5-氨基乙酰丙酸介导的光动力学对裸鼠人胃癌移植瘤的治疗作用

目的观察5-氨基乙酰丙酸（5-ALA）介导的光动力学治疗对裸鼠人胃癌移植瘤的治疗作用。探索5-ALA介导的光动力学（PDT）治疗胃癌的可能机制。方法以MGC-803人胃癌细胞制备裸鼠人胃癌移植瘤模型：整体荧光成像系统下观察动物瘤体发出的荧光信号：测量荷瘤对照组、单纯激光组、单纯5-ALA组和5-ALA介导的PDT治疗组治疗后第1、3、7、14、21天的裸鼠肿瘤大小并计算肿瘤体积：末次测量后切除肿瘤行病理检查和透射电镜观察，TUNEL法检测组织细胞凋亡。结果常规方法培养MGC一803人胃癌细胞并接种裸鼠．可以成功制备出裸鼠人胃癌移植瘤的模型：荷瘤裸鼠肿瘤组织在特定波长的荧光激发下可发出特有的红色荧光。荷瘤对照组、单纯激光组、单纯5-ALA组和5-ALA介导的PDT治疗组治疗后第1、3、7、14、21天4组间的肿瘤体积差异有统计学意义（肚1003．086，P=0．000），PDT治疗组明显小于其他3组；病理检查提示．PDT治疗后肿瘤细胞大片凝固性坏死；透射电镜观察提示，PDT治疗组肿瘤组织存在大量死亡细胞及部分凋亡细胞：TUNEL法检测发现，PDT治疗组的肿瘤组织凋亡指数显著高于其他3组（χ^2=18．237．P=0．000）。结论5-ALA介导的PDT对于裸鼠人胃癌移植瘤具有明显的治疗效果．而单纯给予5-ALA或激光辐射对肿瘤无明显抑制作用；肿瘤细胞的坏死及凋亡是5-ALA介导的PDT产生细胞毒作用的重要机制。     

Protoporphyrin IX fluorescence photobleaching is a useful tool to predict the response of rat ovarian cancer following hexaminolevulinate photodynamic therapy

OBJECTIVE: Accurate dosimetry was shown to be critical to achieve effective photodynamic therapy (PDT). This study aimed to assess the reliability of in vivo protoporphyrin IX (PpIX) fluorescence photobleaching as a predictive tool of the hexaminolevulinate PDT (HAL-PDT) response in a rat model of advanced ovarian cancer. MATERIALS AND METHODS: Intraperitoneal 10(6) NuTu 19 cells were injected in 26 female rats Fisher 344. Peritoneal carcinomatosis was obtained 26 days post-tumor induction. Four hours post-intraperitoneal HAL (Photocure ASA, Oslo, Norway) injection, a laparoscopic procedure (D-light AutoFluorescence system, Karl Storz endoscope, Tuttlingen, Germany) and a fluorescence examination were made for 22 rats. The first group (LASER group, n=26) was illuminated with laser light using a 532 nm KTP laser (Laser Quantum, Stockport, UK) on 1 cm(2) surface at 45 J/cm(2). The second group (NO LASER group, n=26) served as controls. Biopsies were taken 24 hours after PDT. Semi-quantitative histology was performed and necrosis value was determined: 0--no necrosis to 4--full necrosis. Fluorescence was monitored before and after illumination on complete responders (NV=3-4; n=20) and non-responders (NV=0-2; n=6). RESULTS: High PpIX photobleaching corresponded with complete responders whereas low photobleaching corresponded with non-responders (P<0.05). A direct linear correlation was shown between photobleaching and necrosis (R(2)=0.89). CONCLUSION: In vivo PpIX fluorescence photobleaching is useful to predict the tissue response to HAL-PDT.     

Time-dependent hexaminolaevulinate induced protoporphyrin IX distribution after topical application in patients with cervical intraepithelial neoplasia: A fluorescence microscopy study

BACKGROUND AND OBJECTIVES: Compared to the conventional management of cervical intraepithelial neoplasia (CIN) the potential advantage of photodynamic therapy (PDT) for the treatment of cervical human papilloma virus (HPV)-related disease encompasses a minimal invasive procedure with reduced risk of profuse bleeding as a consequence of conization, and possibly more favorable long-term results avoiding cervical stenosis. At present little is known about the precise time-dependent distribution and histological localization of hexaminolaevulinate (HAL) induced protoporphyrin IX (PPIX) fluorescence in healthy tissue and in CIN. The aim of this study was to use ex vivo fluorescence microscopy to determine whether PPIX is selectively induced by neoplastic cells of the cervical epithelium at various times after topical application. STUDY DESIGN/MATERIALS AND METHODS: Cold cream containing 0.5% HAL was applied by means of cervical cap over various periods of time. We analyzed 52 healthy cervical mucosa and 84 CINs. RESULTS: At time delay 100 (+/-10) minutes, high epithelial fluorescence and a significant selectivity between epithelium and underlying lamina propria was found. By contrast, no significant difference between healthy and neoplastic tissues, or between low and high-grade epithelial dysplasia (P > or = 0.05), was observed at any time point. CONCLUSIONS: Application of HAL 0.5% cream to the cervix induced selective fluorescence in epithelial cells. The optimal ratio with a homogeneous PPIX distribution was obtained after 100 ( +/- 10) minutes cream application, which should be evaluated further for PDT.     

Fluorescence staining of laryngeal neoplasms after topical application of 5-aminolevulinic acid: preliminary results

BACKGROUND AND OBJECTIVE: The prognosis of patients suffering from laryngeal carcinomas can be improved by early diagnosis. Exact demarcation of tumor margins could contribute to an optimum preservation of the larynx. Therefore, the aim of the present study was the evaluation of 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PPIX) fluorescence as a new diagnostic procedure for the detection of laryngeal cancer. STUDY DESIGN/MATERIALS AND METHODS: Sixteen patients with suspected malignancies of the larynx received 0.6 wt% 5-ALA-NaCl solution by means of a medical nebulizer. After a period of 1-2 hours, the patients underwent microlaryngoscopy under white light and fluorescence illumination (lambda(ex) = 375-440 nm). A quantitative analysis of the fluorescence contrast between neoplastic and surrounding tissue was performed using an optical multichannel analyzer. RESULTS: Carcinoma, carcinoma in situ, and dysplasia showed red fluorescence that could be attributed to the 5-ALA-induced formation of PPIX. The surrounding normal tissue exhibited autofluorescence in the green spectral range, which was greatly reduced within the tumor. The results of macroscopic red fluorescence staining were correlated with the histologic diagnosis. CONCLUSION: According to these preliminary results, the presented method seems to be a promising adjunct diagnostic procedure for the early identification of malignant neoplasms in the larynx. The aim of further investigations is the assessment of sensitivity and specificity and an evaluation of fluorescence-guided laser resections of laryngeal cancer. Lasers Med. Surg. 25:414-420, 1999.     

5-ALA for photodynamic photorejuvenation--optimization of treatment regime based on normal-skin fluorescence measurements

BACKGROUND AND OBJECTIVES: Photodynamic therapy using 20% 5 aminolevulinic acid (5-ALA) has recently been introduced as a new tool in optical skin rejuvenation. The primary objective of this study was to optimize incubation time, the topical delivery mechanism (vehicle) and the concentration of 5-ALA by detecting the dynamic changes of normal skin after 5-ALA application. The secondary objective was to develop a treatment regime which minimizes post-treatment photosensitivity. STUDY DESIGN/MATERIALS AND METHODS: Skin fluorescence distribution patterns after topical application of low concentrations of 5-ALA (0.5% and 1% preparations encapsulated in liposomes), were investigated. Twenty percent 5-ALA in moisturizing cream was used as a control. Ten healthy volunteers participated, and skin fluorescence was documented by fluorescent photography. The fluorescent intensity was measured in % of maximum obtained fluorescence after 3 hours 5-ALA application. RESULTS: Skin fluorescence intensity after topical application of 0.5% and 1% non-occluded liposome-encapsulated 5-ALA application was heterogeneous distributed and reached saturation level after approximate 2 hours. The maximal fluorescence for 0.5% and 1% 5-ALA treated areas was 4.2% (SD: 3.5%) and 2.4% (SD: 2%), respectively, and this difference was statistically significant (P = 0.036). The fluorescence decayed linearly shortly (within 15 minutes) after end of application and was back to baseline within 8 hours. In contrast, the fluorescence of areas treated more than 1 hour with 20% 5-ALA was very uniform and a linear relationship (r2 = 0.998) to the incubation time (0-3 hours) was registered. Furthermore, fluorescence intensity (15.2-57.9%) continued to increase after the end of 5-ALA application. The maximum fluorescence reach a level of 1.6-9 times the fluorescence measured by end of the 5-ALA application and occurred 8:13 hours (SD: 0:49 hours) after the end of 20% 5-ALA application. The average skin surface fluorescence induced by the liposome-encapsulated 0.5% 5-ALA applied for longer than 2 hours, was found to be statistically equal (P = 0.47) to the average measured skin surface fluorescence (4.2%) obtained after 30 minutes exposure to 20% 5-ALA cream (4.3%). CONCLUSION: Changing the 5-ALA vehicle from a moisturizing cream to liposome encapsulation, the 5-ALA concentration can be lowered by a factor of 40, and still induce the same skin fluorescence and at the same time eliminates the need for occlusion. The low post-treatment fluorescence also suggests a significantly reduced risk of post-treatment phototoxicity.     

Multiphoton excitation fluorescence microscopy of 5-aminolevulinic acid induced fluorescence in experimental gliomas

BACKGROUND AND OBJECTIVE: The clinical usefulness of 5-ALA guided detection of tumor tissue has been demonstrated for a number of malignancies. However, current techniques of intraoperative detection of protoporphyrin IX fluorescence in situ do not offer subcellular resolution. Therefore, discrimination of non-specific 5-ALA induced fluorescence remains difficult. MATERIALS AND METHODS: In this study we have used an orthotopic glioma model to analyze PpIX fluorescence in tumor tissue and normal brain by multiphoton excitation microscopy after intraperitoneal administration of 5-ALA. A DermaInspect in vivo imaging system was used for autofluorescence measurements at 750 nm excitation and detection in the green channel of a standard photomultiplier module. For detection of PpIX fluorescence at different excitation wavelengths a red sensitive version of the photomultiplier and a filter combination of short pass filters and a color glass long pass filter was used restricting the sensitivity in the red channel to a range of 580-700 nm. RESULTS: Multiphoton microscopy allowed a higher structural definition of tumor tissue based on the excitation of 5-ALA induced PpIX fluorescence compared to autofluorescence imaging. The high resolution of multiphoton microscopy allowed discrimination of fluorescence from the cytoplasm of tumor cells and 5-ALA induced PpIX fluorescence of normal brain parenchyma adjacent to tumor. Fluorescence lifetime imaging showed significantly longer fluorescence lifetimes of 5-ALA induced PpIX fluorescence in tumor tissue compared to normal brain. This allowed definition and visualization of the tumor/brain interface based on this parameter alone. CONCLUSION: Multiphoton microscopy of 5-ALA induced PpIX fluorescence in brain tumor tissue conceptually provides a high resolution diagnostic tool, which in addition to structural information may also provide photochemical/functional information.     

Sensitization and photodynamic therapy of normal pancreas,duodenum and bile ducts in the hamster using 5-aminolaevulinic acid

Photodynamic therapy (PDT) using 5-aminolaevulinic-acid-(ALA)-induced protoporphyrin IX (PPIX) increases survival in hamsters with pancreatic cancer. However, experiments with other photosensitizers on this model show a high risk of duodenal perforation. In this paper, the pharmacokinetics and PDT effects of ALA on normal tissues in the pancreatobiliary region are presented. Using quantitative fluorescence microscopy, maximum PPIX fluorescence was seen in the bile ducts, less in the duodenal mucosa and least in the muscularis propria and pancreas. For PDT, light was delivered either using a bare fibre touching the tissue (single-point illumination), or irradiating a 1.5 cm diameter circular area. Single-point PDT (50 J) produced only localized reversible damage without perforation. Surface irradiation of the whole periampullary region (50 J cm^–2) caused extensive damage, sometimes with perforation. Before PDT can be used safely to treat tumours of the pancreas and bile duct, further studies are necessary to understand its effect on larger areas of normal tissue.     

Photoirradiation therapy of experimental malignant glioma with 5-aminolevulinic acid

OBJECT: Accumulation of protoporphyrin IX (PPIX) in malignant gliomas is induced by 5-aminolevulinic acid (5-ALA). Because PPIX is a potent photosensitizer, the authors sought to discover whether its accumulation might be exploited for use in photoirradiation therapy of experimental brain tumors, without injuring normal or edematous brain. METHODS: Thirty rats underwent craniotomy and were randomized to the following groups: 1) photoirradiation of cortex (200 J/cm2, 635-nm argon-dye laser); 2) photoirradiation of cortex (200 J/cm2) 6 hours after intravenous administration of 5-ALA (100 mg/kg body weight); 3) cortical cold injury for edema induction; 4) cortical cold injury with simultaneous administration of 5-ALA (100 mg/kg body weight) and photoirradiation of cortex (200 J/cm2) 6 hours later; or 5) irradiation of cortex (200 J/cm2) 6 hours after intravenous administration of Photofrin II (5 mg/kg body weight). Tumors were induced by cortical inoculation of C6 cells and 9 days later, magnetic resonance (MR) images were obtained. On Day 10, animals were given 5-ALA (100 mg/kg body weight) and their brains were irradiated (100 J/cm2) 3 or 6 hours later. Seventy-two hours after irradiation, the brains were removed for histological examination. Irradiation of brains after administration of 5-ALA resulted in superficial cortical damage, the effects of which were not different from those of the irradiation alone. Induction of cold injury in combination with 5-ALA and irradiation slightly increased the depth of damage. In the group that received irradiation after intravenous administration of Photofrin II the depth of damage inflicted was significantly greater. The extent of damage in response to 5-ALA and irradiation in brains harboring C6 tumors corresponded to the extent of tumor determined from pretreatment MR images. CONCLUSIONS: Photoirradiation therapy in combination with 5-ALA appears to damage experimental brain tumors selectively, with negligible damage to normal or perifocal edematous tissue.     

重复光动力疗法治疗鼠C6胶质瘤

目的 观察5-氨基乙酰丙酸(5-ALA)作为光敏剂对大鼠C6胶质瘤移植瘤进行光动力疗法(PDT)的治疗作用.方法 建立大鼠胶质瘤模型,随机分成4组:A组按剂量20 mg/kg在瘤内注射5-ALA,2 h后在肿瘤局部行激光照射(单次PDT);B组操作与A组相同,但于第4、8天注射同等剂量5-ALA,重复PDT;C组给予单纯激光照射,不予任何光敏剂;D组为荷瘤对照组,不给任何治疗.治疗后测量肿瘤体积,绘制肿瘤生长曲线,以观察PDT及重复PDT对肿瘤生长的抑制情况.结果 重复光动力治疗组(B组)肿瘤体积与对照组(C、D组)比较明显缩小,比单次PDT(A组)亦较小,差异有统计学意义(P<0.05).结论 重复PDT比传统的单次PDT对大鼠C6胶质瘤移植瘤肿瘤的抑制效果更加明显.     

Ex vivo photodynamic diagnosis to detect malignant cells in oral brush biopsies

In this study we proved the efficiency of the fluorimetric detection of a minimum number of malignant cells ex vivo. The goal of this work was to investigate whether the combination of photodynamic diagnosis (PDD) with oral brush biopsy might become a suitable chair-side tool to detect early oral carcinoma. Small numbers (100–500) of established human tumour cells—small cell lung carcinoma (OAT 75), transitional cell carcinoma of the bladder (SW1710) and human embryonic kidney cells (HEK293)—were incubated with 2 mM 5-aminolaevulinic acid (5-ALA). In addition, 50 brush biopsies from volunteers were prepared. After 2 h and 3 h of incubation, all samples were investigated by spectrofluorometry. Measurements were performed in capillaries. For excitation (405 nm) and detection of fluorescence spectra, a fibre microprobe–spectrofluorometer system (fibre 400 μm) was used. A minimum of 100 malignant cells and 3 h of incubation with ALA were needed to detect a typical spectrum for protoporphyrin IX (PPIX). Some epithelial samples from brush biopsy showed strong (bacteria related) PPIX autofluorescence, which increased after the addition of 5-ALA. From the testing of various antibiotics and antiseptics it emerged that 0.4 mM chlorhexidine strongly reduced fluorescence in brush biopsies from healthy volunteers, whereas the fluorescence signal of established cancer cell lines decreased only a little. The experiments revealed that, by means of an optical microprobe, very few cancer cells (100) can be detected. The addition of chlorhexidine before the incubation of brush biopsies with 5-ALA increases the reliability of the test by largely reducing the autofluorescence signal due to the presence of bacteria. Chair-side diagnostics of epithelial carcinoma seem feasible.     

Interstitial photodynamic therapy of nonresectable malignant glioma recurrences using 5-aminolevulinic acid induced protoporphyrin IX

BACKGROUND AND OBJECTIVE: Limited knowledge of the light and temperature distribution within the target volume in combination with non-selective accumulation of the applied photosensitizers (PS) has hampered the clinical relevance of interstitial photodynamic therapy (iPDT) for treatment of malignant glioma patients. The current pilot study focused on the development and the clinical implementation of an accurate and reproducible irradiation scheme for iPDT using 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PPIX) as a selectively working PS. STUDY DESIGN/MATERIALS AND METHODS: Monte Carlo simulations of fluence rate and heat transport simulations were performed using the optical properties of normal brain tissue infiltrated by tumor cells (absorption coefficient micro(a) = 0.2 cm(-1), reduced scattering coefficient: micro'(s) = 20 cm(-1)). A modified 3-D treatment-planning software was used to calculate both, the treatment-volume and the exact position of the light diffusers within the lesion. The feasibility and the risk of iPDT were tested in 10 patients with small and circumscribed recurrent malignant gliomas. RESULTS: The optimum distance between the implanted light diffusers was determined to be 9 mm with regard to both fluence rate and temperature distribution. For this distance a temperature increase above 42 degrees C was not expected to occur. Up to six cylindrical light diffusers were stereotactically implanted to achieve a complete irradiation of the tumor volume, which was possible in every single patient (mean tumor volume: 5.9 cm3). The total applied light fluence was between 4,320 J and 11,520 J. Side effects of iPDT were not observed. Median survival was 15 months. CONCLUSION: 5-ALA iPDT in combination with a 3-D treatment-planning (which was based on optical and thermal simulations) is a safe and feasible treatment modality. The clinical impact of these findings deserves further prospective evaluation.     

Fluorescence diagnosis: a novel method to guide radical inguinal lymph node dissection in penile cancer

In penile cancer there is still a diagnostic dilemma between over treatment of lymph node-negative patients and the missing of occult metastases by watchful waiting. In the current study the value of fluorescence diagnosis during radical inguinal lymph node dissection was evaluated. Five patients with penile cancer were elected to undergo groin dissection. All patients received 5-aminolevulinic acid (5-ALA) orally before the operation for fluorescence diagnosis. Intraoperatively, fluorescence detection of the lymph nodes was performed by visual detection and spectroscopy. Two of the five patients had positive inguinal lymph nodes. Fluorescence in tumor-bearing tissue was detectable in the exposed lymph nodes. Protoporphyrin IX (PPIX) is accumulated in tumor-positive lymph nodes, making fluorescence diagnosis in penile cancer possible. More studies with higher patient numbers are necessary to evaluate optimal dosage and excitation conditions to detect tumor-bearing nodes in vivo.     

Strong 5-aminolevulinic acid-induced fluorescence is a novel intraoperative marker for representative tissue samples in stereotactic brain tumor biopsies

Stereotactic biopsies represent a routine neurosurgical procedure for the diagnosis of intracranial lymphomas and selected diffusely infiltrating gliomas. Acquisition of tissue samples that do not allow correct tumor typing and grading is, however, not uncommon. Five-aminolevulinic acid (5-ALA) has been shown to accumulate in malignant tumor tissue. The aim of this study was to prospectively investigate the clinical usability of 5-ALA for intraoperative detection of representative tissue in stereotactic tumor biopsies. Fifty consecutive patients underwent frameless stereotactic biopsy for a suspected brain tumor. 5-ALA was administered 4 h before anesthesia. Serial biopsy samples were obtained and intraoperatively checked for 5-ALA fluorescence (strong, vague, or none) using a modified neurosurgical microscope. All samples were examined for the presence of representative tumor tissue according to neuroimaging (MRI, positron emission tomography, and/or chemical shift imaging) and histopathological parameters. Visible 5-ALA fluorescence was observed in 43/50 patients (strong in 39 and vague fluorescence in four cases). At biopsy target, 52/53 samples of glioblastomas, 9/10 samples of gliomas grade III, and 14/16 samples of lymphomas revealed strong 5-ALA fluorescence. Samples with strong 5-ALA fluorescence were only observed at, but not outside the biopsy target. All tissue samples with strong 5-ALA fluorescence were representative according to our neuroimaging and histopathological criteria (positive predictive value of 100%). Our data indicate that strong 5-ALA fluorescence is a reliable and immediately available intraoperative marker of representative tumor tissue of malignant gliomas and intracranial lymphomas in stereotactic biopsies. Thereby, the application of 5-ALA in stereotactic brain tumor biopsies may in future reduce costs for operating room and neuropathology and may decrease procedure-related morbidity.     

Fluorescence-guided resection of glioblastoma multiforme by using 5-aminolevulinic acid-induced porphyrins: a prospective study in 52 consecutive patients

OBJECT: It has been established that 5-aminolevulinic acid (5-ALA) induces the accumulation of fluorescent porphyrins in glioblastoma multiforme (GBM), a phenomenon potentially exploitable to guide tumor resection. In this study the authors analyze the influence of fluorescence-guided resection on postoperative magnetic resonance (MR) imaging and survival in a series of patients who underwent surgery in the authors' department. METHODS: Fifty-two consecutive patients with GBM received oral doses of 5-ALA (20 mg/kg body weight) 3 hours before induction of anesthesia. Intraoperatively, tumor fluorescence was visualized using a modified operating microscope. Fluorescing tissue was removed whenever it was considered safely possible. Residual enhancement on early postoperative MR imaging was quantified and related to each patient's characteristics to determine which factors influenced resection. Survival was analyzed using the Kaplan-Meier method and multivariate analysis was performed in which the Karnofsky Performance Scale (KPS) score, residual fluorescence, patient age, and residual enhancement on MR images were considered. Intraoperatively, two fluorescence qualities were perceived: solid fluorescence generally reflected coalescent tumor, whereas vague fluorescence mostly corresponded to infiltrative tumor. Complete resection of contrast-enhancing tumor was accomplished in 33 patients (63%). Residual intraoperative tissue fluorescence left unresected for safety reasons predicted residual enhancement on MR images in 18 of the 19 remaining patients. Age, residual solid fluorescence, and absence of contrast enhancement in MR imaging were independent explanatory factors for survival, whereas the KPS score was significant only in univariate analysis. No perioperative deaths and one case of permanent morbidity were encountered. CONCLUSIONS: The observations in this study indicate the usefulness of 5-ALA-induced tumor fluorescence for guiding tumor resection. The completeness of resection, as determined intraoperatively from residual tissue fluorescence, was related to postoperative MR imaging findings and to survival in patients suffering from GBM.     

Adriamycin enhanced in vitro and in vivo photodynamic therapy of mesothelioma.

The ability of Adriamycin (AD) to enhance the known in vitro and in vivo tumoricidal effects of photodynamic therapy (PDT) on the H-MESO-1 human malignant mesothelioma cell line was investigated. In vitro cytotoxicity was determined by incubating H-MESO-1 cells in microtiter plates (2 x 10(5) cells/well, 6 wells/group) with the photosensitizer Photofrin II (PF) and varying concentrations of AD (0, 2.5, 5.0, and 10.0 micrograms/ml) for 24 hr followed by exposure to gold vapor laser light (GVL) at a fluence of 6000 J/M2. [3H]Thymidine (1 microCi) was added to each well 24 hr after treatment. Cells were harvested and counted for thymidine incorporation 24 hr later. PDT alone resulted in a decrease in thymidine incorporation of 23% while the addition of AD to PDT at AD concentrations of 2.5, 5.0, and 10.0 micrograms/ml resulted in decreases of 62, 85, and 69%, respectively (P = 0.005) as compared to untreated controls. H-MESO-1 tumor bearing nude mice (n = 5) were injected ip with PF (5 mg/kg) and AD (5 mg/kg) 24 hr prior to illumination of the tumor site with GVL (120 J/cm2). Control groups (n = 5) received PDT, AD, and/or GVL alone. Tumor surface area was measured as the product of the greatest perpendicular dimensions every 5 days for 30 days. Administration of PDT without AD resulted in a decrease in tumor surface area of 50% on Day 10 with regrowth of tumor by Day 30 while AD alone with or without GVL had no impact on tumor growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of porphyrin fluorescence accumulation in pediatric brain tumor cells incubated in 5-aminolevulinic acid

Background

Fluorescence-guided surgery with 5-aminolevulinic acid (5-ALA) enables more complete resections of tumors in adults. 5-ALA elicits accumulation of fluorescent porphyrins in various cancerous tissues, which can be visualized using a modified neurosurgical microscope with blue light. Although this technique is well established in adults, it has not been investigated systematically in pediatric brain tumors. Specifically, it is unknown how quickly, how long, and to what extent various pediatric tumors accumulate fluorescence. The purpose of this study was to determine utility and time course of 5-ALA–induced fluorescence in typical pediatric brain tumors in vitro.

Methods

Cell cultures of medulloblastoma [DAOY and UW228], cPNET [PFSK] atypical teratoid rhabdoid tumor [BT16] and ependymoma [RES196] were incubated with 5-ALA for either 60 minutes or continuously. Porphyrin fluorescence intensities were determined using a fluorescence-activated cell sorter (FACS) after 1, 3, 6, 9, 12 and 24 hours. C6 and U87 cells served as controls.

Results

All pediatric brain tumor cell lines displayed fluorescence compared to their respective controls without 5-ALA (p?<?0.05). Sixty minutes of incubation resulted in peaks between 3 and 6 hours, whereas continuous incubation resulted in peaks at 12 hours or beyond. 60 minute incubation peak levels were between 52 and 91 % of maxima achieved with continuous incubation. Accumulation and clearance varied between cell types.

Conclusions

We demonstrate that 5-ALA exposure of cell lines derived from typical pediatric central nervous system (CNS) tumors induces accumulation of fluorescent porphyrins. Differences in uptake and clearance indicate that different application modes may be necessary for fluorescence-guided resection, depending on tumor type.     

In vitro and in vivo photosensitizing capabilities of 5-ALA versus photofrin in vascular endothelial cells

BACKGROUND AND OBJECTIVE: The objective of the present study was to evaluate the feasibility of photodynamic therapy (PDT) for complicated hemangiomas. The photosensitizing activities of 5-aminolevulinic acid (5-ALA) and Photofrin were evaluated in vitro with human dermal microvascular endothelial cells (MEC) and in vivo with the chicken cox comb. STUDY DESIGN/MATERIALS AND METHODS: The in vitro absorption and photosensitizing activities of 5-ALA and Photofrin were examined in a MEC culture system. The percentages of MEC killed by different drug concentrations at a wavelength of 630 nm were measured by either live/dead or lactate dehydrogenase-released assays. Similarly, the in vivo biological activities of 5-ALA and Photofrin exposed to different total light dosages at 630 nm were studied by determining the amount of necrosis produced in chicken combs. RESULTS: MEC incubated with 5-ALA at a concentration of 35 microg/ml and exposed to laser light at 630 nm at a power density of 100 mW/cm2 showed a 50% cell kill. MEC incubated with Photofrin at a concentration of 3.5 microg/ml and exposed to laser light at 630 nm at a power density of 100 mW/cm2 showed a 50% cell kill. Chicken combs that received 200 mg/kg of 5-ALA exposed to laser light at 630 nm at a power density of 100 mW/cm2 had an injury depth of 362.5+/-27.6 microm at histologic examination. Combs exposed to a power density of 100 or 120 mW/cm2 showed injury depths of 732.5+/-29.1 and 792.5+/-36.0 microm, respectively. Chicken combs that received 2.5 mg/kg of Photofrin exposed to laser light at 630 nm at a power density of 80 mW/cm2 had an injury depth of 535.6+/-22.3 microm at histologic examination. Combs exposed to a power density of 100 or 120 mW/cm2 showed injury depths of 795.8+/-32.5 and 805.2+/-49.1 microm, respectively. CONCLUSION: Both 5-ALA and Photofrin have the capability to destroy MEC in vitro and vasculature in vivo. However, Photofrin achieved a higher degree of cell kill and tissue destruction at lower drug concentrations and at lower power densities.     

Clinical experience with intravesical instillations of 5-aminolevulinic acid (5-ALA) for the photodynamic diagnosis using fluorescence cystoscopy for bladder cancer

PURPOSE: To report our clinical experience with intravesical instillations of 5-aminolevulinic acid (5-ALA) for the photodynamic diagnosis of bladder cancer and to assess any side-effects of the diagnostic method. MATERIALS AND METHODS: Photodynamic diagnosis was performed in 18 patients of which 14 were men and 4 women with a median age of 71 years (range 44-84), 7 were primary cases and 11 were recurrent cases with bladder cancer. Two to two and half hours prior to endoscopy 1.5 g 5-ALA dissolved in 50 ml of 8.4% sodium hydrogen carbonate (NaHCO3) solution was instilled intravesically. For fluorescence excitation a blue light source (D-LIGHT System, Karl Storz Endoscopy Japan K. K.) was used. Under white and fluorescence light guidance, tumor locations were recorded, cold cup biopsies were taken and tumors were resected. The levels in images of the 5-aminolevulinic acid-induced fluorescence were compared with the pathological results. The area under the receiver operative characteristic (ROC) curve (AUC) in blue light endoscopy was also compared with that in white light endoscopy. RESULTS: Among the 129 specimens obtained by transurethral biopsy 45 were obtained from polypoid lesion and 84 from non-polypoid lesion, and among the 76 malignant diseases 36 were obtained from polypoid lesion and 40 from non-polypoid lesion (including 19 carcinoma in situ), and 21 patients with dysplasia were detected pathologically, with a sensitivity of 89.5% and specificity of 58.5% with a predictive accuracy of 77.0%. The AUC in blue light endoscopy was more than that in white light endoscopy in not only all cases (p = 0.010) but also in cases with non-polypoid lesion (p = 0.007) and recurrent cases (p = 0.002). Duration of 5-ALA instillation with a median time of 80 (range 30-150) min. did not seem to affect the accuracy of photodynamic diagnosis. Procedures were well tolerated by all patients with mild bladder irritability but no systemic side effect. CONCLUSION: Photodynamic diagnosis with intravesically applied 5-ALA is more effective than observation by conventional cystoscopy in detecting bladder cancer without additional risk or complication, and is expected to become a golden standard in the detection program.     

Correlation of photodynamic activity and fluorescence signaling for free and pegylated mTHPC in mesothelioma xenografts

BACKGROUND/OBJECTIVES: Correlation of photodynamic activity (PDT) and fluorescence signaling for free and pegylated meta-tetrahydroxyphenylchlorin (mTHPC) in nude mice with mesothelioma xenografts. STUDY DESIGN/MATERIALS AND METHODS: Twelve animals received light delivery (20 J/cm(2), 150 mW/cm(2), spot size 1.2 cm) on the tumor and the hind leg 3 days after sensitization with 0.15 mg/kg free mTHPC (n = 6) or equimolar-dosed pegylated mTHPC (n = 6). Groups of three animals each were sensitized with 0.15 and 0.5 mg/kg free mTHPC or equimolar dosed pegylated mTHPC followed after 3 days by fluorescence microscopy measurements. RESULTS: Pegylated mTHPC resulted in a similar extent of PDT-related tumor necrosis but in lower skin phototoxicity than free mTHPC. Both mTHPC formulations were heterogeneously distributed in the tumor and were mainly localized in perivascular areas. Pegylated mTHPC revealed a higher tumor to skin fluorescence intensity ratio than free mTHPC (P<0.001). CONCLUSIONS: Fluorescence signaling measurement has the potential to predict the photodynamic activity for both mTHPC formulations in mesothelioma xenografts.     

Photodynamic therapy mediated induction of accelerated re-endothelialisation following injury to the arterial wall: implications for the prevention of postinterventional restenosis.

OBJECTIVE: Accelerated re-endothelialisation may inhibit the development of restenosis. Basic Fibroblast Growth Factor (bFGF) plays a key role for early proliferative activity in the artery following injury. Therefore, this study was devised to examine the effect of photodynamic therapy (PDT) on post-injury re-endothelialisation in vivo, and bFGF-mRNA expression in endothelial cells (EC) in vitro. MATERIALS AND METHODS: Rat carotid arteries were balloon-injured prior to PDT. Arteries were analysed after 1, 3, 5, 14 and 30 days. Morphometric measurements were undertaken following injection of 0.5% Evans Blue which stains non-endothelialised surfaces only. To identify EC, immunohistochemistry (CD-31) was performed. Proliferation was assessed by fluorescence cell counting. PCR quantification of bFGF-mRNA expression and proliferation were assessed in bovine aortic EC which were plated on isolated, PDT-treated EC-derived extracellular matrix at (12), 24, 48 (72 h). RESULTS: Three days following PDT, arteries displayed significantly increased endothelial lining (p = 0.02), which was more pronounced at 5 (p = 0.03) and 14 days (p = 0.02). At 30 days no relevant differences between PDT and control were noted. EC proliferation on PDT-treated matrix was significantly increased at 24, 48, and 72 h (p = 0.0004), whereas bFGF-mRNA expression was significantly increased at 24 h only (p = 0.007). CONCLUSION: Post-injury PDT appears to accelerate re-endothelialisation. Expression of bFGF-mRNA, however, although increased shortly after PDT, may not be responsible for a constant stimulation of EC proliferation.