Macro-microscopic fluorescence of human bladder cancer using hypericin fluorescence cystoscopy and laser confocal microscopy

The early detection of carcinoma is very essential for the diagnosis and prognosis of a bladder cancer patient. In this study we have investigated the use of hypericin as a fluorescent tumour marker and laser confocal microscopy as a diagnostic tool to aid the diagnosis of such cancers. Both cellular and clinical studies have been conducted. In the cellular studies, we have compared two bladder cell lines for the uptake and sub-cellular localization of hypericin. It was found that there was a rapid uptake and clearance of hypericin and significant localization in mitochondria and lysosomes. The study also revealed that there was a time-dependent increase in fluorescence intensity in bladder cells. The optimum localization was found to be 2-4 h post drug incubation. In the clinical study, consisting of 30 patients, both white light and fluorescence cystoscopy were performed after hypericin instillation. Biopsies taken from fluorescing regions were imaged using the confocal microscope. The order of fluorescence was detected to be as follows: normal < inflammation < grade 1 < grade 2 < CIS < grade 3. It was also found that the fluorescence intensity increased with the stage of the disease thereby enabling the determination of the degree of invasiveness of cancer. This enables the use of hypericin as a prognostic marker and laser confocal microscopy as a tool to aid in diagnosis of bladder cancer.     

Fluorescence detection of bladder cancer using urine cytology

Bladder cancer is the fourth most common malignant disease worldwide, accounting for 4% of all cancer cases. In Singapore, it is the ninth most common form of cancer. The high mortality rate in bladder cancer can be reduced by early treatment following pre-cancerous screening. Currently, the gold standard for screening bladder tumors is histological examination of biopsy specimens, which is both invasive and time-consuming. In this study, ex vivo urine fluorescence cytology was investigated to offer an alternative timely and biopsy-free means for detecting bladder cancers. Sediments in patient urine samples were extracted and incubated with a novel photosensitizer, hypericin. Laser confocal microscopy was used to capture the fluorescence images at an excitation wavelength of 488 nm. Images were subsequently processed to single out the exfoliated bladder cancer cells from the other cells based on the cellular size. Intensity histograms of each targeted cell and feature vectors, derived from the histogram moments, were used to represent each sample. A difference in the distribution of the feature vectors of normal and low-grade cancerous bladder cancer cells were observed. A diagnostic algorithm for discriminating between normal and low-grade cancerous cells is elucidated in this report. This study suggests that the fluorescence intensity profiles of hypericin in bladder cells can potentially provide an automated quantitative means of early bladder cancer diagnosis.     

Photodynamic selectivity of 5-aminolevulinic acid to prostate cancer cells

OBJECTIVE: To determine the selectivity of 5-aminolevulinic acid (5-ALA) as a photosensitizer to malignant prostatic cells in men undergoing radical retropubic prostatectomy. PATIENTS AND METHODS: Nineteen patients with localized prostate cancer were included in the study. Eighteen patients received 5-ALA and one patient did not receive it and was used as a control. The dose was 20mg /kg body weight, 15 patients received 5-ALA 4 hours before radical prostatectomy, two patients received it 2 hours before prostatectomy through a Ryle tube, and one patient received 5-ALA 12 hours before the operation. The removed prostates were examined for protoporphyrin IX (PpIX) fluorescence macroscopically, by fluorescence microscopy and by light microscopy. RESULTS: All carcinomas showed a clear evidence of PpIX-enrichment except in the control case. The enrichments were strong (++) in 15 cases and weak (+) in 3 cases. Two of those three cases were given 5-ALA two hours through a Ryle tube before excision of the prostate as well as the patient who was given 5-ALA 12 hours preoperatively. No PpIX enrichment was observed in the stroma of the prostate gland or in the benign tissue sections in any case (0/19). CONCLUSION: Oral 5-ALA is selectively concentrated in malignant cells of the prostate. This may lead to the clinical application of photodynamic therapy for localized prostate cancer.     

Photodynamic therapy of vulvar intraepithelial neoplasia using 5-aminolevulinic acid

Photodynamic (PDT) therapy is a relatively new technique with unique properties that make it attractive for the local treatment of superficial epithelial disorders. The objective of this study was to investigate the clinical response of PDT with the photosensitizing agent 5-aminolevulinic acid (5-ALA) in patients with vulvar intraepithelial neoplasia (VIN) grades 1 to 3. Twenty-five patients with 111 lesions of VIN 1-3 were topically sensitized with 10 ml of a 20% solution of 5-ALA and treated with 57 cycles of laser light at 635 nm (100 J/cm(2)). Seventy (64%) of the 111 VIN lesions regressed after various PDT cycles. A complete response was achieved in 13 patients (52%) with 27 lesions. All patients with VIN 1 and mono- and bifocal VIN 2-3 showed complete clearance. However, a complete response could be achieved in only 4 (27%) of 15 women with multifocal VIN 2-3, whereas a partial response was noted in 9 of these patients with a total of 70 lesions, out of which 44 (63%) lesions disappeared. No response was seen in 2 patients with multifocal VIN 3. Histological assessment of the fluorescence-directed biopsies revealed that increased pigmentation and hyperkeratosis of the lesions were associated with low response rates. PDT using 5-ALA represents an alternative treatment modality for VIN which is easy to perform and has the advantage of minimal tissue destruction, low side effects and excellent cosmetic results. However, multifocal VIN disease with pigmented and hyperkeratinic lesions remains difficult to treat.     

Clinical significance of macroscopic 5-aminolevulinic acid (ALA)-inducer fluorescence in transurethral resection of non-invasive bladder cancer

Macroscopic fluorescence which is induced with aminolevulinic acid (ALA) allows visualizing of small flat tumors, carcinoma in situ, true neoplasm margins and dysplasias of the bladder. Following ALA instillation, cystoscopy was performed under both standard and blue light illumination. In a prospective randomized multicenter study, 102 patients underwent TUR of bladder tumor(s) either with white light or ALA-fluorescence. Significant reduction in the number of residual tumours was detected in 59% (p = 0.005) after 8 weeks, 3 months--in 58% (p = 0.002) and 6 months in 38% (p = 0.01) respectively.     

A study of 5-aminolevulinic acid and its methyl ester used in in vitro and in vivo systems of human bladder cancer

The use of 5-aminolevulinic acid and its esters to induce endogenous porphyrins for the purpose of detection of epithelial cancers is being studied extensively in many centres around the world. The challenge is to prepare an efficacious formulation for the purpose of cancer detection. Photodynamic diagnosis of cancer using 5-aminolevulinic acid (ALA) and its ester derivatives is being actively investigated. In this study, we compared ALA with ALA methyl ester (AME) derivative in terms of PpIX fluorescence intensity in in vitro and in vivo systems of bladder carcinoma. For the in vivo system consisting of RT112 xenografts, the modes of drug administration compared were intravenous administration and topical application. The Karl Storz fluorescence endoscopy system was used to obtain macroscopic fluorescence images. The macroscopic images were further analysed for fluorescence intensity distribution. For the intravenous administration, over all time points studied (1, 3, 6 h), AME-PpIX fluorescence was lower than ALA-PpIX fluorescence and was cleared at a faster rate than the ALA-PpIX when administered intravenously. Topical application with two different polymers, Gantrez and Polyvinyl pyrrolidone (PVP) which are fast releasing polymers was found to be comparable in inducing PpIX fluorescence. Topical AME-PpIX fluorescence was found to be comparable with ALA-PpIX fluorescence. The results of this study suggest that the AME can also be used as a good diagnostic agent.     

Comparison between intravesical and oral administration of 5-aminolevulinic acid in the clinical benefit of photodynamic diagnosis for nonmuscle invasive bladder cancer

BACKGROUND:

This study was undertaken to evaluate the clinical value of photodynamic diagnosis (PDD) with intravesical and oral instillation of 5‐aminolevulinic acid (ALA) (ALA‐PDD), and transurethral resection of bladder tumor (TURBT) guided by ALA‐PDD (PDD‐TURBT) for nonmuscle invasive bladder cancer.

METHODS:

Of all 210 cases, 75 underwent PDD with intravesically applied ALA, and 135 cases underwent PDD with orally applied ALA. Diagnostic accuracy was evaluated by comparing the level on images of ALA‐induced fluorescence with the pathological result. PDD‐TURBT was performed in 99 completely resectable cases corresponding to 210 ALA‐PDD cases. To evaluate the abilities of PDD‐TURBT, survival analysis regarding intravesical recurrence was retrospectively compared with the historical control cases that underwent conventional TURBT.

RESULTS:

The diagnostic accuracy and capability of ALA‐PDD were significantly superior to those of conventional endoscopic examination. Moreover, 72.1% of flat lesions, including dysplasia and carcinoma in situ, could be detected only by ALA‐PDD. The recurrence‐free survival rate in the cases that underwent PDD‐TURBT was significantly higher than that of conventional TURBT. Moreover, multivariate analysis revealed that the only independent factor contributing to improving prognosis was PDD‐TURBT (hazard ratio, 0.578; P = .012). Regardless of the ALA administration route, there was no significant difference in diagnostic accuracy, ability of PDD, or recurrence‐free survival. All procedures were well tolerated by all patients without any severe adverse events.

CONCLUSIONS:

This multicenter study is likely to be biased, because it is limited by the retrospective analysis. This study suggests that regardless of the ALA administration route, ALA‐PDD and PDD‐TURBT are remarkably helpful in detection and intraoperative navigation programs. Cancer 2012;. © 2011 American Cancer Society.     

Subcellular localization of sigma-2 receptors in breast cancer cells using two-photon and confocal microscopy

Sigma-2 receptor agonists have been shown to induce cell death via caspase-dependent and caspase-independent pathways. Unfortunately, there is little information regarding the molecular function of sigma-2 receptors that can explain these results. In this study, two fluorescent probes, SW107 and K05-138, were used to study the subcellular localization of sigma-2 receptors by two-photon and confocal microscopy. The results indicate that sigma-2 receptors colocalize with fluorescent markers of mitochondria, lysosomes, endoplasmic reticulum, and the plasma membrane in both EMT-6 mouse and MDA-MB-435 human breast cancer cells. The fluorescent probe, K05-138, was internalized rapidly, reaching a plateau of fluorescent intensity at 5 min. The internalization of K05-138 was reduced approximately 40% by phenylarsine oxide, an inhibitor of endocytosis. These data suggest that sigma-2 ligands are internalized, in part, by an endocytotic pathway. The localization of sigma-2 receptors in several organelles known to have a role in both caspase-dependent and caspase-independent pathways of cell death supports the conclusions of previous studies suggesting that sigma-2 receptor ligands should be evaluated as potential cancer chemotherapeutic agents.     

Comparisons of 5-aminolevulinic acid photodynamic therapy and after-loading radiotherapy in vivo in cervical cancer

Objective

To compare the differences between 5-aminolevulinic acid photodynamic therapy (5-ALA-PDT) with traditional after-loading radiotherapy in aspects of efficacies and side effects.

Materials and methods

MTT assay was adopted to detect the inhibitive effects of 5-ALA-PDT on Hela cells proliferation. Flow cytometry was used to analyze cell apoptosis. After establishment of human cervical cancer xenograft model, the comparisons between 5-ALA-PDT with radiotherapy were performed with respect to treatment efficacies (survival rate, body weight, and tumor volume) and side effects (appearance and behavior, ovarian endocrine functions, and skin lesion around the tumor).

Results

5-Aminolevulinic acid photodynamic therapy exerted killing effects on cervical cancer cells. Morphological changes and flow cytometric analyses indicated apoptosis to be one of the mechanisms for tumor growth suppression. Both proliferation inhibition and cell apoptosis showed dependency on photosensitizer concentration and irradiation intensity. Repeated photodynamic therapy presented stronger inhibitive effects on tumor growth compared to after-loading radiotherapy, while producing milder impairment of ovarian endocrine functions and skin lesions around the tumors.

Conclusions

5-Aminolevulinic acid photodynamic therapy has great potential to be an alternative treatment modality for cervical cancer.     

A novel detection strategy for living circulating tumor cells using 5-aminolevulinic acid

Most circulating tumor cell (CTC) detection methods have technical limitations, allowing the detection of only cells expressing epithelial antigens, and they cannot identify if the CTCs are alive or dead. Herein, we constructed a novel CTC detection system comprised of filter separation and 5-aminolevulinic acid (5-ALA)-based labeling, termed “Fs-ALA”. Blood specimens (7.5 mL) were subjected to this method. Cells enriched on the filter were incubated with 5-ALA and Hoechst 33342 as positive markers for CTCs. Images of the whole filter surface were obtained using a fluorescence microscope. No 5-ALA positive cells were detected in healthy blood specimens. The Fs-ALA method was capable of detecting not only EpCAM-positive, but also EpCAM-negative tumor cells. In the Fs-ALA method, one or more CTCs were detected in samples from 13 of 18 (72.2%) colorectal cancer patients. The Fs-ALA method had a significantly higher CTC detection rate than CellSearch™ in colorectal cancer patients (P < 0.05), and only the former was capable of identifying live cells. This method is highly efficient for detecting CTC populations having undergone phenotypic changes, such as epithelial–mesenchymal transition.     

Pathological analysis of the surgical margins of resected glioblastomas excised using photodynamic visualization with both 5-aminolevulinic acid and fluorescein sodium

During glioma resection, 5-aminolevulinic acid (5-ALA) and fluorescein sodium (Fl-Na) are used for photodynamic tumor visualization. The objective of this study was to evaluate the pathological findings of the boundary zone between the tumor and adjacent normal brain in glioblastoma patients undergoing simultaneous double staining with 5-ALA and Fl-Na during surgery. Eight patients received 5-ALA (20 mg/kg orally) before the induction of general anesthesia, and Fl-Na (20 mg/kg) was administered intravenously before the dural incision was performed. The tumor bulk was removed under the guidance of Fl-Na staining alone using conventional white light. Subsequently, residual tumor was removed under the guidance of both fluorescent agents within functionally safe limits until both were visibly undetectable. Twenty specimens exhibiting different staining intensities of both agents were obtained. The vessel index (VI) was calculated from CD31 immunohistochemistry (IHC) samples. Boundary zone tumor cells were detected by IHC for olig2, and were expressed as the olig2 index (OLI). The VI was significantly higher in Fl-Na-positive areas than in Fl-Na-negative areas (p?=?0.0005). In contrast, the OLI was significantly higher in 5-ALA-positive areas than in 5-ALA-negative areas (p?=?0.0149). 5-ALA-positive/Fl-Na negative areas were observed in 7 patients. These findings indicate that Fl-Na accumulates in areas with a disrupted blood–brain barrier, and that 5-ALA fluorescence is dependent on tumor cell protoporphyrin IX metabolism. In conclusion, 5-ALA was better for detecting tumor cells in the boundary zone than was Fl-Na. Of note, tumor cells existed outside the fluorescence-stained boundaries of both agents.     

GFP image analysis in the mouse orthotopic bladder cancer model

Precise and objective measurements of tumor response have yet to be standardized in the mouse orthotopic bladder cancer model. In this study, we used image analysis and green fluorescent protein (GFP) to objectively measure tumor size in response to chemotherapy. KU-7 human bladder cancer cells transfected with GFP were intravesically inoculated into 8-week-old female nude mice. Fourteen days after tumor cell inoculation, the mice were assigned into a control (PBS) group or a doxorubicin (conc. 1.0 mg/ml) treatment group and received a single instillation of treatment. Fourteen days after treatment, the bladders were surgically exposed and fluorescent images were captured and later analyzed using image analysis. Bladders were processed for histological examination. Tumor incidence determined by GFP expression and histology was 100 and 80%, respectively, in the doxorubicin treatment group. A 9-fold (histology) vs. 12-fold (GFP expression) difference in tumor regression measured by tumor area (P<0.05) and a 5-fold (histology) vs. 9-fold (GFP expression) difference in tumor regression measured by the percent of tumor area in the bladder (P<0.001) were observed in the doxorubicin treatment group. Our findings suggest that using image analysis provides a precise, sensitive and objective means to measure tumor growth and treatment response in the mouse orthotopic bladder cancer model in lieu of histological methods. Consequently, the number of mice required in an experiment can be reduced since tissue samples are not needed for histology, thus making tissue samples readily available for additional assays in both a labor-effective and cost-effective manner.     

Photodynamic therapy in women with cervical intraepithelial neoplasia using topically applied 5-aminolevulinic acid

Photodynamic therapy (PDT) is a novel treatment modality that produces local tissue necrosis with laser light after prior administration of a photosensitizing agent. We performed a study of topically applied 5-aminolevulinic acid (5-ALA) in the photodynamic treatment of women with high-grade cervical intraepithelial neoplasia (CIN) using fixed 5-ALA doses and application protocols derived from previous in vitro and in vivo results. Three to 5 hr prior to PDT, 10 ml of a 20% solution of 5-ALA was topically applied using a cervical cap. PDT was performed with irradiation of 100 J/cm2 at an irradiance of 100-150 mW/cm2 with an argon-ion-pumped dye laser at 635 nm. For the endocervix, a specifically designed cylindrical applicator was used. Ten treatment cycles of PDT using 5-ALA were performed in 7 patients with high-grade CIN. Non-thermal laser treatment with 100-150 mW/cm2 was well tolerated. Local toxicity was minor as several patients reported burning sensations and vaginal discharge, but no necrosis, sloughing or scarring occurred. After 3 months, a significant reduction in the size of the ectocervical CIN lesions was noted in only 3 patients, who underwent a second PDT cycle. However, no significant improvement in CIN lesions was noted since cold knife conization revealed persistent CIN in all 7 cases. Therefore, PDT after topical application of 5-ALA using an irradiation of 100 J/cm2 produces only minimal side effects. However, it does not appear to be effective in treating CIN.     

Antifungal effect of 5-aminolevulinic acid PDT in Trichophyton rubrum

In the present investigation, we have shown for the first time that the onychomycosis-inducing dermatophyte Trichophyton rubrum was able to metabolize 5-aminolevulinic acid (ALA) to protoporphyrin IX (PpIX) in liquid culture medium. We have established and optimized the culture conditions and could show the typical PpIX-induced red fluorescence which was evaluated qualitatively by Wood's light examination and fluorescent microscopic analysis. The optimum concentration of ALA was in the range of 1-10 mmol l(-1). If used in higher concentrations, ALA leads to a significantly reduced growth rate and absence of PpIX formation due to highly acidic conditions. The first observation of red fluorescence was detected between 10 and 14 days poststimulation with ALA, increasing thereafter. Fluorescent microscopic examinations demonstrated that formation of PpIX was restricted to selected parts of the fungal mycelium. Repeated application of ALA in order to achieve the highest formation of PpIX in T. rubrum failed, probably due to the sustained low pH values. ALA treatment and irradiation of T. rubrum clearly demonstrated the growth-inhibiting effect of ALA PDT, either leading to reduced numbers of colonies or reduced diameters of single fungal colonies. Summarizing our results, ALA PDT might be a promising approach in the reduction of T. rubrum colonization in onychomycosis.     

Photodynamic therapy of residual or recurrent basal cell carcinoma after radiotherapy using topical 5-aminolevulinic acid or methylester aminolevulinic acid

This study was conducted to assess the efficacy of aminolevulinic acid-based photodynamic treatment for residual or recurrent basal cell carcinomas after radiotherapy. Photodynamic therapy with either topical 5-aminolevulinic acid 20% and dimethylsulfoxide 99% pretreatment or topical methylester aminolevulinic acid 20% w/w in cream and subsequent light illumination of 50-200 J/cm² was performed after an initial skin shaving procedure in 20 patients with 22 residual or recurrent basal cell carcinomas. Three lesions were treated once, while twelve, three, one and two lesions received two, three, four and five treatment sessions respectively. At examination, 6-40 months (mean 22 months) after the last treatment, 18 lesions were in complete remission. All lesions were considered excellent or good with regard to cosmetic outcome. Three lesions responded only partially to photodynamic therapy and a fourth lesion recurred 21 months after photodynamic treatment. Two of these four lesions were confirmed as the morpheaform type of basal cell carcinoma.     

Photobleaching of protoporphyrin IX in cells incubated with 5-aminolevulinic acid

Protoporphyrin IX (Pp IX) is the main photosensitizer in photochemotherapy with 5-aminolevulinic acid (ALA). Pp IX is photolabile and the present work shows that 70–95% of Pp IX in cells is degraded by clinically relevant light exposures (40–200 J cm⁻² at 630 nm). During light exposure a small yield of photoprotoporphyrin, which is also photolabile, is formed. A substantial fraction of Pp IX in cells incubated with ALA is bound to proteins. During light exposure these binding sites are destroyed, those close to tryptophan residues being the most sensitive. The rate of photodegradation of Pp IX in the cells is dependent on the initial concentration of Pp IX. The degradation mechanisms are therefore not only first order processes. Different degradation rates appear to be related to different types of binding sites. During light exposure, Pp IX molecules appear to move to different binding sites, evidently sites that are more vital for cell survival. Thus, the yield of photoinactivation of the cells, as measured per emitted photon of Pp IX fluorescence, increased during light exposure. © 1997 Wiley-Liss, Inc.     

5-aminolevulinic acid photodynamic therapy for the treatment of high-grade gliomas

Journal of Neuro-Oncology - In the original article, the author names were published incorrectly. The names are correct in this publication.