R. van Hillegersberg

Biochemical basis of 5-aminolaevulinic acid-induced protoporphyrin IX accumulation : a study in patients with (pre)malignant lesions of the oesophagus

Administration of 5-aminolaevulinic acid (ALA) leads to porphyrin accumulation in malignant and premalignant tissues, and ALA is used as a prodrug in photodynamic therapy (PDT). To understand the mechanism of porphyrin accumulation after the administration of ALA and to investigate whether ALA-induced protoporphyrin IX might be a suitable photosensitizer in Barretts oesophagus and adenocarcinoma, we determined the activities of porphobilinogen deaminase (PBG-D) and ferrochelatase (FC) in various malignant and premalignant as well as in normal tissues of the human oesophagus. A PDT power index for ALA-induced porphyrin accumulation, the ratio of PBG-D to FC normalized for the normal squamous epithelium of the oesophagus, was calculated to evaluate intertissue variation in the ability to accumulate porphyrins. In malignant and premalignant tissue a twofold increased PBG-D activity and a marginally increased FC activity was seen compared with normal squamous epithelium. A significantly increased PDT power index in Barretts epithelium and adenocarcinoma was found. Our results suggest that, after the administration of ALA, porphyrins will accumulate in a greater amount in Barretts epithelium and adenocarcinoma of the oesophagus because of an imbalance between PBG-D and FC activities. The PDT power index here defined might be a useful indicative parameter for predicting the susceptibility of these tissues to ALA-PDT.

Interstitial Nd:YAG laser coagulation with a cylindrical diffusing fiber tip in experimental liver metastases

Interstitial laser coagulation as a means of destructing hepatic metastases was investigated. Colon carcinoma CC531 was implanted in the liver of 42 Wag/Rij rats; 20 days later, tumors (5.5±0.2 mm) were exposed to 1,064 nm Nd:YAG laser light at 4 W/cm and either 600, 1,200, 2,400, 3,400, or 4,800 J/cm from a 0.5 cm Helioseal® coated cylindrical diffuser. Temperature and fluence rate were measured at the tumor boundary. Lesions were studied on day 2 and 36 posttreatment by light microscopy. Tumor proliferative activity was assessed by bromodeoxyuridine incorporation. Liver damage and function were determined by serum liver enzymes and antipyrine clearance. Fluence rate increased during laser treatment up to 170%; mean temperature increased logarithmically up to 69.7°C. Short‐term light microscopy showed coagulation necrosis of 7–11 mm without charring. Lesion size and liver enzymes increased logarithmically with laser energy applied. No deterioration in liver function was found. At 4,800 J/cm complete tumor remission occurred in three of four animals. This study shows the ability of interstitial laser coagulation to produce selective destruction of colonic tumor within the liver.

Porphyrin biosynthesis in human Barrett's oesophagus and adenocarcinoma after ingestion of 5-aminolaevulinic acid

5-Aminolaevulinic acid (ALA)-induced porphyrin biosynthesis, which is used for ALA-based photodynamic therapy (ALA-PDT), was studied in tissues of 10 patients with Barretts oesophagus (BE) and adenocarcinoma of the oesophagus (AC) undergoing oesophagectomy at a mean time interval of 6.7 h after the ingestion of ALA (60 mg kg–1). In BE, AC, squamous epithelium (SQ) and gastric cardia, the activities of the haem biosynthetic enzymes porphobilinogen deaminase (PBG-D) and ferrochelatase (FC) and the PDT power index – the ratio between PBG-D and FC in BE and AC in comparison with SQ – were determined before ALA ingestion. Following ALA administration, ALA, porphobilinogen, uroporphyrin I and PPIX were determined in tissues and plasma. The PDT power index did not predict the level of intracellular accumulation of PPIX found at 6.7 h. In BE, there was no selectivity of PPIX accumulation compared to SQ, whereas in half of patients with AC selectivity was found. Higher haem biosynthetic enzyme activities (i.e. PBG-D) and lower PPIX precursor concentrations were found in BE and AC compared to SQ. It is therefore possible that PPIX levels will peak at earlier time intervals in BE and AC compared to SQ.

Fractionated Illumination for Oesophageal ALA-PDT: Effect on Blood Flow and PpIX Formation

Abstract. The effect of fractionating the 633 nm illumination of 5-aminolaevulinic (ALA)-based photodynamic therapy (PDT) of the normal rat oesophagus was studied. Fractionation of the illumination could enhance the PDT effect in two ways: (a) delay of the vascular shutdown or relaxation of the vasoconstriction induced by ALA-PDT and (b) use of newly formed protoporphyrin IX (PpIX), produced during the dark interval. Forty rats were randomly allocated to two groups of 20 animals each. To study vascular effects, in group 1 illumination with 633 nm (100 mW/cm) was performed at 3 h after oral ALA administration (200 mg/kg) either continuously with 20 J/cm diffuser length (n=5) or fractionated 2×10 J/cm with a 150 s interval (n=5), five animals served as controls. Blood flow was measured with a laser Doppler flowmeter. To study the effect of renewed PpIX forming, animals in group 2 were illuminated continuously at 3 h after ALA with 20 J/cm (n=5) or 40 J/cm (n=5) or fractionated 2×20 J/cm with a 3 h interval (n=5), five animals served as controls. In all animals the in vivo fluence rate and PpIX fluorescence were measured during illuminations and animals were killed at 48 h after PDT.ALA-PDT did not cause any significant vasoconstriction. Fluorescence measurements and dosimetric results in group 1 did not differ between animals illuminated continuously or fractionated with a 150 s interval. In group 2, during a 3 h dark interval, PpIX fluorescence increased and was bleached during the second illumination. The tissue optical properties changed during the 3 h dark interval, resulting in a lower in vivo fluence rate (p≤0.001). Fractionation did not result in more oesophageal damage. It was concluded that a 150 s interval during illumination in ALA-PDT does not increase oesophageal blood flow. During an interval of 3 h new PpIX is formed. In the present study, fractionated illumination using short or long time intervals did not result in more damage. Thus, this study shows no evidence for improved PDT effect with fractionated light delivery.

Timing of illumination is essential for effective and safe photodynamic therapy: a study in the normal rat oesophagus

Summary5-Aminolaevulinic acid (ALA)-induced, protoporphyrin IX (PpIX)-mediated photodynamic therapy (PDT) is an experimental treatment modality for (pre)malignant oesophageal lesions. This study aimed to optimize the time of illumination after ALA administration. Six groups of eight rats received 200 mg kg–1ALA orally, eight rats served as controls. Illumination was performed at 1, 2, 3, 4, 6 or 12 h after ALA administration with a 1-cm cylindrical diffuser placed in a balloon catheter (laser parameters: 633 nm, 25 J radiant energy, power output 100 mW). During illumination, fluorescence measurements and light dosimetry were performed. Animals were sacrificed at 48 h (n= 4) or 28 days (n= 4) after PDT. At day 28, an oesophagogram was performed. Largest PpIX fluorescence was found at 3 h after ALA administration. In vivo fluence rate was three times higher than the calculated incident fluence rate. At 48 h after PDT, major epithelial damage was found in all animals illuminated at 2 h, whereas less epithelial damage was found at 3–6 h and none at 1 and 12 h. In animals illuminated at 4, 6 and 12 h, but not at 2 h, oesophagograms showed severe dilatations and histology showed loss of Schwann cells. These results demonstrate that the choice of time interval between ALA administration and illumination is critical for achieving epithelial damage without oesophageal functional impairment. A short interval of 2–3 h seems to be most appropriate.

Adjuvant intraoperative photodynamic therapy diminishes the rate of local recurrence in a rat mammary tumour model

The use of photodynamic therapy (PDT) as an adjunct to curative tumour resection was investigated in a tumour recurrence model, using rat mammary adenocarcinoma BN472. Tumours were inoculated subcutaneously in 60 animals and resected after 21 days of growth. Immediately after removal, the operation site was exposed to 320-450 nm light of 0.1 W cm-2 and 60 J cm-2 after photosensitisation with either Photofrin (5 mg kg-1 i.v. 48 h before illumination) or 5-aminolaevulinic acid (ALA) (2 mg ml-1 in drinking water for 9 days). Porphyrin concentrations were measured in tissue samples. After 28 days, animals treated with adjunctive PDT had a significantly longer tumour-free interval than controls (P < 0.01); median 25 days (Photofrin), 18 days (ALA), 14 days (controls). Moreover, in the PDT groups significantly fewer rats had lymph node metastasis. A prophyrin concentration ratio between tumour and mammary tissue of 2:1 was found after Photofrin and 4:1 after ALA. The results indicate that adjuvant intraoperative PDT may be a safe and effective method of destroying residual tumour, thereby preventing locoregional tumour recurrence.

Interstitial photodynamic therapy in a rat liver metastasis model.

Photodynamic therapy (PDT) of hepatic tumours has been restricted owing to the preferential retention of photosensitizers in liver tissue. We therefore investigated interstitial tumour illumination as a means of selective PDT. A piece of colon carcinoma CC531 was implanted in the liver of Wag/Rij rats. Photofrin was administered (5 mg kg-1 i.v.) 2 days before laser illumination. Tumours with a mean (+/- s.e.) diameter of 5.7 +/- 0.1 mm (n = 106, 20 days after implantation) were illuminated with 625 nm light, at 200 mW cm-1 from a 0.5 cm cylindrical diffuser and either 100, 200, 400, 800 or 1600 J cm-1. Control groups received either laser illumination only, Photofrin only or diffuser insertion only. Short-term effects were studied on the second day after illumination by light microscopy and computer-assisted integration of the circumference of damaged areas. Long-term effects were studied on day 36. To determine the biochemistry of liver damage and function, serum ASAT and ALAT levels were measured on day 1 and 2, and antipyrine clearance on day 1. Tumour and surrounding liver necrosis increased with light dose delivered (P < 0.001). Best long-term results were obtained at 800 J cm-1 with complete tumour remission in 4 out of 6 animals. No deterioration in liver function was found. The results of this study show the ability of interstitial PDT to cause major destruction of tumour tissue in the liver combined with minimal liver damage.

Photodynamic Therapy for Gastrointestinal Tumors

Photodynamic therapy is based on the administration of a compound that is preferentially accumulated by a tumor and which causes tumor destruction after exposure to light of a specific wavelength. The photosensitizers most commonly used in treating tumors of the gastrointestinal tract are porphyrins--hematoporphyrin derivative and dihematoporphyrin ether. These compounds have been used with success to produce reduction in tumor size of esophageal, gastric, and colorectal cancers. In some instances long-lasting complete remission have been observed after photodynamic therapy. New developments include photosensitizers that react to light of a longer wavelength, which is able to penetrate tissue to a greater depth, the use of 5-aminolevulinic acid, which is preferentially converted to porphyrin in malignant cells, and combination of photodynamic therapy with thermic laser, radiotherapy, or chemotherapy.

Interstitial Laser Photocoagulation with Four Cylindrical Diffusing Fibre Tips: Importance of Mutual Fibre Distance

Abstract. Simultaneous application of multiple fibres could increase the volume of coagulation produced with interstitial laser photocoagulation (ILP) for solid tumours. To take full advantage of the presumed synergistic thermal effect between the fibres, the optimal combination of laser power and distance between the fibres was investigated. Four fibres with a cylindrical diffusing tip of 2 cm length were used, coupled to an optical beamsplitter for Nd:YAG light (four channels, maximal variation 9.5%, transmission >85%). The distance between the fibres was 1, 1.5, 2, 2.5 , 3 or 4 cm with a power output of either 4, 5, 6 or 7 W/fibre; energy per fibre was constant at 1800 J by adjusting exposure time. After laser application, dimensions of the coagulated lesions were measured.The optimal mutual fibre distance was 2 cm ( p<0.01) at all power levels. This resulted in lesions with a mean (SD) volume of 44.5 (2.1) cm3 and a largest diameter of 5.1 (0.4) cm at 7 W/fibre. Smaller distances between the fibres resulted in smaller lesions with central carbonisation, whereas larger distances resulted in four separate zones of coagulation. It was concluded that simultaneous application of four interstitial fibres may result in a considerable increase of volume of coagulation. Fibre position and mutual fibre distance determines whether synergism of the coagulative effect occurs.