Marie-Ange D'Hallewin

Hypericin-based fluorescence diagnosis of bladder carcinoma

Objective To determine the use of hypericin instillation for the fluorescent detection of papillary bladder cancer and carcinoma in situ.

Biodistribution of hypericin in orthotopic transitional cell carcinoma bladder tumors: Implication for whole bladder wall photodynamic therapy

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 μM) 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 μM) 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.

Whole bladder wall photodynamic therapy of transitional cell carcinoma rat bladder tumors using intravesically administered hypericin

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.

Fluorescence detection of flat transitional cell carcinoma after intravesical instillation of aminolevulinic acid.

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.

Cellular Photodestruction Induced by Hypericin in AY-27 Rat Bladder Carcinoma Cells

Abstract In a recent clinical study we showed that hypericin accumulates selectively in urothelial lesions following intravesical administration of the compound to patients. In the present study the efficacy of hypericin as a photochemotherapeutic tool against urinary bladder carcinoma was investigated using the AY-27 cells (chemically induced rat bladder carcinoma cells). The uptake of hypericin by the cells increased by prolonging the incubation time and increasing the extracellular hypericin concentration. Photodynamic treatment of the cells incubated with 0.8 and 1.6 μM hypericin concentrations resulted in remarkable cytotoxic effects the extent of which depended on the fluence rates. Photoactivation of 1.6 μM hypericin by 0.5, 1.0 or 2.0 mW/cm2 for 15 min resulted in 3, 30 and 95% of the antiproliferative effect, respectively. Increasing the photoactivating light dose from 0.45 to 3.6 J/cm2 resulted in a five-fold increase in hypericin photodynamic activity. Irrespective of the fluence rates and irradiation times incubation of the cells with 10 μM hypericin induced rapid and extensive cell death in all conditions. The type of cell death (apoptosis or necrosis) induced by photoactivated hypericin depended largely on the hypericin concentration and the postirradiation time. At lower hypericin concentrations and shorter postirradiation times apoptosis was the prominent mode of cell death; increasing the hypericin concentration and/or prolonging the postirradiation time resulted in increased necrotic cell death. Cell pretreatment with the singlet oxygen quencher histidine, but not with the free-radical quenchers, significantly protected the cells from photoactivated hypericin–induced apoptosis, at least when a relatively low concentration (1.25 μM) was used. This result suggests the involvement of a Type-II photosensitization process. However, cells treated with higher hypericin concentrations (2.5–5 μM) were inadequately protected by histidine. Since hypericin is thus shown to be a potent and efficient photosensitizer, and since the conditions used were the same as when hypericin is used clinically to locate early-stage urothelial carcinoma lesions, hypericin may well become very important for the photodynamic treatment of superficial bladder carcinoma.

A Novel Orthotopic Bladder Tumor Model with Predictable Localization of a Solitary Tumor

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 pre-conditioning 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.

Fluorescence characteristics of atherosclerotic plaque and malignant tumors

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.

Tissue characterization in some clinical specialities utilizing laser-induced fluorescence

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.

The Human Umbilical Vein Graft in Below-Knee Femoropopliteal and Femorotibial Surgery: an Eight Year Experience

The authors present a series of 175 femoropopliteal (below-knee) and 65 femorotibial reconstructions with the human umbilical vein (HUV) graft performed over an eight year period. With a mean follow-up of 36.7 months (range one month to 84 months) the early patency rate of 89% decreased to 54% after five years. Long-term patency was found to be primarily related to the location of the distal anastomosis and the quality of the outflow, showing a statistically significant decrease after repeat revascularization. Early thrombosis, even in the absence of a technical failure and late aneurysmal degeneration remain the major problems associated with the use of the HUV graft. It is therefore recommended that these reconstructions be preserved for patients with advanced ischemia or a limited life-expectancy.

Clinical fluorescence diagnosis of human bladder carcinoma following low-dose photofrin injection

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.