I Wang

Photodynamic therapy of non‐melanoma malignant tumours of the skin using topical δ‐amino levulinic acid sensitization and laser irradiation

Eighty basal cell carcinomas (BCCs) in 21 patients, 10 lesions of Bowens disease in three patients, and four lesions of cutaneous T‐cell lymphoma in two patients, were treated with photodynamic laser therapy (PDT), using topical application of the haem precursor δ‐amino levulinic acid (ALA). The diagnoses were confirmed histologically prior to treatment. Fifty‐five of the BCCs were superficial lesions, and 25 were nodular. Of the 80 BCCs, 39 (49%) were located on the trunk, 36 (45%) on the head and neck region, four (15%) on the leg and one on the arm. The two principal locations of the 10 Bowens disease lesions were the leg (50%) and the trunk (40%). The T‐cell lymphoma lesions were located on the shoulder and on the arm. A water‐in‐oil based cream containing 20% ALA was applied to the lesions, with a margin of about 10–20 mm beyond the visible tumour border, 4–6 h before the laser procedure. During this period of time the highly fluorescent and photodynamically active substance protoporphyrin IX (Pp IX) is synthesized via the haem cycle. Laser‐induced fluorescence (LJF) was used for real‐time monitoring of the Pp IX distribution in the tumour and in the normal surrounding skin, before and after treatment in all patients. Before laser treatment the Pp IX distribution demonstrated by LJF showed a demarcation between tumour and normal skin of about 15:1 for BCC and Bowens disease, and 5:1 for T‐cell lymphomas. Laser light from a pulsed frequency‐ doubled Nd: YAG laser pumping a dye laser with light emission at 630 nm was used for the therapy. The power density in the irradiation was kept below 110 mW/cm2, in order to avoid hyperthermal effects. A total energy of 60 J/cm2 was delivered for 10–20 min, depending on the tumour size. A complete response rate of 100% in superficial BCCs and 64% in nodular BCCs occurred after a single laser treatment, and a response rate of 100% was achieved after one additional treatment in the nodular BCCs. In the Bowens disease lesions a complete response of 90% was obtained with a single treatment. Two of the four T‐cell lymphomas resolved completely. The follow‐up time was between 6 and 14 months.

Photodynamic therapy vs. cryosurgery of basal cell carcinomas: results of a phase III clinical trial

Background A previously reported randomized clinical trial showed treatment of Bowens disease using photodynamic therapy (PDT) with topically applied δ‐aminolaevulinic acid (ALA) to be at least as effective as cryosurgery and to be associated with fewer adverse effects.

Preliminary evaluation of two fluorescence imaging methods for the detection and the delineation of basal cell carcinomas of the skin

Fluorescence techniques can provide powerful noninvasive means for medical diagnosis, based on the detection of either endogenous or exogenous fluorophores. The fluorescence of δ‐aminolevulinic acid (ALA)‐induced protoporphyrin IX (PpIX) has already shown promise for the diagnosis of tumors. The aim of the study was to investigate the localization of skin tumors after the topical application of ALA, by detecting the PpIX fluorescence either in the spectral or in the time domain.

Clinical multi-colour fluorescence imaging of malignant tumours - initial experience

Purpose: the detection of malignant tumours relies on a variety of diagnostic procedures including X-ray images and, for hollow organs, endoscopy. the purpose of this study was to present a new technique for non-invasive tumour detection based on tissue fluorescence imaging Material and Methods: A clinically adapted multi-colour fluorescence system was employed in the real-time imaging of malignant tumours of the skin, breast, head and neck region, and urinary bladder. Tumour detection was based on the contrast displayed in fluorescence between normal and malignant tissue, related to the selective uptake of tumour-marking agents, such as haematoporphyrin derivative (HPD) and δ-amino levulinic acid (ALA), and natural chromophore differences between various tissues. in order to demarcate basal cell carcinomas of the skin, ALA was applied topically 4–6 h before the fluorescence investigation. for urinary bladder tumour visualisation (transitional cell carcinoma of different stages including carcinoma in situ), ALA was instilled into the bladder 1–2 h prior to the study. Malignant and premalignant lesions in the head and neck region were imaged after i.v. injection of HPD (Photofrin). Finally, the extent of in situ and invasive carcinomas of the breast was investigated in surgically excised specimens from patients that received a low-dose injection of HPD 24 h prior to the study. the tumour imaging system was coupled to an endoscope. Fluorescence light emission from the tissue surface was induced with 100-nslong optical pulses at 390 nm, generated from a frequency-doubled alexandrite laser. with the use of special image-splitting optics, the tumour fluorescence, intensified in a micro-channel plate, was imaged in 3 selected wavelength bands. These 3 images were processed together to form a new optimised-contrast image of the tumour. This image, updated at a rate of about 3 frames/s, was mixed with a normal colour video image of the tissue Results: A clear demarcation from normal surrounding tissue was found during in vivo measurements of superficial bladder carcinoma, basal cell carcinoma of the skin, and leukoplakia with dysplasia of the lip, and in in vitro investigations of resected breast cancer Conclusions: the initial clinical experience of using multi-colour fluorescence imaging has shown that the technique has the potential to reveal malignant tumour tissue, including non-invasive early carcinoma and also precancerous tissue. Further investigations are needed to fully develop the method

Kinetic fluorescence studies of 5-aminolaevulinic acid-induced protoporphyrin IX accumulation in basal cell carcinomas.

Laser-induced fluorescence (LIF) investigations have been performed in connection with photodynamic therapy (PDT) of basal cell carcinomas and adjacent normal skin following topical application of 5-aminolaevulinic acid (ALA) in order to study the kinetics of the protoporphyrin IX (PpIX) build-up. Five superficial and 10 nodular lesions in 15 patients are included in the study. Fluorescence measurements are performed prior to the application of ALA, 2, 4 and 6 h post ALA application, immediately post PDT (60 J cm-2 at 635 nm), and 2 h after the treatment. Hence, the build-up, photobleaching and re-accumulation of PpIX can be followed. Superficial lesions show a maximum PpIX fluorescence 6 h post ALA application, whereas the intensity is already the highest 2-4 h after the application in nodular lesions. Immediately post PDT, the fluorescence contribution at 670 nm from the photoproducts is about 2% of the pre-PDT PpIX fluorescence at 635 nm. Two hours after the treatment, a uniform distribution of PpIX is found in the lesion and surrounding normal tissue. During the whole procedure, the autofluorescence of the lesions and the normal skin does not vary significantly from the values recorded before the application of ALA.

Pharmacokinetic studies on 5-aminolevulinic acid-induced protoporphyrin IX accumulation in tumours and normal tissues

Laser-induced fluorescence (LIF) for in vivo point monitoring and fluorescence microscopy incorporating a CCD camera were used to study the fluorescence distribution of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) in tumours. Fluorescence in a chemically-induced adenocarcinoma in the liver of rats and in an aggressive basal cell carcinoma in a patient were studied after intravenous injection of ALA at a dose of 30 mg/kg body weight. The LIF technique demonstrated slightly more ALA-induced PpIX fluorescence in the tumour than in the surrounding normal liver and abdominal muscle of rats. The visible parts of the human basal cell carcinoma exhibited strong ALA-induced fluorescence, while this fluorescence was much weaker in the necrotic areas of the tumour and in the surrounding normal skin.

Superficial blood flow following photodynamic therapy of malignant non–melanoma skin tumours measured by laser Doppler perfusion imaging

Laser Doppler perfusion imaging offers a new modality for in vivo monitoring of the superficial blood perfusion in biological tissue. In this study, the superficial blood perfusion of malignant nonmelanoma skin tumours and the surrounding normal skin was measured in conjunction with photodynamic therapy (PDT) using topical ò–aminolaevulinic acid (ALA)–induced protoporphyrin IX as a photosensitizer. The results clearly show that, in contradiction to PDT with the intravenously administered photosensitizer photofrin. no direct vascular damage can be seen. With the topical sensitization the blood perfusion is increased immediately after the treatment irradiation. The increased blood flow is seen up to a week after treatment, in a similiar way as for an inflammatory reaction. Despite this, all basal cell carcinoma and squamous cell carcinoma in situ lesions in this study healed without any sign of residual tumour after the treatment, suggesting an efficient direct tumour cell destruction induced by PDT.

Blood perfusion studies on basal cell carcinomas in conjunction with photodynamic therapy and cryotherapy employing laser-Doppler perfusion imaging

Superficial blood perfusion was monitored using laser-Doppler perfusion imaging in connection with a phase III clinical trial comparing photodynamic therapy, utilizing topically applied delta-aminolevulinic acid, with cryotherapy of basal cell carcinomas. A total of 526 images were recorded before and immediately after the treatment and during the follow-up period. Before treatment, the lesions exhibited a blood perfusion 3+/-2 times that in normal tissue. Both treatment modalities induced an increased blood perfusion inside the lesions, which slowly approached normal values in conjunction with successful treatments. The blood perfusion in successfully treated lesions approached normal values 2 months after photodynamic therapy, and about 1 year after cryotherapy. The tissue perfusion in recurrent lesions did not decrease to normal values after the treatment, suggesting that the laser-Doppler perfusion imaging technique can be used to follow the healing process and discover possible persistent tumour growth.

Photodynamic therapy using intravenous delta-aminolaevulinic acid-induced protoporphyrin IX sensitisation in experimental hepatic tumours in rats.

The efficacy of photodynamic therapy (PDT) using delta-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) sensitisation and laser light at 635 nm was investigated in the treatment of experimental hepatic tumours. The model of liver tumours was induced either by local inoculation or by administration of tumour cells through the portal vein in rats. ALA at a dose of 60 mg kg(-1) b.w. was intravenously administered 60 min before PDT. PpIX accumulation in tumour, normal liver and abdominal wall muscle was detected by means of laser-induced fluorescence (LIF). Laser Doppler imaging (LDI) was used to determine changes in the superficial blood flow in connection with PDT. Histopathological examinations were performed to evaluate the PDT effects on the tumour and the surrounding liver tissue, including pathological features in the microvascular system. The accumulation of PpIX, as monitored by LIF, showed high fluorescence intensities at about 635 nm in both the hepatic tumour tissue and normal liver and low values in the abdominal wall. LDI demonstrated that the blood flow in the treated tumour and its surrounding normal liver tissue decreased immediately after the PDT, indicating an effect on the vascular system. A large number of thrombi in the irradiated tumour were found microscopically 3 h after the PDT. The tumour growth rate showed a marked decrease when evaluated 3 and 6 days after the treatment. These results show that the ALA-PDT is effective in the inhibition of growth of experimental hepatic tumours.

Laser Doppler perfusion imaging: New technique for determination of perfusion and reperfusion of splanchnic organs and tumor tissue

Several investigations indicated that laser Doppler flowmetry on the liver surface reflects relative changes of the total liver blood flow. In this study, Laser Doppler Perfusion Imaging (LDI), monitoring the surface only, was used for measurements of tissue perfusion of normal and/or impaired liver, pancreas, spleen, stomach and intestine, and the blood flow of hepatic tumors in rats.