Detection of squamous cell carcinoma of the oral cavity by imaging 5-aminolevulinic acid-induced protoporphyrin IX fluorescence

OBJECTIVES: Early cancer detection is the best way to improve the prognosis of patients with oral cancer. Therefore this study presents quantitative fluorescence measurements and results in the visualization of cancerous oral mucosa with 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PPIX). METHODS: Time progression and type of porphyrin accumulation were analyzed in neoplastic and surrounding healthy tissue of 58 patients with a suspected cancer of the oral cavity by measuring emission spectra of 5-ALA-induced PPIX fluorescence. Fluorescence images in the red and green spectral range from the tumor tissue were recorded with a charge-coupled device camera. RESULTS: After topical application of 0.4% 5-ALA and incubation for 1 to 2.5 hours, all patients revealed higher intensities of red fluorescence in neoplastic tissue compared with the surrounding normal tissue. Maximum contrast was reached after 1.5 hours of incubation. In 13.8% (n = 8) of the patients, additional findings like dysplasia, carcinoma in situ, primary tumor, secondary carcinomas, and tumor branches were found by means of fluorescence marking in contrast to white light examination. An evaluation of the biopsy specimens resulted in a specificity of 60% and a sensitivity of 99%. CONCLUSIONS: As a fluorescent marker, PPIX could represent a possible new diagnostic tool to detect early malignant and secondary lesions in the oral cavity. In addition, 5-ALA-induced PPIX fluorescence is promising as a useful intraoperative tool for determining adequate surgical margins of resection. Further investigations aim to assess this diagnostic procedure as a sensitive and clinically reliable method for patients with oral cancer.     

��-Aminolaevulinic acid-induced photodynamic therapy inhibits protoporphyrin IX biosynthesis and reduces subsequent treatment efficacy in vitro

Recently, considerable interest has been given to photodynamic therapy of cancer using δ-aminolaevulinic acid to induce protoporphyrin IX as the cell photosensitizer. One advantage of this modality is that protoporphyrin IX is cleared from tissue within 24 h after δ-aminolaevulinic acid administration. This could allow for multiple treatment regimens because of little concern regarding the accumulation of the photosensitizer in normal tissues. However, the haem biosynthetic pathway would have to be fully functional after the first course of therapy to allow for subsequent treatments. Photosensitization of cultured R3230AC rat mammary adenocarcinoma cells with δ-aminolaevulinic acid-induced protoporphyrin IX resulted in the inhibition of porphobilinogen deaminase, an enzyme in the haem biosynthetic pathway, and a concomitant decrease in protoporphyrin IX levels. Cultured R3230AC cells exposed to 0.5 mM δ-aminolaevulinic acid for 27 h accumulated 6.07 × 10⁻¹⁶ mol of protoporphyrin IX per cell and had a porphobilinogen deaminase activity of 0.046 fmol uroporphyrin per 30 min per cell. Cells cultured under the same incubation conditions but exposed to 30 mJ cm⁻² irradiation after a 3-h incubation with δ-aminolaevulinic acid showed a significant reduction in protoporphyrin IX, 2.28 × 10⁻¹⁶ mol per cell, and an 80% reduction in porphobilinogen deaminase activity to 0.0088 fmol uroporphyrin per 30 min per cell. Similar effects were evident in irradiated cells incubated with δ-aminolaevulinic acid immediately after, or following a 24 h interval, post-irradiation. There was little gain in efficacy from a second treatment regimen applied within 24 h of the initial treatment, probably a result of initial metabolic damage leading to reduced levels of protoporphyrin IX. These findings suggest that a correlation may exist between the δ-aminolaevulinic acid induction of porphobilinogen deaminase activity and the increase in intracellular protoporphyrin IX accumulation. © 1999 Cancer Research Campaign     

Split-face-study using two different light sources for topical PDT of actinic keratoses:non-inferiority of the LED system

Background: Photodynamic therapy (PDT) with 5-amino-4-oxo-pentanoate (methylaminolevulinate, MAL) is an effective and safe treatment option for actinic keratoses. Light-emitting diodes (LED) are suitable light sources for topical PDT. To evaluate the efficacy, painfulness, patient satisfaction and cosmesis of LED-based PDT a prospective, randomized and controlled split-face study was performed. Methods: Topical ALA-PDT was administered to 17 patients whose actinic ker-atoses (n = 131) were symmetrically distributed and suitable for a two-side comparison. After incubation with MAL (16%), irradiation was performed with the incoherent lamp (160 mW cm⁻²; 100 J cm⁻², PDT 1200L, Waldmann Medizintechnik, Villingen-Schwenningen, Germany) on one side and the LED system (120 mW cm⁻²; 40 J cm⁻², LEDA, WaveLight AG, Erlangen, Germany) on the other side. The patients were followed by re-evaluation up to 6 months. Results: Six months following treatment there was no significant difference between the infiltration and keratosis scores in both treatment regimes (p = 0.812). The remission rate was 78.5% (LED system) vs.80.3% (incoherent lamp). There was no significant difference between both light sources regarding the pain during therapy (p = 0.988). There was no significant difference between both treatment regimes regarding patient satisfaction (p = 1). Conclusions: LEDA-based MAL-PDT is an effective alternative for the treatment of atinic keratoses. The remission rates and cosmetic results are not inferior to PDT using incoherent light systems. Both treatment regimes are similarly painful.     

Carrier analysis of a moderately affected haemophilia B family.

Here we report the successful genetic diagnosis of a pregnant caucasian female patient whose family has a history of moderate haemophilia B. While restriction fragment length polymorphism (RFLP) analysis was not informative, nucleotide sequencing of the factor IX genes of the patient's family members determined that her mother and one of her two sisters were carriers of the mutation C31008T, which causes a Thr296Met transition. In contrast, the pregnant female herself and her other sister were found to carry only normal alleles. Plasma factor IX activity and antigen levels supported these findings.     

Novel heterozygous missense mutation in the platelet glycoprotein Ib beta gene associated with isolated giant platelet disorder.

The glycoprotein (GP) Ib/IX/V complex plays an important role in primary hemostasis, serving as the platelet receptor for von Willebrand factor (vWF). Recent studies have shown that the phenotype caused by mutations in the subunits of the GPIb/IX complex spans a wide spectrum; from the normal phenotype, to isolated giant platelet disorders (GPD), and to the full-blown bleeding disorder, the Bernard-Soulier syndrome (BSS). We characterize here a novel missense mutation of the GPIb beta gene associated with isolated GPD. In the patient's platelets, the expression level of the GPIb/IX complex was moderately reduced compared with that of the GPIIb/IIIa complex, whereas the latter was expressed at higher levels than in a normal control. Immunoblot analysis showed normal electrophoretic mobility of GPIb alpha, GPIb beta, and GPIX. However, the amount of GPIb beta was approximately 66% of the normal value. DNA sequencing analysis revealed a novel heterozygous missense mutation in the GPIb beta gene that converts Arg (CGC) to Cys (TGC) at residue 17. Transient transfection studies demonstrated that mutant GPIb beta protein was not detected in transfected 293T cells. These findings indicated that null expression of the abnormal GPIb beta causes decreased expression of the complex and results in the GPD phenotype in the patient, and suggested that homozygosity of the mutation may lead to a BSS phenotype in vivo.     

绞股蓝总皂甙对氧化修饰低密度脂蛋白损伤小牛主动脉内皮细胞的防治作用

目的 :探讨绞股蓝总皂甙 (GYP)对氧化修饰低密度脂蛋白 (oxidativelymodifiedlowdensitylipopro tein ,oxLDL)损伤小牛主动脉内皮细胞的防治作用及可能机制。方法 :在体外培养的小牛主动脉内皮细胞 (bovineaorticendothelialcell ,BAEC )中加入oxLDL(终浓度为 0 .1gpr·L- 1)及GYP低、中、高剂量组 (5 .0 ,10 .0 ,2 0 .0mg·L- 1) ,培养 2 4h后 ,采用细胞毒试验、丙二醛 (MDA)测定及细胞计数等方法 ,分别对其贴壁细胞及培养液进行检测。结果 :①细胞毒试验发现 ,oxLDL对BAEC的生长增殖有明显的抑制作用 (2 0 .6 3% ) ,GYP组 (10 .0 ,2 0 .0mg·L- 1)为9.14 %和 6 .5 6 % (P <0 .0 1) ;②加oxLDL组和GYP组BAEC粘附的HL6 0细胞数明显多于对照组 (P <0 .0 1) ;③MDA检测发现oxLDL促进BAEC脂质过氧化物生成 ,并高于GYP组和对照组 (P <0 .0 1)。结论 :绞股蓝总皂甙具有对氧化修饰低密度脂蛋白损伤小牛主动脉内皮细胞的防治作用。     

A comparison of 5-aminolaevulinic acid- and its heptyl ester: dark cytotoxicity and protoporphyrin IX synthesis in human adenocarcinoma WiDr cells and in athymic nude mice healthy skin

Abstract:  5-aminolevulinic acid heptyl ester was investigated in human adenocarcinoma WiDr cells and in healthy skin of athymic nude mice in comparison with 5-aminolevulinic acid (ALA). Incubation of WiDr cells with ALA and ALA heptyl ester resulted in production of protoporphyrin IX (PpIX). Concentrations higher than 0.01 m m of ALA heptyl ester and higher than 1 m m of ALA were cytotoxic. The dark cytotoxicity was not related to PpIX. Intracellular localization, photocytoxicity and photobleaching rate of PpIX were the same for both drugs, although a 100 times lower concentration of ALA heptyl ester (0.01 m m ) was needed in comparison with ALA (1 m m ) to induce the same level of PpIX. ALA heptyl ester, topically (but not systemically) applied, is a promising candidate for fluorescence diagnosis and photodynamic therapy. Special attention must be focused on the concentrations of ALA heptyl ester; as excess may lead to cytotoxicity and inefficient PpIX generation.     

Influence of treatment-induced changes in tissue absorption on treatment volume during interstitial photodynamic therapy

Interstitial photodynamic therapy on thick skin lesions has been shown to induce changes in tissue light transmission as a direct consequence of variations in total blood volume and oxygen saturation. A finite element method was used in order to simulate the fluence rate distribution and total light dose throughout the target tissue for two cases. The first case constitutes a pre-treatment model where the tissue optical properties are assumed constant during the entire treatment. The second situation takes into account observed changes in tissue light transmission, small deviations in fiber insertion depth and a few cases of almost complete loss of source fiber output power possibly as a result of blood accumulation in front of the fiber tip. The pre- and post-treatment models from six clinical treatments are compared in terms of simulated treatment volumes. We conclude that real-time monitoring of the delivered fluence is necessary in order to ascertain a pre-determined light dose to the target tissue. Finally, we speculate on how to also include the sensitizer fluorescence level and tissue oxygenation in the real-time treatment feedback.