epithelium. Conclusions Hypericin-induced fluorescence has a high sensitivity and specificity for detecting bladder cancer. After 4 months there are few false-positive results in patients treated with BCG.

In a recent clinical study, we reported a selective uptake of hypericin in superficial bladder tumors. The results suggested that hypericin, a potent photosensitizer, could be used not only for diagnosis but also for photodynamic therapy (PDT) of superficial bladder tumors. In the present study, we investigated the biodistribution of hypericin in an orthotopic rat bladder tumor model by assessing the extent of hypericin penetration and the kinetics of accumulation into rat bladder tumors and normal bladder wall. Hypericin (8 or 30 microM) was instilled into the bladder via the catheter for 1, 2 or 4 hr. The fluorescence of hypericin in the bladder tumors and normal bladder was documented using fluorescence microscopy. In situ quantification of hypericin fluorescence in the tumor or normal bladder was performed using the laser-induced fluorescence technique. There was much more hypericin fluorescence in the tumor than in the normal bladder, with the tumor-to-normal-bladder ratio mounting to 12:1 after 4 hr of hypericin (30 microM) instillation. Moreover, hypericin was retained in the tumor for at least 1 hr before it was gradually lost from the tissue. Microscopically, the fluorescence of hypericin was restricted to the urothelial tumor and normal urothelium without fluorescence in the submucosa and the muscle layers. Subsequently no hypericin was detected in plasma, indicating that under these conditions systemic side effects should not be expected. Because the conditions used in this study were similar to those used in our previous clinical study, it is therefore likely that whole bladder wall PDT in the clinic under these conditions will produce selective urothelial tumor destruction without causing damage to the underlying muscle layers.

International audience; A prerequisite for starting a clinical trial is evidence of a positive impact of the technique or drug used in animals. The choice of the animal models is thus very important and should mimic as closely as possible the human situation. A variety of animal models has been validated for pharmaceutical trials. The situation in phototheranostics however is not fully clear. This is due to the very complex interplay of various elements such as vascularisation, oxygenation, drug availability and biodistributrion, light absorption and scattering etc. The present paper will give general information on aspects of animal models that have to be considered in phototheranostics as well as highlight some typical animal models that are useful for the investigation of light tissue interactions.

International audience; mTHPC is a non-polar photosensitizer used in photodynamic therapy. To improve its solubility and pharmacokinetic properties, liposomes were proposed as drug carriers. Binding of liposomal mTHPC to serum proteins and stability of drug carriers in serum are of major importance for PDT efficacy, however neither was reported before. We studied drug binding to human serum proteins using size-exclusion chromatography. Liposomes destruction in human serum was measured by nanoparticle tracking analysis (NTA). Inclusion of mTHPC into conventional (Foslip®) and PEGylated (Fospeg®) liposomes does not affect equilibrium serum protein binding compared to solvent-based mTHPC. At short incubation times the redistribution of mTHPC from Foslip® and Fospeg® proceeds by both drug release and liposomes destruction. At longer incubation times the drug redistributes only by release. The release of mTHPC from PEGylated vesicles is delayed compared to conventional liposomes, alongside with greatly decreased liposomes destruction. Thus, for long circulation times the pharmacokinetic behavior of Fospeg® could be influenced by a combination of protein- and liposome-bound drug. The study highlights the modes of interaction of photosensitizer-loaded nanovesicles in serum to predict optimal drug delivery and behavior in vivo in pre-clinical models, as well as the novel application of NTA to assess the destruction of liposomes.

What's known on the subject? and What does the study add? Fluorescence cystoscopy with hexylaminolevulinate (h-ALA, Hexvix®) is known to improve tumour detection in non-muscle-invasive bladder cancer. However, specificity is relatively low and the intensity of the observed fluorescence signal decreases over time due to protoporphyrin IX (PpIX) efflux. This study evaluates in an in vivo model the use of a dendritic 5-aminolevulinic acid compound for fluorescence diagnosis. Fluorescence ratios between tumour and urothelium as well as muscle were significantly better as compared with h-ALA. Sustained synthesis of PpIX accounts for preservation of fluorescence for24 h.• To overcome the relative lack of tumour selectivity of fluorescence-guided cystoscopy using 5-aminolevulinic acid (ALA) or its ester derivative (e.g. hexylaminolevulinate, h-ALA; Hexvix®), we evaluated the use of dendrimers bearing different ALA loads in rats bearing orthotopic bladder tumours.• Rat bladders were instilled with h-ALA or ALA dendrimers and fluorescence ratio between tumour and normal urothelium, as well as tumour and muscle and depth of fluorescence were determined with Image J software. • Quantification of ALA and/or esters systemic reabsorption was evaluated by high-performance liquid chromatography.• Slow hydrolysis of ALA from dendrimers as observed in vitro implies a higher initial ALA load and longer resting times in vivo. Sustained synthesis of protoporphyrin IX (PpIX) explains persistence of fluorescence for24 h. • There were significantly better fluorescence ratios with dendrimers, as well as higher penetration depths and absence of systemic reabsorption.• The prolonged and sustained PpIX synthesis, the improved tumour selectivity with a deeper penetration and the absence of systemic reabsorption are primary indicators that ALA dendrimers could be an alternative to h-ALA in fluorescence-guided cystoscopy.

International audience; We used the phenomenon of previously described photoinduced fluorescence quenching and fluorescence polarization to evaluate the transfer of meta-tetra(hydroxyphenyl)chlorin (m-THPC) from commercial high-drug load liposomes to plasma proteins and model membranes. Fluorescence quenching of m-THPC in liposomes by iodide indicates that part of m-THPC in PEGylated liposomes is localized in the PEG shell, while the rest is bound to the lipid bilayer. It was shown that the two molecule pools in the commercial PEGylated liposomal formulation Fospeg® condition the characteristics of the m-THPC release kinetics. A substantial percentage of m-THPC from Fospeg® is released much faster than from the conventional liposomal formulation Foslip®. Using the technique of resonance light scattering, it was shown that partial m-THPC aggregation is present in liposomes with very high drug loads, higher in PEGylated liposomes compared to conventional ones.

published in Photodiagnosis and Photodynamic Therapy, 8(2):156, 2011; International audience; A serious limitation of photodynamic therapy (PDT) is the absence of specific cancer targeting, resulting in an excessive tissue destruction, which can provoke life threatening situations. To partially overcome these constraints, incorporation of the active compound into liposomal nanoparticles can be proposed. Embedding of active drugs in liposomes favours passive targeting of tumors through Enhanced Permeability Retention (EPR) effect. Liposomal formulations of mTHPC (Foslip®) enable a more selective and faster accumulation of drugs in the tumors, with a faster clearance, together with at least similar to liposomes-free mTHPC therapeutic efficacy. Best Foslip® - photoinduced response was obtained from the study of spatial intratumoral Foslip distribution rather than from bulk pharmacological tissues pharmacokinetics. Comprehension of the transport kinetics of the photosensitizer from the vessels towards extravascular structures is essential for optimizing clinical protocols. We have observed different redistribution patterns of mTHPC to plasma components according to liposomal composition (conventional vs. pegylated liposomes) in vitro. The visualization of these processes in real time in vivo using the chick chorio allantoic membrane (CAM) model corroborate with in vivo results. Future directions consist in aptamer-mediated targeted delivery of chlorin types photosensitizers to tumor tissues.

published in Photodiagnosis and Photodynamic Therapy, 8(2):149, 2011; International audience; Bladder PDT, the first FDA indication, has been abandoned in favour of photodiagnosis with ALA or derivatives. Major drawback of this treatment modality is prolonged and severe irritative bladder symptoms, due to lack of selectivity of the drug and generalized urothelial necrosis. Low fluence rates are known to potentiate therapeutic efficacy due to an oxygen sparing regime. We have evaluated the effect of low fluence rate PDT on therapeutic outcome and side effects in rat tumor bearing bladders. Bladders were instilled with 8 mM hexylaminolevulinate and illuminated with 635 nm light to a total light dose of 20 or 15 J/cm2 at 100 or 15 mW/cm2, 5 rats per group. Fluence rates of 15 mW/cm2 did not provoke enhancement of cardiac and respiratory rhythms as opposed to 100 mW/cm2, neither was hematuria observed post treatment. Macroscopic aspects of bladders at 100 mW/cm2 show thickened bladders with hemorrhagic areas whereas 15 J/cm2 does not alter macroscopic features. Pathology revealed the absence of viable superficial tumors, irrespective of irradiation conditions. Inflammatory volume was calculated to be 96 a.u. for high fluence high light dose, reduced to 18 a.u. when reducing the light dose and 12 a.u. for the low fluence rates, irrespective of the light dose. Normal urothelial is preserved over the total bladder in case of 15 mW/cm2, whereas major portions of the bladder show absence of reduction of cell layers at 100 mW/cm2. Low fluence rate PDT does not hamper therapeutic efficacy and minimizes local side effects in rats.

published in Photodiagnosis and Photodynamic Therapy, 8(2):175, 2011; International audience; Background: Fluorescence guided cystoscopy with 5-aminolevulinic acid (5-ALA) or its hexyl derivative (h-ALA) is used in bladder cancer diagnosis. However, the lack of specificity and the fast photobleaching of the induced sensitizer protoporphyrin IX (PpIX) impair the complete resection of the tumor during cystoscopy. Methods: To overcome these drawbacks, a dendrimer containing 18 ALA molecules (18-ALA) was synthesized. These dendrimers induce a sustained synthesis of PpIX following intraperitoneal administration. We investigated the in vitro hydrolysis of ALA and the PpIX synthesis rate with ALA equimolar concentrations of h-ALA and 18-ALA. We further instilled prodrugs intravesically in rats bearing orthotopic bladder tumors and analyzed the PpIX distribution on frozen sections by fluorescence microscopy. Results: A slow mono-exponential hydrolysis of the dendrimers was observed, compared with the rather fast release of ALA from h-ALA. This result was attributed to steric hindrance and limited access to esterases explaining the observed continuous synthesis of PpIX during 72 h. In vivo, the best tumor vs. muscle specificity of 18-ALA dendrimers was obtained after 1 h instillation following 4 h resting time. By comparison with h-ALA or smaller dendrimers, 18-ALA dendrimers presented an increased tumor vs. urothelium ratio. Furthermore, the continuous synthesis of PpIX has ensured a prolonged observation of PpIX fluorescence for 24 h after the instillation of 18-ALA. Conclusion: The prolonged and sustained PpIX synthesis and the improved selectivity are primary indicators that ALA dendrimers could possibly overcome drawbacks of ALA/h-ALA fluorescence guided cystoscopy.

International audience; Three approaches to analyzing the rate of release of the photosensitizer meta-tetrahydroxyphenylchlorin (mTHPC) from unilaminar lipid vesicles (ULV) in model biological systems are studied: by excitation energy transfer from probe diphenylhexatriene to mTHPC, by the fluorescence anisotropy of mTHPC, and by photoinduced quenching of the fluorescence of mTHPC. Each of these methods has its characteristic range of sensitivity for measurements of the local concentration of mTHPC in ULV. Fluorescence anisotropy can be used for quantitative determination of the mTHPC yield from ULV for mTHPC:lipid ratios of 1:100 to 1:1000, determining the efficiency of fluorescence quenching of diphenylhexatriene for ratios

International audience; The incidence of oesophageal cancer continuously increases and 400 000 new cases/year are diagnosed worldwide. Total oesophagectomy is recommended when multifocal disease, especially in case of Barrett's oesophagus [1]. However, selected cases may benefit from subtotal oesophagectomy, with 5-year survival rates ranging from 20% to 50% [1] G. Decker, W. Coosemans and P. De Leyn et al., Minimally invasive oesophagectomy for cancer, Eur J Cardiothorac Surg 35 (2009), pp. 13–20.[1]. Local recurrence after subtotal oesophagectomy is relatively high (30–40%) and regular endoscopic surveillance is mandatory. Early stage lesions are eligible for endoscopic minimally invasive surgery such as endoscopic mucosal resection or dissection (EMR, EMD), argon plasma photocoagulation, radiofrequency ablation or photodynamic therapy (PDT). Photodynamic therapy is now commonly applied to eradicate dysplasia and Barrett's oesophagus but can also achieve destruction of squamous cell carcinoma. In the present paper, we report a case of multifocal high grade dysplasia, in a patient with prior subtotal oesophagectomy, treated with PDT.

We report on the preliminary results of 12 patients with multifocal carcinoma in situ of the bladder treated with whole bladder wall photodynamic therapy. The total light dose (scattered plus nonscattered light) measured in situ was 100 joules per cm.2 in the first 6 patients (group 1) and 75 joules per cm.2 in the remaining 6 (group 2). These light doses correspond on the average to 27 joules per cm.2 and 15.5 joules per cm.2 nonscattered light as reported by other investigators. Followup ranged from 6 to 22 months (average 11.5). In group 1, 2 tumors recurred after 6 and 9 months, respectively, and 2 other patients had a permanently shrunken bladder without evidence of disease. In group 2, 1 tumor recurred 5 months after photodynamic therapy. In this group the bladder capacity increased on the average to 135% of the pretreatment value 3 months after photodynamic therapy. All recurrences were in patients with a history of invasive bladder cancer (stages T1 and T2). These preliminary results demonstrate the importance of in situ scattered light dosimetry for minimizing local side effects of whole bladder photodynamic therapy.

We determined the sensitivity and specificity of detecting flat bladder carcinoma in situ through fluorescent detection after intravesical hypericin instillations.The study included 40 patients, of whom 26 presented with macroscopic visible tumor, 9 had a positive cytology without visible tumor and 5 underwent cystoscopy after bacillus Calmette-Guerin instillations (4) or radiotherapy (1). We instilled 40 ml. of a 8 microM. solution of hypericin intravesically for at least 2 hours. Fluorescence excitation with blue light was effective up to 16 hours after termination of the instillation.All visible papillary tumors showed red fluorescence. In addition, 134 flat fluorescent areas were detected. Analysis of 281 biopsies from flat bladder wall indicated 93% sensitivity and 98.5% specificity for detecting carcinoma in situ. Visible lesions resulting from radiotherapy, chemotherapy or immunotherapy did not show any fluorescent signs and, therefore, did not induce false-positive readings. There were no signs of photobleaching during inspection and resection.We report a simple yet comprehensive endoscopic method for early detection of bladder cancer, including carcinoma in situ. Hypericin induced fluorescence has a high sensitivity and specificity for detection of bladder transitional cell carcinoma, papillary and flat carcinoma in situ. When carcinoma in situ is suspected, this technique is highly recommended.

The prevalence of bladder cancer is very high, due to its high recurrence rate in superficial bladder cancer (30 to 85%), which is the staging of approximately 80% of the patients at first diagnosis. Risk of recurrence and progression is associated with grade, stage, presence of concomitant carcinoma in situ, size and number of lesions, as well as time to first recurrence. Recurrences can be partly attributed to new occurrences but also to residual tumors after resection. Incomplete tumor removal has been observed in 30 to 50% of TUR's, especially when dealing with T1 or poorly visible malignant or pre-malignant disease1. Fluorescence guided resection with 5 amino levulinic acid (ALA) or its hexyl ester derivative (Hexvix, has now unequivocally been demonstrated to increase detection rate and a growing number of studies indicate this has a positive impact on recurrence and progression ratesImplantation of viable tumor cells, dispersed during resection, is a third factor influencing bladder cancer recurrence. The aim of early intravesical therapy is to interfere with cell viability and thus reduce implantation risks.

International audience; Foslip is a recently designed third generation photosensitiser based on unilamellar dipalmitoylphosphatidylcholine /dipalmitoylphosphatidylglycerol (DPPC/DPPG) liposomal formulations of meta-tetra(hydroxyphenyl)chlorine (mTHPC). The present study investigates Foslip behaviour and its photodynamic efficiency in EMT6 xenografted nude mice at different times following i.v. administration of 0.3 mg kg-1 mTHPC in a Foslip formulation. Plasma pharmacokinetics and biodistribution were studied by high performance liquid chromatography and were described by a three compartments analysis with half-lifes of 0.13, 4.31 and 35.7 h. The highest tumour to muscle ratios were observed at 6 and 15h post-administration. Intratumoral distribution was carried out using two photon excitation confocal microscopy. Progressive efflux from the vascular compartment was noted in favour of tumour parenchyma, which was almost completed at 15h. The best tumour response was obtained for a drug-light interval of 6 h, interval for which mTHPC was present in both endothelial and parenchyma cells. Tumour and plasma concentrations however were far below their maximal values. Based on these observations, we assume that the presence of mTHPC in both vasculature and tumour cells is required for optimal PDT efficacy.

Fluorescence guided transurethral resection has gained acknowledgment from the urological community and it is progressively becoming more applied. It has been shown to decrease the recurrence rate of nonmuscle invasive bladder cancer due to incomplete resection due to lack of visualization. The implantation of viable tumor cells seeded during transurethral resection is another reason for recurrence. We investigated whether applying photodynamic therapy on sensitized tumor cells would decrease the amount of viable intraluminal cells and tumor cell implantation.Two models were designed to mimic the situation after fluorescence guided transurethral resection, including partly or fully de-epithelialized bladders and circulating tumor cells loaded with protoporphyrin IX. Photodynamic therapy was performed. Controls consisted of no drug with no light, light only and drug only. Immediately after photodynamic therapy the intravesical contents were retrieved and clonogenic assays were performed on cells. Bladders were harvested 10 days after cell administration and subjected to pathological analysis.In the photodynamic therapy and control groups tumor volume was proportional to the instilled cell load. Clonogenic assays showed that viable cells were decreased a tenth of the initial administered amount. Tumor implantation decreased to less than a fifth of control values.Photodynamic therapy can effectively decrease the amount of viable tumor cells in the bladder lumen. This results in a significant decrease in tumor implantation. This technique could possibly be used to further decrease the recurrence rate of nonmuscle invasive bladder cancer.

International audience; Liposomal formulations of meta-tetra(hydroxyphenyl)chlorin (mTHPC) have already been proposed with the aim to optimize photodynamic therapy. Spectral modifications of these com- pounds upon irradiation have not yet been investigated. The objective of this study was to evaluate photobleaching properties of mTHPC encapsulated into dipalmitoylphosophatidylcholine (DPPC) liposomes, Foslip. Fluorescence measurements in DPPC liposomes with different DPPC:mTHPC ratios demonstrated a dramatic decrease in fluorescence anisotropy with increasing local mTHPC concentration, thus suggesting strong interactions between mTHPC molecules in lipid bulk medium. Exposure of Foslip suspensions to small light doses (

Generalized skin sensitization is a main drawback of photodynamic therapy with systemic administration of photosensitizers. We have evaluated the potential use of an intratumoral injection of a liposomal formulation of mTHPC (Foslip) in a mouse model of local recurrence of breast cancer.Mice were directly injected into the tumor (IT) with 25 microl of a Foslip suspension (0.15 mg/ml) and illumination (652 nm, 20 J/cm(2)) was performed at different time points with pathological assessment after 48 hours. In a parallel mice series plasma samples were obtained at different endpoints after IT Foslip injection for HPLC analysis and the tumors were subjected in toto to macrofluorescence imaging. Fluorescence polarization measurements were conducted in vitro to estimate the rate of sensitizer redistribution from liposomes.Optimal, albeit partial, cure rates were obtained at 24 hours post-sensitizer and uninistration. Inhomogeneous and weak fluorescence was observed at early time points and became maximal at 24 hours. Plasma levels of mTHPC increased until 15 hours. Fluorescence polarization measurements showed a slow sensitizer transfer from liposomes to model membranes.The weak intratumoral fluorescence at early time points could be explained by concentration quenching within the liposomes as evidenced from fluorescence polarization studies. Progressive mTHPC redistribution from liposomes and its further incorporation into tumor tissue resulted in fluorescence build-up over time with a maximum at 24 hours post-injection. This correlates perfectly with the best therapeutic effect at this time point. The absence of total cure can be attributed to inhomogeneous photosensitizer distribution. mTHPC is reabsorbed into the blood stream but the total administered amount is much reduced as opposed to systemic administration so that repeated PDT sessions might be favorable in terms of side effects and tumor response.

International audience; Hexyl ester of 5-aminolevulinic acid (HAL) is a very efficient precursor of the photosensitizer protoporphyrin IX (PpIX) for photodynamic therapy (PDT). Our previous study, performed in rat orthotopic bladder tumors, indicated an opposite effects of HAL/PpIX-PDT according to HAL concentration. The present study investigated possible reasons for this differentiate effect considering the impact of extracted amounts of PpIX in normal and tumor bearing bladders along with PpIX distribution in distinctive histopathological layers. High performance liquid chromatography (HPLC) analysis of tumor and normal bladder tissues after 8 mM and 16 mM HAL instillation showed that PpIX was the main porphyrin species. The PpIX production in tumor bladders instilled with 8 mM HAL was significantly higher than after 16 mM HAL. Fluorescence confocal microscopy demonstrated a punctuate bright fluorescence pattern in tumor zones of bladders instilled with 8 mM HAL, whereas a more diffuse cytoplasmatic fluorescence distribution was observed after 16 mM HAL instillation. Immunofluorescence staining together with transmission electron microscopy showed severe mitochondrial damage in tumor zones of bladders treated with 8 mM HAL/PpIX PDT, with intact mitochondria in tumor zones of bladders treated with 16 mM HAL/PpIX PDT. We conclude that the differential response to HAL/PpIX PDT in function of HAL concentrations could be attributed to diminished PpIX synthesis and differential intracellular localisation of PpIX. Mitochondria were shown to be the critical photodamged sites of HAL/PpIX PDT and as such tissue sensitivity to treatment can be estimated through investigation of intracellular PpIX distribution.

International audience; The present study addresses the impact of different aggregation states of meta-tetra(hydroxyphenyl)chlorin (mTHPC) on the photoinactivation of cells. Measurements of the photophysical properties of mTHPC in MCF-7 cells showed progressive sensitizer aggregation with increasing incubation time. Reconstructed absorption spectra of intracellular mTHPC showed a significant decrease in the molar extinction coefficient and broadening of the Soret band at 24 h incubation compared to 3 h. Intracellular photobleaching of mTHPC slowed down, and the profile changed from mono- to bi-exponential upon incubation. Fluorescence lifetime imaging (FLIM) measurements revealed a substantial decrease in the lifetime of mTHPC fluorescence at 24 h compared to 3 h. In addition, the intracellular localization of mTHPC as observed by fluorescence microscopy changed from a diffuse homogeneous fluorescence pattern at short incubation times to a punctiform pattern at 24 h. The efficiency of photodynamic therapy (PDT) assessed by a clonogenic assay was three times greater at 24 h. However, when the survival curves were replotted as a function of the number of absorbed photons, the efficiency was 1.8 times greater at 3 h than at 24 h. The loss of photosensitizing efficiency at higher mTHPC concentrations was attributed to self-quenching of the triplet states of the sensitizers.

An ideal bladder tumor model consists in an orthotopic solitary tumor with a well-defined localization and stage, as well as unaltered normal mucosa. None of the existing models covers all these requirements. We have created a new model, suitable for diagnostic and therapeutic purposes. Female Fisher rats were divided into different groups according to bladder preconditioning and tumor cell (AY27) administration. Generalized desepithelialization was obtained by an intravesical instillation of HCl, neutralized by NaOH. Localized desepithelialization of the bladder fundus was the result of application of a micro-swab, imbibed with the same chemicals. Tumor cells administration was either generalized (intravesical instillation) or focal (micro swab). No bladder perforations were observed. Generalized desquamation always produced multifocal tumors, whereas focal application of HCl/NaOH resulted in solitary tumors of the bladder fundus, irrespective of the method of tumor cell administration. Only hyperplasia could be detected at day 3. AY27 cells were covered by umbrella cells at day 5 and subepithelial AY 27 tumor nests, covered by full thickness epithelium were observed day 7.

Purpose We evaluated the possibility of performing endoscopic fiber-optic confocal microscopy in a rat bladder model and we distinguished different cell types. Material and Methods Rhodamine 123 (Molecular Probes, Eugene, Oregon) (100 μM) was instilled for 30 minutes in 5 tumor bearing rat bladders (AY27). Five normal rats served as controls. A Cell-vizio™ confocal microscopy fiber was placed transurethrally in contact with normal or transformed bladder wall. Frozen sections were obtained from the same spots and subjected to conventional fluorescence microscopy and anatomical-pathological analysis. Results The different cells types present in rat epithelium (umbrella, intermediate and basal cells) could easily be identified with the Cell-vizio™ device due to their differences in morphology and fluorescence intensity. Individual AY-27 cells could not be demarcated due to the strong fluorescence signal but the entire tumor appeared as a brightly homogenous fluorescent blot surrounded by small inflammatory cells. Conclusions We report the feasibility of endoscopic, in vivo, fiber-optic confocal microscopy in the rat bladder. We distinguished tumors from normal epithelium and visualized the different epithelial cell types in nontransformed rat bladder epithelium.

The present study investigates the interaction of the second generation photosensitizer Foscan® with plasma albumin and lipoproteins. Spectroscopic studies indicated the presence of monomeric and aggregated Foscan® species upon addition to plasma protein solutions. Kinetics of Foscan® disaggregation in albumin-enriched solutions were very sensitive to the protein concentration and incubation temperature. Kinetic analysis demonstrated that two types of Foscan® aggregated species could be involved in disaggregation: dimers with a rate constant of k 1 = (2.30 ± 0.15) × 10 −3 s −1 and higher aggregates with rate constants varying from (0.55 ± 0.04) × 10 −3 s −1 for the lowest to the (0.17 ± 0.02) × 10 −3 s −1 for the highest albumin concentration. Disaggregation considerably increased with the temperature rise from 15 °C to 37 °C. Compared to albumin, Foscan® disaggregation kinetics in the presence of lipoproteins displayed poorer dependency on lipoprotein concentrations and smaller variations in disaggregation rate constants. Gel-filtration chromatography analysis of Foscan® in albumin solutions demonstrated the presence of aggregated fraction of free, non-bound to protein Foscan® and monomeric Foscan®, bound to protein.

In the present study, the systemic absorption of hypericin was investigated after intravesical instillation of the compound in nine patients with superficial transitional cell carcinoma (TCC) bladder tumors. Hypericin (8 microM) was instilled in the bladder for 2-3 h before photodynamic diagnosis of bladder tumors. Blood was then collected from a peripheral vein 1 h after termination of the instillation. Solid phase extraction with ammonium acetate buffer and methanol was used to extract hypericin from the plasma. A reversed-phase high performance liquid chromatographic method with fluorescence detection was used to identify and quantify hypericin in the extracts from plasma samples. Analysis of standard plasma samples, which were spiked with known amounts of hypericin, indicated that the pH of the buffer was a determining factor in the extraction yield. The results obtained using ammonium buffer (pH 3.5) and methanol showed the mean extraction recovery of hypericin to be 64% (RSD=12%, n=6). The limits of detection and quantification were 6 and 20 nM, respectively. Extraction and analysis of the plasma of patients after intravesical administration showed hypericin concentrations below the detection limit (6 nM). In addition, photodynamic treatment of in vitro cultured HeLa cells incubated with 1-100 nM hypericin concentrations showed that lower concentrations (1-20 nM) of hypericin do not induce significant photocytotoxic effects. Taken together, these results imply that photosensitization or other systemic side effects in patients are not to be expected after photodynamic diagnosis of TCC bladder tumors with hypericin.

In a recent clinical study, we showed that hypericin accumulates selectively in urothelial lesions of the bladder following intravesical administration of the compound in patients. This observation infers that hypericin, a potent photosensitizer, could be used as a selective photodynamic therapy (PDT) tool against superficial bladder cancer. In the present study we investigated the in vivo PDT activity of hypericin in transition cell carcinoma (TCC) tumors of the bladder. Both the distribution and tumor PDT response were carried out using subcutaneous heterotopic AY-27 TCC tumors in syngeneic rats. For both PDT and distribution studies, hypericin (1 or 5 mg/kg) was injected intravenously 0.5, 6 or 24 h before PDT or distribution evaluation. The data show that hypericin is a potent photosensitizer in the treatment of TCC tumors in vivo and that the interval between drug administration and photo-irradiation has a dramatic effect on the PDT outcome. Using a 0.5 h interval between drug administration and photo-irradiation the tumor regrowth study indicated that no tumor mass could me measured 9-10 days after PDT. On the contrary, lengthening the time interval between drug administration and photo-irradiation resulted in a gradual loss of PDT efficiency in these tumors. For instance, while the 6 h drug interval protocol produced a moderate PDT activity in which the tumor sizes decreased to about 50% of their original sizes 11-16 days after photo-irradiation, the 24 h interval protocol was even less effective. The distribution data indicate that the PDT efficiency of hypericin in TCC tumors corresponded to the plasma concentrations rather than to the over all concentrations in the tumor. It is therefore conceivable that the mechanism of PDT efficacy of hypericin in TCC tumors is through indirect (vascular effects) rather than through direct effects (cellular destruction) of hypericin in these tumors. In conclusion, our data indicate that hypericin is a potent photosensitizer against AY-27 TCC tumors and that the PDT efficacy of hypericin is largely determined by photosensitizer distribution in the tumor at the time of photo-irradiation.

Topical instillations with Epirubicine were performed in vivo in patients with macroscopically visible tumours. Within 8 h Epirubicine fluorescence could be detected in normal mucosa. After 60 to 72 h no fluorescence was detected in normal mucosa but could still be found in tumour areas, though not at a constant time interval. It is suggested that photosensitizers could be used topically provided that they are not only tumour specific but also time specific.

Carcinoma of the bladder (CaB) is a common tumor of the genitourinary tract. In the United States in 1997, CaB was second in frequency of occurrence and third in mortality among genitourinary tumors. This tumor has a well-documented history of environmental and industrial causative factors. The strongest etiologic risk factors include the use of tobacco, which is thought to be responsible for half of the CaB diagnosed in men in the United States, and some arylamines. In the past 30 years, there has been major improvement in the survival of patients with this disease. Multiple factors were responsible for this accomplishment and they include: 1) better understanding of the natural history of CaB, 2) development of immunohistochemical analysis helpful in defining prognostic factors, 3) improved imaging and nonimaging diagnostic modalities helpful in making earlier diagnosis and better defining the true anatomical extent of the tumor, 4) development of more effective therapy for carcinoma in situ, 5) major improvement in surgical techniques resulting in better treatment outcomes, and 6) the wide use of adjuvant chemotherapy. Major stress has been placed on the quality of life of patients treated for CaB. Quality of life was improved by optimizing surgical, radiation, and medical treatment techniques. The two most important factors producing this quality-of-life improvement include: 1) the use of organ-preserving therapy in properly selected patients that involves the use of a multimodality therapeutic approach with transurethral resection, radiation therapy, and chemotherapy; and 2) the ability to treat selected men and women with radical cystectomy followed by orthotopic reconstruction that allows patients nearly physiologic voiding. Current research efforts are directed toward better patient selection for appropriate therapy which is expected to increase patient survival and improve quality of life. Of particular importance in the selection of this optimal therapy in patients with CaB is a wide application in the clinical practice of important recent advances in molecular genetics.

Carcinoma in situ (CIS) of the bladder is a confounding disease that is difficult to recognize endoscopically because it is a flat cancer. Many studies have suggested its relationship with subsequent invasive disease. Early recognition of CIS therefore is essential in offering the patient the most appropriate treatment and the highest cure rate. Because white light cystoscopic examination is not sufficient to reveal areas of dysplasia or CIS, random biopsies are recommended. The authors evaluate whether amino levulinic acid (ALA) fluorescence detection could be helpful in diagnosing CIS and if the specificity could be enhanced by reducing the ALA dose. Sixteen patients with papillary bladder cancer, and CIS and dysplasia were given low-dose ALA. Fluorescence detection of the metabolized ALA was performed 3 hours later, with the naked eye, after blue light illumination. Carcinoma in situ or dysplasia was found in 50 biopsies. The sensitivity for detecting CIS was 94% with a specificity of 54%. Carcinoma in situ can be diagnosed with a very high accuracy through fluorescence detection after ALA instillation. Fluorescence detection can be achieved with the naked eye and does not necessitate either complex equipment or specially trained personnel.

Carcinoma in situ (CIS) of the bladder is a treacherous entity, that will develop into invasive cancer. Early treatment is mandatory in order to prevent progression. When conservative measures, such as Bacillus Calmette Querin (BCG) instillations have failed, radical cystectomy and urinary diversion is recommended. Whole bladder wall photodynamic therapy (PDT) with Photofrin II has been shown to be effective in eradicating carcinoma in situ, but often resulted in bladder shrinking. We wanted to evaluate the effects of PDT after aminolevulinic acid (ALA) sensitization. Six patients with refractory carcinoma in situ of the bladder were treated with whole bladder wall photodynamic therapy, after intravesical sensitization with aminolevulinic acid. The total light dose (scattered plus non scattered) was 75 J/cm2. No skin sensitization occurred, nor loss of bladder capacity. One patient did not respond and was successfully treated with BCG. Another patient developed distant metastases. Carcinoma in situ was completely absent after 3 months in four patients (66%).© (1997) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Carcinoma in situ (CIS) of the bladder is a confounding disease that is difficult to recognize endoscopically since it is a flat cancer. Many studies have suggested its relationship with subsequent invasive disease. Early recognition of CIS therefore is essential in order to offer the patients the most appropriate treatment and the highest cure rate. Since white light cystoscopic examination is not sufficient to reveal areas of dysplasia or carcinoma in situ random biopsies are recommended. We wanted to evaluate whether amino levulinic acid (ALA) fluorescence detection could be helpful in diagnosing carcinoma in situ and if the specificity could be enhanced by reducing the ALA dose. Sixteen patients with papillary bladder cancer and carcinoma in situ and dysplasia were instilled with low dose ALA. Fluorescence detection of the metabolized ALA was performed three hours later, with the naked eye, after blue light illumination. CIS or dysplasia was found in 50 biopsies. The sensitivity for detecting CIS was 94% with a specificity of 89%. Carcinoma in situ can be diagnosed with a very high accuracy through fluorescence detection after ALA instillation. Fluorescence detection can be achieved with the naked eye and does not necessitate complex equipment neither specifically trained personnel.© (1997) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Objectives This article evaluates the results of whole bladder wall photodynamic therapy (PDT) for multifocal carcinoma in situ after a mean follow-up time of 3 years. Methods Photofrin II was used as a photosensitizer (2 mg/kg) and in situ dosimetry to obtain the best possible central positioning of the light diffuser as well as to know exact dosimetry data (scattered plus nonscattered light). Results Major classical drawbacks of PDT, such as severe bladder irritative symptoms and bladder shrinking, can be minimized with the help of in situ dosimetry. The success rate after 3-year follow-up is 60%. Fifty percent of the recurrences occurred in the prostatic urethra without evidence of disease of the bladder. Conclusions A success rate of 60% is comparable to the results obtained after bacille Calmette-Guerin (BCG). Side effects such as loss of bladder capacity can be minimized with adequate light dosimetry but they are still higher than with BCG (9% versus 1 % cystectomy).

Bladder tissue autofluorescence spectra are obtained in vivo at two excitation wavelengths (334 nm and 365 nm) with a cystoscopic fiber- optic device based on a small mercury arc lamp. Upon 365 nm excitation, both normal and cancerous bladder tissue have nearly identical fluorescence spectra, characterized by a broad peak at 455 nm. However, the fluorescence yield from malignant tissue is approximately a factor 3 lower compared to normal tissue. A similar decrease in fluorescence yield is observed upon 334 nm excitation. More importantly, at this excitation wavelength, the spectra from normal and malignant tissue have different lineshape. Normal tissue shows two distinct fluorescence peaks (at 385 nm and 455 nm), while malignant tissue only shows the 455 nm peak. Based on these insights, we have developed a simple spectroscopic algorithm, to differentiate normal from malignant bladder tissue with our device. The main underlying biophysics will be addressed. The integration of the diagnostic method with a reliable therapeutic technique for tumor cell destruction, may open the way for cost- effective preventive care of high-risk patients.

The diagnosis of transitional cell bladder carcinoma and especially carcinoma in situ of the bladder in spinal cord disordered persons is often made difficult by catheters, infections or stone-induced chronic inflammation. Fluorescence tagging of tumors by sensitizing agents such as hematoporphyrin derivatives enhances visualization but presents a number of drawbacks for the patients, even at low doses. We have developed a cystoscopic fiber optic instrument, based on a mercury arc lamp, for in vivo detection of human bladder carcinoma without sensitizing agents. The tissue autofluorescence upon UV excitation (365 nm) is detected and a demarcation contrast ratio for carcinoma in situ and transitional cell carcinoma of 2.6 and 3.2 respectively is obtained. This demarcation ratio is 60 percent higher than the contrast ratio obtained after photosensitizer injection. The integration of a reliable diagnostic method with a known efficient therapeutic technique (Nd YAG laser irridiation) opens the way for cost-effective preventive care of high-risk patients.

Laser-induced fluorescence (LIF) can be used for noninvasive spectroscopic identification of biological tissue and is of special interest in early tumor detection. The basis for this optical biopsy method is the interaction of the laser light with tissue chromophores, such as tryptophan, collagen, elastin, NADH, beta-carotene and hemoglobin. The UV-excited fluorescence that arises from the native chromophores, the autofluorescence, has a broad distribution, peaking at about 490 nm with a lower intensity in tumor compared to normal tissue. The tumor detection potential is enhanced with exogenously administrated tumor- marking agents, such as hematoporphyrin (HPD, commercial name Photofrin), with two fluorescence peaks at about 630 and 690 nm. We have developed clinical instrumentation both for tissue point monitoring and for full real-time image processing. Seventy-one patients were investigated in vivo and surgical samples from additional 20 patients. In 46 patients the autofluorescence only was monitored. In 45 patients low-dose Photofrin injection was used. The in vivo investigations included different kinds of lung tumors, urinary bladder tumors, and malignant gliomas. The in vitro measurements were performed in breast tumors and prostatic tumors. Invasive and early tumors and also precancerous lesions can be revealed utilizing LIF in low-dose Photofrin injected patients.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Transitional bladder cell carcinoma (TCC) is easily recognizable, but for the diagnosis of severe dysplasia and carcinoma in situ (CIS) one can only rely on randomly taken biopsies. Fluorescence tagging of tumors by sensitizing agents such as hematoporphyrin derivatives (HpD) is possible but presents, even at low doses, a number of serious drawbacks for the patient. We demonstrate a cystoscopic fiber optic instrument, based on a small mercury arc lamp, for in vivo demarcation of human bladder carcinoma. The instrument detects the tissue autofluorescence upon UV excitation (365 nm), thus eliminating the need for sensitizing agents. The average demarcation contrast obtained for CIS and TCC is respectively 2.6 and 3.2, which is about 60% higher than what can be expected from photodynamic imaging with low-dose HpD. The main underlying biophysics are derived from a spectral analysis of the observed autofluorescence signals. The integration of the diagnostic method with a reliable therapeutic technique for tumor cell destruction, opens the way for cost-effective preventive care of high-risk patients.

A point-monitoring fluorescence diagnostic system based on a low-energy pulsed laser, fiber transmission optics, and an optical multichannel analyzer was used for diagnosis of patients with bladder malignancies. Twenty-four patients with bladder carcinoma, carcinoma in situ, and/or dysplasia were injected with hematoporphyrin derivative, Photofrin, 0.35 or 0.5 mg/kg body weight, forty-eight hours prior to the investigation. The ratio between the red sensitizer emission and the bluish tissue autofluorescence provided excellent demarcation between papillary tumors and normal bladder wall. Certain cases of dysplasia also could be differentiated from normal mucosa. Benign exophytic lesions such as malakoplakia appeared different from malignant tumors in fluorescence. Flat suspicious bladder mucosa such as seen in infectious diseases or after radiation therapy appeared normal on fluorescence.

Fluorescence guided cystoscopy with 5-amino levulinic acid (ALA) or its hexyl derivative (h-ALA, Hexvix®) has been shown to increase bladder cancer diagnosis and offer the possibility to perform a more complete resection, thereby reducing the recurrence and progression rate. However, aspects of this procedure could be further improved. The fast photobleaching of the fluorophore protoporphyrin IX (PpIX) limits the visualization during cystoscopy and the specificity is low. We thus investigated the possibility to use dendrimers ALA in order to overcome these drawbacks. We have previously synthesized a dendrimer containing 18 ALA molecules (18-ALA) that provoked a sustained synthesis of PpIX following IP administration. We investigated the in vitro hydrolysis of ALA, the PpIX synthesis rate as well as the photobleaching rate of equimolar concentrations of ALA, h-ALA and 18-ALA. We further instilled 18-ALA intravesically in rats, bearing orthotopic bladder tumors. Frozen sections were analyzed with fluorescence microscopy. We observed a very slow mono-exponential hydrolysis of the dendrimers (k 6.87 h−1), as opposed to the rather fast and bi-exponential liberation of free ALA from Hexvix® (k1 32.06 h−1, k2 3.88 h−1). This can be attributed to steric hindrance and limited access to esterases. Only ALA esters induce PpIX synthesis after withdrawal of the produg. Due to the slow hydrolysis of dendrimers, a minimal concentration is needed to obtain a sustained release. PpIX synthesis is continuous for the first 24 H for both h-ALA and 18-ALA but rapidly decreases thereafter for h-ALA, whereas PPIX is still synthesized after several days for dendrimers. This is compatible with the very slow but prolonged hydrolysis and production of free ALA. This continuous synthesis of PpIX in itself would thus ensure a more prolonged presence of fluorescence during cystoscopy since the tumor cells having endocytosed the dendrimer keep producing the fluorophore. Furthermore, when comparing in vitro photobleaching of h-ALA and 18-ALA, we observe a slower photobleaching of dendrimer induced PpIX. We futher investigated PpIX synthesis following intravesical instillation of 18-ALA. One hour instillation of dendrimers is sufficient to enable endocytosis of the prodrug but a minimum of 3 H resting time is required to observe fluorescence. Unlike ALA or other derivatives administered to rats, dendrimers induce a tumor vs. normal urothelium fluorescence ratio of 3, with a tumor to muscle ratio superior to 7. Intravesical administration of ALA dendrimers in rats results in a PpIX synthesis which is much more selective towards the tumor as opposed to ALA or h-ALA. In vitro experiments show that there is a prolonged and sustained PpIX synthesis with a reduced photobleaching as compared to h-ALA induced PpIX. These are primary indicators that ALA dendrimers could possibly overcome drawbacks of ALA / h-ALA fluorescence guided cystoscopy Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 498.

Neodymium-Yag (Nd:YAG) laser irradiation for superficial bladder cancer is a valuable and safe treatment modality. The local recurrence rate is significantly reduced as compared to transurethral resection (TUR) alone (2.6 % versus 14.3 %). Remote recurrences can also significantly be reduced in solitary and multiple tumors if biopsies of the tumor are avoided. We state that the macroscopic appearance of a tumor is sufficiently reliable since only 0.67 % of the pathology proven Ti tumors were understaged macroscopically. The recurrence free interval for solitary tumors after Nd:YAG laser irradiation alone without biopsy was 19.i months as compared to i6 months after TUR with adjuvant intravesical instilations and in multiple tumors i3.6 months for Nd YAG laser irradiation alone without biopsies as compared to iO.4 months after TUR.

We report on 15 patients with multifocal carcinoma in situ of the bladder, treated with whole bladder wall photodynamic therapy (PDT). The total light dose, measured in situ (scattered plus nonscattered light) was 100 J/cm2 in the first six patients and 75 J/cm2 in the remaining nine patients. Follow-up ranges were from 6 to 27 months (average 15 months). Two cystectomies had to be performed in the first treatment group because of permanent shrunk bladders. Pathology of the resection specimens showed extensive granulation and fibrosis throughout the whole bladder wall. In the second treatment group, the maximal bladder capacity measured three months after PDT had increased on the average of 63% compared to the initial pretreatment values. No increased fibrosis could be detected on microscopical examination of random biopsies. Four recurrences necessitated cystectomy after 5 to 9 months, two in each treatment group. Three out of these originated in patients with a previous history of invasive bladder cancer. The preliminary data demonstrate the importance of in-situ light dosimetry for minimizing local side effects of PDT as well as the importance of strict inclusion criteria to optimize the therapeutic ratio.

A point-monitoring fluorescence diagnostic system based on a low-energy pulsed laser, fiber transmission optics, and an optical multichannel analyzer was used for diagnosis of patients with bladder malignancies. Twenty-four patients with bladder carcinoma, carcinoma in situ, and/or dysplasia were injected with Photofrin (0.35 or 0.5 mg/kg body weight) 48 hours prior to the investigation. The ratio between the red sensitizer emission and the bluish tissue autofluorescence provided excellent demarcation between papillary tumors and normal bladder wall. Certain cases of dysplasia could be also be differentiated from normal mucosa. Benign exofytic lesion such as malakoplakia appeared different from malignant tumors in fluorescence. Flat suspicious bladder mucosa such as that seen in infectious diseases or after radiation therapy appeared normal in terms of fluorescence.© (1991) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Two series of investigations utilizing laser-induced fluorescence (LIF) in characterizing diseased tissue are presented. In one in vitro investigation the fluorescence from normal and atherosclerotically diseased arteries are studied. In another clinical study the fluorescence in vivo from superficial urinary bladder malignancies in patients who had received a low-dose injection of Hematoporphyrin Derivative (HpD) is investigated. Additionally, the fluorescence properties of L-tryptophan, collagen-I powder, elastin powder, nicotinamide adenine dinucleotide and (beta) -carothene were investigated and compared with the spectra from the tissue samples. A nitrogen laser (337 nm) alone or in connection with a dye laser (405 nm) was used together with an optical multichannel analyzer (OMA) to study the fluorescence spectra. The fluorescence decay characteristics of atherosclerotic plaque were examined utilizing a mode locked argon ion laser, synchronously pumping a picosecond dye laser. A fast detection system based on photon counting was employed. The fluorescence decay curves were evaluated on a PC computer allowing up to three lifetime components to be determined. A fluorescence peak at 390 nm in fibrotic plaque was identified as due to collagen fibers, while a fluorescence peak at 520 nm was connected to (beta) -carotene. The in vivo measurements of urinary bladder malignancies were performed with the optical fiber of the OMA system inserted through the biopsy channel of a cystoscope during the diagnostical procedure. The spectral recordings from urinary bladders, obtained at 337 nm and 405 nm excitation, revealed fluorescence features which can be used to demarcate tumor areas from normal mucosa. The fluorescence emission might also be useful to characterize different degrees of dysplasia.

Purpose: We analyzed the value of the Bard bladder tumor antigen (BTA* *Bard Urological, Covington, Georgia.) test for the diagnosis of stage Ta superficial bladder cancer and carcinoma in situ, and compared it to the highly sensitive bladder washing cytology.Materials and Methods: The BTA test is a latex agglutination test that qualitatively detects the presence of basement membrane complexes in the urine. A total of 60 patients with superficial bladder cancer underwent voided urine BTA analysis and bladder washing cytologies.Results: Of the patients 65 percent were correctly diagnosed with the BTA test compared to 32 percent with bladder washings, which is statistically significant (p less than 0.001).Conclusions: The BTA test is a noninvasive diagnostic tool that is superior to bladder washing cytology for diagnosing superficial bladder cancer.