Photodiagnosis and Photodynamic Therapy (2004) 1, 211—223

REVIEW

Photodynamic therapy in dermatology: Dundee clinical and research experience S.H. Ibbotson, MD, FRCPa,∗, H. Moseleya, L. Brancaleona, M. Padgettb, M. O’Dwyerb, J.A. Woodsa, A. Lesara, C. Goodmana, J. Fergusona a

Photobiology Unit, Barbara Stewart Cancer Trust, Scottish PDT Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD19SY, UK b Optics Group, Department of Physics and Astronomy, University of Glasgow, Glasgow, UK

KEYWORDS Photobiology; PDT; T-cell lymphoma

Summary Topical photodynamic therapy (PDT) is increasingly accepted and used as a highly effective treatment for superficial non-melanoma skin cancer and dysplasia. We describe the developments in topical PDT for the treatment of skin diseases in our own PDT Centre in Dundee, both clinically and from a research base. Improvements in PDT could be achieved by optimisation of photosensitiser and light delivery, and these goals underpin the aims of our centre. We hope to facilitate the dissemination of use of PDT in dermatology throughout Scotland and outline some of the progress in these areas. © 2004 Elsevier B.V. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

212

Photosensitisers and light sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

212

Topical PDT in dermatology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

213

Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

214

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

214

Photodiagnostics in the photobiology unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

215

Fluorescence diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Description of instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

215 215 216 216

* Corresponding author. Tel.: +44 1382 425717; fax: +44 1382 633925.

E-mail address: [email protected] (S.H. Ibbotson).

1572-1000/$ — see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/S1572-1000(04)00045-6

212

S.H. Ibbotson et al.

In vivo detection of PpIX fluorescence in non-melanoma skin cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Patient selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

217 217 217 217 218 218

The study of photosensitisers in in vitro cell culture models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

218

Nursing/technician management in photodynamic therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

220

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

220

Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

221

The Scottish Photodynamic Therapy Centre based at Ninewells Hospital & Medical School, Dundee, Tayside. . . .

221

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction Every Teaching Hospital Department faces treatment development dilemmas. At what point do they become involved in a new therapy, which is still at the speculative stage and yet clearly has promise? In the early 1990s, the Photobiology Unit within the Department of Dermatology in Dundee discussed whether to actively become involved in the development of PDT for skin cancers. At that time we felt PDT was just too new to invest significant resources without a fairly certain outcome. By 1998, the position had changed and enough good quality data existed to suggest that, for pre-malignant and malignant skin lesions, treatment outcomes justified becoming involved in the development of both PDT and photodiagnosis (PD). From a clinical research and therapeutic point of view, the skin has two huge advantages. Firstly, it can be easily examined with the naked eye, and secondly, it is the most accessible organ for investigation, biopsy and treatment. Although PDT firmly has its roots at the beginning of the last century, it is only over the last 15 years that it has gained considerable popularity as a topical treatment of great promise for the treatment of skin cancers [1]. The Photobiology Unit (photobiology = the study of light on living systems) has the purpose in Scotland of diagnosing light sensitive skin disease (the photodermatoses) and the development of new forms of light therapy (phototherapy). This Centre, which has been in existence since 1973, has always combined clinical skills (photodermatology) with a strong scientific base (photophysics) and laboratory biology (photobiology). This combination of applied science and clinical service in the same unit has pro-

vided exciting research opportunities. Applied photophysics, through the Medical Physics Department, has dedicated members of staff whose only role is optical physics. The necessary expertise in light delivery and measurement is essential for predictable PDT and PD.

Photosensitisers and light sources Most early reports of clinical applications of PDT used Argon laser-pumped dye lasers, although several different types of lasers have actually been used over the years (Table 1). Dye lasers have the flexibility of variable wavelength, generally between 500 and 750 nm but they require a considerable amount of technical support. Diode lasers are semi-conductor devices, extremely compact and easy to use. Unfortunately our experience in the Scottish PDT Centre has been mixed. Three laser diode devices have been used; one of these, a Diomed® 630 nm, suffered terminal failure after only 400 h use. The advantage of the diode laser compared to a non-laser source is that it can be used for treating both systemic cancer and skin lesions. The photosensitisers in common use are characterised by a large absorption band between 400 and 430 nm (Soret band) and smaller absorption bands (Q-band) above 550 nm [2,3]. Q-bands above 600 nm are normally targeted for PDT because of the much higher penetration of longer wavelength light into tissue. Although producing a much broader spectralemission than lasers, filtered lamps have been used successfully to treat Bowen’s disease (BD), superficial basal cell carcinoma (BCC) and actinic keratosis

Photodynamic therapy in dermatology

Table 1

213

Lasers commonly used for PDT.

Source

Wavelength (nm)

Sensitiser

Reference

Flashlamp-pumped dye laser Copper vapour-pumped dye laser Argon ion-pumped dye laser Helium-neon laser Argon ion-pumped dye laser Copper vapour-pumped dye laser

673 630 630 633 680 628, 648, 652, 653, 656 628 675 675 675 510 779 630

Zn-phthalocyanine Haematoporphyrin derivative Photofrin Haematoporphyrin derivative Aluminium phthalocyanine Various porphyrins and chlorines

[37] [38] [39] [40] [41] [42]

Haematoporphyrin derivative Aluminium phthalocyanine Aluminium phthalocyanine Aluminium phthalocyanine Haematoporphyrin derivative Bacteriochlorin Photofrin

[43] [44] [44] [44] [45] [46] [47]

Gold vapour laser Copper vapour-pumped dye laser Argon ion-pumped dye laser Flashlamp-pumped dye laser Copper vapour laser AlGaAs/GaAs diode laser Frequency-doubled NdYAGpumped dye laser

(AK) using 5-aminolaevulinic acid (ALA) [4,5]. Both tungsten filament quartz halogen lamps and Xenon arc lamps have been utilised in these systems. They are broad-spectrum sources filtered to emit light between 600 and 700 nm. Metal halide lamps have also been used and are available commercially (Waldmann® 1200). These lamps have a unique feature of producing a very large diameter beam (up to 25 cm in diameter). Potentially, they may be used for treating field changes or multiple lesions over a large area. Fluorescent lamps with a maximum at 417 ± 5 nm are used in a commercial system (DUSA), which targets the Soret band for treatment of AK. Light Emitting Diode (LED) arrays are now widely used for PDT of skin lesions using both ALA and its methyl ester (MAL). Power output is available up to 150 mW/cm2 but with many of the systems in practice the surface irradiance is much lower (20—50 mW/cm2 ). A comparative analysis was performed in the Scottish PDT Centre for cases treated using a home built Xenon arc lamp system, laser, metal halide and halogen sources [6]. Patients were not randomised to different light sources. Nonetheless, clearance of BD was achieved within two treatments in the majority of cases, whichever lamp was used (range 92—100%). Similar results were also recorded for superficial BCC, although the range in this case was slightly larger (88—100%). Statistically significant differences were noticed with respect to the pain associated with each source. The lower irradiance Xenon lamp was associated with mild/no pain in 79% of cases compared to 38% laser, 49% metal halide and 35% halogen. The concept of total effective fluence was developed to predict efficiency of different light sources

[7]. Application of the model shows that green light is more effective than red for superficial lesions down to a depth of approximately 2 mm, whereas red light gives a much more uniform effective fluence with depth and is more appropriate for treating lesions situated below approximately 2 mm. Laser and non-laser light sources for PDT have been reviewed elsewhere [8].

Topical PDT in dermatology Topical PDT in dermatology using ALA as pro-drug and red light irradiation was first introduced as a therapy for dermatological conditions in 1990 and has been extensively studied since then [1,9]. Topical PDT using either ALA or MAL is increasingly used to treat superficial non-melanoma skin cancers and dysplasia, in particular, superficial BCC, BD and AK [1]. As we have become more comfortable with its use in the treatment of these lesions, PDT has also been applied to a wide range of other skin diseases, and in particular, has been studied in some detail for its use in the treatment of recalcitrant viral warts, psoriasis, cutaneous T-cell lymphoma (CTCL) and acne vulgaris [10]. As discussed, a variety of light sources have been used for topical PDT: either red light, or blue light for more superficial lesions such as AK. Lasers, noncoherent sources and, more recently, LEDs have been used with no obvious differences in treatment outcomes [6,11]. Topical PDT is at least as effective as conventional therapies for the treatment of superficial BCC, BD and AK, and the advantages are of

214 improved cosmesis and healing, thus allowing treatment at cosmetically difficult sites or sites associated with poor healing such as on lower leg [1]. Many centres are now undertaking topical PDT as part of their routine treatments offered for nonmelanoma skin cancers (NMSCs). In order to establish the place of topical PDT in dermatology, comparisons with proven therapies and adequate follow-up periods are essential. We commenced the use of topical PDT for skin diseases in the Photobiology Unit in Dundee in 1998. Referrals are received from dermatologists in our own department, from other regional dermatology centres and from local plastic surgeons and oncologists. When we established the PDT service, a single clinic, one day per week, was performed. However, due to demand, we now carry out PDT five days per week and treatment is increasingly technicianled. We have a two-month waiting list for treatment and perform approximately 60—80 treatments per month.

Methods Patients are assessed for PDT, photographic documentation of lesions is undertaken, consent obtained and treatment is performed once the histological diagnosis is confirmed. If multiple lesions are to be treated then representative biopsies are taken. The surface of the lesion is prepared using light curettage without local anaesthetic, to remove crusting. ALA (20% in Unguentum Merck; Crawford’s Pharmaceuticals, UK) is applied to the lesion under occlusion for 4 h (AK, BD) or 6 h (superficial BCC up to 2 mm in histological thickness). After removal of ALA surface fluorescence is assessed using Wood’s light examination and a visual grading system for intensity and specificity of fluorescence. The patient is then prepared for the irradiation procedure and covered up so that only the site to be treated is exposed. Irradiation is performed using a standard light dose of 125 J/cm2 for all laser or non-coherent light sources or 37.5 J/cm2 for LED light sources. If MAL PDT is performed, MAL is applied for 3 h prior to irradiation at 37.5 J/cm2 using the Actilite® LED. We have a variety of light sources for use (Diode 630 nm laser (Diomed® ), PDT 1200® , tungsten filament halogen lamp (PhotoCure), short arc xenon lamp (Phototherapeutics) and LEDs (Omnilux® ) (Phototherapeutics) and Actilite® (PhotoCure/Galderma)). During irradiation approximately 20% of patients report treatment as being severely painful, and therefore we use a range of methods of pain relief including, at the simplest

S.H. Ibbotson et al. level, chatting to the patient and reassuring them. We also use a cooling fan, and more recently, forced cooling using a jet of chilled air (Cynosure® ) or Xylocaine spray. If the patient has experienced severe problems with pain in previous treatments or has a large area for treatment, for example, a whole scalp, then we apply lidocaine local anaesthetic gel for an hour before irradiation, although a recent study has not shown this to be superior to placebo for pain relief during PDT to small lesions [12]. It is unknown whether Ametop® may be effective for pain relief for larger areas. Injectable local anaesthetic is required in approximately 1% of cases but is not always totally effective in providing pain relief for PDT. Following treatment, all patients experience an inflammatory reaction, which appears to be at its peak within a few hours of treatment and subsides thereafter. We advise patients to keep covered up from natural sunlight for two days and to expect inflammation and subsequent crusting of the lesion over about a one-week period. Healing is usually not problematic, and ulceration or erosion of the lesions occurs in less than 1% of cases, with pigmentary change and minor scarring apparent in about 1—2% of lesions treated. Cosmetic results are excellent.

Results Our patients are routinely treated on two separate occasions eight weeks apart. Treatments are carried out by photobiology technicians, although medical review is performed six months after the first treatment, and follow-up is for three years for BD and AK and five years for superficial BCC, with photographic documentation on each occasion. Our preliminary three-year experience of topical ALA PDT has recently been published on the first 688 treatments on 483 lesions in 207 patients [6]. The vast majority of patients treated had diagnoses of superficial BCC, BD or AK and they were treated with a range of broadband and laser light sources. Initial clearance rates of 93% were observed with similar efficacy with the broadband and laser light, and recurrence rates of 5% for AK and BCC and 10% for BD. Treatment was well-tolerated with only minor side-effects of pigmentary change and scarring, although significant discomfort was experienced in 16—21% of treatments. We concluded that topical ALA PDT is effective for superficial BCC, BD and AK, and is well tolerated and readily performed within our routine dermatology service. We now have experience of treatment in 596 patients, with 1355 lesions and a total number of

Photodynamic therapy in dermatology

Fig. 1. Bowen’s disease on an oedematous low leg before and after a single treatment with topical PDT showing clinical clearance.

treatments of 2602. Of these lesions, the vast majority were superficial NMSC or dysplasia (superficial BCC, 436; BD, 657; AK, 164). We have obtained initial clearance rates of 88% for AK, 96% for BD and 95% for superficial BCC (Fig. 1). Of some concern, of the lesions cleared, we have noted a 17% recurrence rate, which is higher than our initial three-year experience, indicating recurrence rates of up to 10%. On review of these recurrences, it was apparent that 75% of these occurred in patients who had only been treated on a single occasion, and therefore, for the last year of treatment, all patients with AK, BD or superficial BCC have routinely received two treatments at an eight-week interval. To date we have no recurrences reported in this group treated with this revised regime. We have experience of treating a range of other skin conditions including viral warts of 15 patients with 37 warts: six patients with 21 warts cleared, 50% with one treatment and nine subjects were treatment failures. However, as approximately six treatments are usually required, we do not routinely offer this treatment because of demand on the service for skin cancer and dysplasia treatment. The treatment of recalcitrant viral warts appears to be particularly painful, and pain relief is essential for these subjects. Our experience in the treatment of psoriasis is disappointing [13]. However, our experience with cutaneous T-cell lymphoma (CTCL) is encouraging, although again, treatment can be painful, and it seems that optimal treatment parameters are not yet established. We are carrying out a range of research studies in order to examine optimal treatment parameters with photosensitisers and light delivery and methods of pain relief. In order to optimise treatment for some of the more difficult conditions, such as nodular BCCs

215 or CTCL, it is essential that long-term follow-up is monitored. It is also essential that comparisons with established therapies are performed, and this is the overall aim of our research programme. In conclusion, the PDT service in dermatology has been an invaluable addition to our dermatology therapeutic repertoire and is ideal for treatment of multiple lesions or those lesions at difficult treatment sites such as below the knee. The requirements for treatment are relatively straightforward and light sources are increasingly available at lower cost. Nursing or technician input is required and, as we have demonstrated, the service can largely be run by technicians or nurses overseen by medical staff with respect to treatment of difficult cases, surface preparation and review of treatment responses. There seems to be every reason for PDT to be introduced to most dermatology centres and, in Dundee, we are hoping to facilitate the introduction of PDT at other centres in Scotland, giving support and advice where necessary.

Photodiagnostics in the photobiology unit Fluorescence diagnosis Fluorescence spectroscopy, or optical biopsy, was first used to study innate human autofluorescence, and is currently used for the early detection of cancer and for monitoring PDT [14—16]. Since Alfano, a number of spectroscopy systems have been developed for detection of both autofluorescence and ALA-induced PpIX fluorescence [17,18]. Early systems tended to be large and difficult to operate, but recently a compact fluorescence spectrometer system has been developed by Gustafsson et al. [19], which utilises newly available GaN diode lasers and miniature spectrometers. We have used a similar system to investigate photobleaching of ALA-induced PpIX in vivo on the skin of healthy volunteers during illumination with coherent red and blue light sources at a variety of fluence rates. Description of instrument The system incorporates a 5 mW GaN diode laser emitting at 405 nm, which corresponds to the highest absorption peak of the PpIX spectrum. This laser is coupled via a 600 ␮m core optical fibre into an electronic shutter containing a BG38 glass filter that prevents small amounts of 635 nm light seemingly emitted by the laser from reaching the tissue (Fig. 2).

216

S.H. Ibbotson et al. tip. In order to prevent ambient light from contaminating the spectra or contributing to photobleaching, all measurements were carried out in a darkened room. The tissue was illuminated for a total of approximately 300 s, and spectra were obtained at the start of the illumination and subsequently at 15-s intervals. The spectra were displayed on the computer monitor and saved for further analysis. The dark spectrum, autofluorescence spectrum, PpIX fluorescence spectrum and photoproduct fluorescence spectrum, obtained through direct measurement, formed the basis spectra for the least squares fitting, carried out on all acquired spectra within the experimental data sets, as the least squares fit coefficients for the component spectra were found to be more reproducible than peak height measurements alone.

Fig. 2. Photograph showing apparatus used to perform photobleaching measurements.

To prevent reflected laser light from saturating the spectrometer, light from tissue was filtered using a 455 nm long-pass filter. The remaining light enters a compact grating spectrometer with a resolution of 10 nm. The excitation and detection arms of the system were connected to the two branches of 600 ␮m core bifurcated optical fibre. Interchangeable fibre optic probes were used to allow decontamination. The spectroscopy system is fully controlled using a laptop PC. Experimental procedure Prior to carrying out the experiments, 50 mg/cm2 of ALA cream (5-Aminolaevulinic Acid HCl 20%, w/w, in Unguentum Merck (Crawford’s Pharmaceuticals, UK)) was applied to the skin on the forearm of healthy volunteers (n = 4) at sites approximately 10 mm in diameter, and occluded under Finn® Chambers (Epitest Ltd Oy, Tuusula, Finland). Six to eight hours later, the discs were removed and excess ALA wiped away. Red or blue illumination light was obtained from a He—Ne laser and GaN diode laser and was applied to one of the photosensitised regions by placing the optical fibre probe in contact with the tissue. In order to provide uniform pressure against the tissue surface the distal end of this fibre was fixed inside a perspex cylinder with a 20 mm diameter, which was flush with the fibre

Results Experiments confirmed, firstly, that lower fluence rate irradiance photobleaches more quickly than would be predicted by the light dose alone, and secondly, that in comparison with red light, the same fluence rate of blue light illumination increases the rate of photobleaching within superficial tissue layers by five to six times. To determine the capacity of a given light source to induce photobleaching, it is useful to define a figure of merit. In this work, we chose the inverse of the product of incident light intensity and the 50% decay time. If the capacity of a fixed light dose to induce photobleaching did not depend upon the wavelength or intensity of the light, then this product would be a constant. Fig. 3 shows the figure of merit plotted against the light intensity, for both red and blue illumination. The

Fig. 3. The reciprocal of the product of the 50% decay time of the PpIX fluorescence and the intensity of the light delivered to the skin, for red and blue light delivered at different intensities.

Photodynamic therapy in dermatology capacity of a fixed dose of either red or blue light to cause photobleaching increases with decreasing intensity. Perhaps a more useful comparison of red and blue illumination is to consider what relative intensities result in the same photobleaching rate. The dotted contour of iso-decay rate shown in Fig. 3 shows that a blue light intensity of 5 mW/cm2 gives the same rate of photobleaching rate as the typical red light PDT intensity of 100 mW/cm2 .

In vivo detection of PpIX fluorescence in non-melanoma skin cancer The use of non-invasive or minimally invasive in vivo fluorescence spectroscopy has been demonstrated in several cases in skin [20—25], and in other organs [26—29]. Although the optical detection of PpIX in tissues is governed by sophisticated models that have to account for the penetration of the excitation and the emitted photons and on the complexity of the optical properties of the tissue in first approximation, one can realistically consider that the fluorescence intensity of PpIX is proportional to the uptake of ALA into the cells [30]. This is even more realistic when the goal of the experimental approach is to quantify relative (instead of absolute) changes in fluorescence intensity [25,31—33]. Under this assumption, we investigated the effect that surface preparation by either gentle abrasion or curettage versus no preparation of NMSC lesions had on the uptake of ALA, and ultimately on the clinical outcome. Instrumentation A home-built instrument was assembled for the excitation and detection of PpIX fluorescence. The instrument employed a bifurcated liquid guidelight. The proximal end of the bifurcated guidelight used to excite PpIX, was coupled to an irradiation monochromator (Bausch & Lomb, 500 mm grating monochromator, Type 33-86-45) through a home built brass adaptor. The proximal end of the bifurcated liquid guidelight used to detect PpIX fluorescence was optically coupled to a microspectrometer (MicroParts VIS-LIGA) with a diode array detector. The distal end of the bifurcated fibre, where the excitation and emission guidelight join, was gently placed against the skin of each patient. A quartz spacer 1 mm thick (Hellma, Southend-onSea, UK) was positioned between the end of the optical fibre and the skin in order to (a) increase the collected signal and (b) protect the fibre. The excitation source was represented by the collected output of the irradiation monochromator. The excitation wavelength and the width of the slits of the

217 monochromator were selected to obtain an excitation beam at 405 ± 5 nm. The fluorescence of PpIX was then recorded between 450 and 700 nm. Several in vitro calibration procedures were carried out to determine the optical and spectroscopic characteristics of the instrument. The potential advantage of this instrument is the fast acquisition time. The combination of the sensitivity of the microspectrometer and the diode array detector allowed the acquisition of the entire in situ fluorescence spectrum in 2.5 s. Patient selection The study was performed with local ethics committee approval. Eleven patients with a total of 13 NMSC lesions were enrolled in the study. Eight lesions were BD and five lesions were superficial BCC. The selected lesions were at least 1.5 cm in diameter and located in flat anatomic areas (back, lower leg, shoulder and arms). Nodular BCCs and lesions that were in regions with high curvature were not investigated. Methods For each lesion the same procedure was followed. (1) Patients were informed and the detail of the study was explained. (2) For the patients who decided to participate in the study, written consent was obtained. (3) Fluorescence spectra were recorded on the selected lesion and on proximal normal skin. Three spectra per region were averaged. (4) The selected lesion was then virtually divided in two halves. One half did not receive any surface preparation, the other half was randomly assigned to be prepared with curettage or abrasion. (5) A second set of fluorescence spectra was taken from each half and adjacent normal skin. Three spectra per region were averaged. (6) A topical preparation containing 20% ALA (w/w) in Unguentum Merck (Crawford’s Pharmaceuticals, UK) was applied and left on the entire lesion under occlusion for 4 h (BD) or 6 h (superficial BCC). (7) After removal of the ALA preparation another set of fluorescence spectra were taken from each half of the lesion and from adjacent normal skin. (8) Patients subsequently underwent routine PDT as described earlier (125 J/cm2 at 120 mW/cm2 using Diomed® 630 nm laser). (9) Immediately after treatment the last set of fluorescence spectra were collected from each half of the lesion and from adjacent normal skin. Three spectra were averaged for each region. Spectra were analysed with the software provided by the manufacturer of the microspectrometer. Intensity was calculated both as the area of the emission between 500 and 700 nm or as the value of the emission maximum.

218 Results At the excitation wavelength selected for this study skin autofluorescence can be entirely attributed to collagen and elastin cross-links [34,35]. The autofluorescence of normal and abnormal skin before any surface preparation shows the tail of the emission of collagen and elastin from 450 to 600 nm. As expected, the autofluorescence intensity of NMSC was, on average, 35% lower than the intensity of the surrounding normal skin [34]. This is consistent with the destruction of the connective tissue by tumour, even when the tumour is classified as superficial [34]. After surface preparation there was a further reduction of the autofluorescence of the abnormal tissue, with a slight further reduction of the half of the lesion that was prepared with abrasion or curettage. After incubation with ALA the autofluorescence appeared to be unchanged and the large emission peak of PpIX appeared at around 635 nm. Using this analysis there was no statistically significant difference in the intensity of the prepared and unprepared halves of the lesions. The prepared half (regardless of the method) showed an intensity of 86 ± 35 a.u. while the unprepared half showed a fluorescence intensity of 81 ± 42 a.u. t-test analysis established that fluorescence was not statistically significant in the two halves (p > 0.5). If, instead, we analyse the data as the ratio between the fluorescence of PpIX at 635 nm and the intensity of the autofluorescence at the same wavelength as measured before the application of ALA, we found a slight difference between the two halves of the NMSC lesions, which is now statistically significant. The ratio calculated for the half of the lesion that was prepared by curettage or abrasion was 8.3 ± 3.4, whereas the ratio calculated for the half of the lesion that was not prepared was 7.1 ± 2.8. In this case the t-test revealed a statistically significant separation between the two (0.01 < p < 0.05). After PDT the intensity of PpIX disappeared completely according to complete photobleaching of the drug [36]. No significant effect on autofluorescence occurred after PDT. The clinical outcome observed at 3 months indicated that there was no difference between the prepared and unprepared halves of the NMSC lesions. Discussion We assumed that the fluorescence intensity of PpIX is proportional to the uptake of the ALA. Under this assumption, our conclusion is that surface preparation of NMSC does not significantly affect the uptake of ALA. There are possible explanations for these findings: (a) ALA is able to penetrate the depth of the tumour regardless of the surface of the lesion; (b) in superficial lesions such as the ones in-

S.H. Ibbotson et al. cluded in this study, the penetration of ALA is always sufficient to produce enough PpIX; (c) only small amounts of ALA, and therefore PpIX, are necessary to produce tumour damage. However, we cannot rule out that ALA (under such long incubation periods) will penetrate at a higher rate in the prepared half of the lesion and then diffuse laterally into the unprepared half of the lesion. Obviously this study should be repeated on a larger number of lesions, however, our preliminary results seem to indicate that surface preparation may not be crucial for an optimal clinical outcome.

The study of photosensitisers in in vitro cell culture models We used primarily skin-derived cell cultures to investigate the effects of different classes of photosensitisers; ranging from the porphyrin-based molecules, to hypericin, which is a potent photosensitiser obtained from the herb Hypericum perforatum. Our group (and others) have shown that PDT results in extensive DNA damage, ranging from simple strand breaks to base modification (Fig. 4). This DNA damage is regardless of the intracellular localisation of the photosensitiser. The mediators of this damage have not yet been identified, but are most likely reactive species generated by type II and type I mechanisms. Work with hypericin in immortalised keratinocyte cultures has demonstrated that chelating iron and using peroxide inhibitors does not greatly reduce this damage, implying a different mechanism from that of exogenously added oxidants such as H2 O2 (Fig. 5). Despite the fact that DNA damage seems to be secondary to, and not involved in the phototoxic effect, the discovery that PDT causes DNA damage in vitro has raised the question as to whether this poses a potential risk; either from light exposure to patients after systemic administration of the drug, or from tumour cells that survive the treatment, but with altered DNA. Factors such as the exact nature of this damage; potential lifetime exposure to photosensitisers; and protective advice issued to photosensitive patients will have to be taken into account in attempting to answer this question. However, individuals with naturally high levels of photosensitisers (e.g. porphyria patients) have not been reported to be at an increased risk of developing skin cancers, compared to the normal population. Many photosensitisers, upon irradiation, can kill cells via apoptosis or necrosis in a dose-dependent fashion. Resistance to apoptosis and development

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Fig. 5. Prevention of hydrogen peroxide (H2 O2 )-induced DNA damage, but not hypericin-PDT induced damage by the metal ion chelator 1,10-phenanthroline (Phen).

affect the relative resistance of a cell to apoptosis. Ongoing work in our laboratory is attempting to answer this question; preliminary study of hsps and enzymes of lipid metabolism, often expressed by the cell in reaction to stress, as a survival response, have shown that the factors governing a cell’s reaction to PDT are complex and multifactorial. However, once these pathways are understood it may be possible to identify markers that could be used to predict the effectiveness of PDT outcome. Moving outward from the study of photosensitisers at the intracellular level, we are also investigating the pharmacokinetics of photosensitisers Fig. 4. Using the comet assay to measure the DNA damage response in HaCaT keratinocytes following treatment with Photofrin and monochromatic red light (630 nm). Nuclei have been stained with the fluorescent DNA binding dye, ethidium bromide. Image A: Undamaged nuclei treated with 630 nm red light, but not with Photofrin. Image B: Damaged nuclei treated with Photofrin and 630 nm red light. Because the broken ends of DNA migrate away from the main body of the nucleus, a typical ‘‘comet’’like image is seen. The extent of migration, and thus DNA damage, can be quantified by image analysis.

of chemoresistance is a characteristic of tumour cells. The fact that PDT can induce apoptosis and be used against chemoresistant tumours makes the modality interesting not only as a method of treatment, but also as a tool to understand how certain intracellular signalling pathways are subverted in cancer cells (Fig. 6). The sensitivity of a cell to undergo apoptosis in response to PDT varies widely depending on the cell type. Recent interest has focussed on whether factors such as p53 status, or levels of stress proteins such as the heat shock proteins (hsp) may

Fig. 6. HaCaT keratinocytes undergoing apoptosis (arrowed) in response to treatment with hypericin and broad-band red light. The cell nuclei have been stained with the fluorescent DNA binding dye Hoechst 33342. Nuclear fragmentation and condensation can be seen clearly.

220 in vivo. By combining this information and that obtained from fluorescence microscopy of tissue samples, we hope to understand more about the bioavailability and behaviour of the photosensitiser in the target organ, which in turn will allow us to plan useful in vitro experiments to test our hypotheses. Finally, although photosensitisers are traditionally considered as having no effect in the absence of light, there is no doubt that many of them (porphyrins, hypericin) influence manifold cellular functions in the dark. This non-photodynamic activity of photosensitisers may influence the outcome of PDT, and may also have advantages in cancer treatment as it may sensitise tumour cells to other forms of chemotherapy. In conclusion, investigation of the mechanisms of action of photosensitisers and preliminary preclinical screening of novel compounds can be performed on cell cultures in vitro. By adopting this approach, the mechanisms underlying PDT action can be identified in individual cell types, or in cocultures of cell types. Combining the information obtained from these model systems, and comparing it to the pharmacokinetics and bioavailability of photosensitisers in vivo can help optimise the therapeutic window and identify target molecules that could be used as markers of efficacy of PDT. Tumour cells have the ability to evolve and evade death in response to some treatments; identifying the mechanisms by which they do this (and how PDT can subvert this) allows us to target them (e.g. by another class of photosensitiser, or other chemotherapeutic agent) in a combinatorial approach to improve the outcome of PDT and reduce the chance of recurrence of disease.

Nursing/technician management in photodynamic therapy Photodynamic therapy (PDT) as a treatment modality presents the Nurse Specialist and Photobiology Technician with a unique challenge. The Scottish Photodynamic Therapy Centre is a truly multi-specialty ‘‘virtual’’ centre with Neurosurgery, Respiratory, Gastroenterology, Hepatobiliary, Head and Neck, Urology, Dermatology and a full research laboratory with our own Photobiologist. The ‘‘virtual’’ unit is so called as it is spread out throughout the various departments in Ninewells Hospital, Dundee. PDT is carried out both topically and systemically, therefore presenting the need for careful preparation, delivery of treatment and close

S.H. Ibbotson et al. follow-up. We have formulated new protocols and established specially darkened ward, anaesthetic, recovery and high dependency unit (HDU) rooms to prevent any unwanted photoreactions occurring whilst the patient is in the hospital. Patient counselling is second only to the treatment, and we have designed and produced patient, staff and support protocols for each of the PDT treatments we carry out. Detailed counselling involves careful assessment of patients’ activities of daily living (ADL) in the sole aim of planning to assist the continuation or improvement of quality of life. We encourage our patients to become part of ‘‘the team’’ in which they willingly assist us in continuing research and phototesting to increase our understanding of photosensitivity issues associated with each of the commonly used photosensitisers. We have established a unique team which meets frequently and enthusiastically shares ideas and treatment possibilities. The NHS staff at Ninewells, Macmillan and community nurses all assist in the continued post procedure support.

Summary This report summarises the varied PDT activities which occur in the Scottish Photodynamic Therapy Centre, and in particular has focused on the use PDT in Dermatology, both for treatment and also with respect to PD. The Centre is well-established and offers routine and experimental treatments to a wide range of different cancers and even nononcological diseases, particularly with respect to the skin. Topical PDT should be readily available to Dermatology Departments, and this can be facilitated by improvements in photosensitisers, light delivery, cost of drug and light. Training courses are centred at the major skin PDT units in Dundee and Falkirk. We see topical PDT as an exciting and increasingly used effective therapy for superficial nonmelanoma skin cancers and dysplasia, and there is now a considerable body of evidence to show that treatment is at least as effective as conventional treatments for these skin diseases, with superior healing and cosmetic outcome. Published guidelines and recent approval by the Scottish Medicines Consortium for the use of topical PDT in selected cases further improves the profile of this readily used outpatient-based therapy. Clinical and laboratory studies are ongoing to clarify the mechanism of PDT, and this is also an exciting area of development.

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Acknowledgement We would like to thank the Barbara Stewart Scottish PDT Centre for supporting photodynamic therapy in Dundee.

Appendix A. The Scottish Photodynamic Therapy Centre based at Ninewells Hospital & Medical School, Dundee, Tayside In the year 2000, we were approached by a charity (Barbara Stewart Cancer Trust). This Trust had been founded by Mrs Barbara Stewart and her husband Mr Alf Stewart, following her personal experience of an uncommon form of lung cancer which was treated successfully with PDT by Professor Keyvan Moghissi in Goole, England. It was the Charity’s intention that such therapy should be available for Scottish patients locally. Having repeatedly travelled to England they decided to develop a Scottish service and invited bids from a range of hospitals in Scotland. Dundee was successful in its bid and established a dedicated centre for provision of both PDT and PD within the Photobiology Unit. Although the original idea was to have a lead clinical endoscopist/oncologist who could treat multiple types of cancer, this appointment was not possible; accordingly, the Photobiology Unit (as part of photodermatology) took on responsibility for the dayto-day organisation and delivery of laser administration, photophysics and nursing advice skills with the development of multiple PDT protocols for different types of internal and skin cancer. The basic plan was to produce a virtual PDT Centre with a core administrative, nursing and photophysics function based within the Photobiology National Service (Fig. 7). These core skills were provided to clinicians with a special interest in a range of internal cancers. The technology for provision of care involves endoscopy skills with PDT beTable 2

Fig. 7.

ing administered by photophysics technicians with patients treated as per written protocols by the Centre-based nurse. The mission statement of the Centre was to provide treatment for patients from throughout Scotland as well as educational and technical support for clinicians and other Scottish Centres who were developing an active interest in this area. Postgraduate educational activities have extended to provide local, national and international educational events. An active programme of research study has taken place within the Centre in relation not only to technical and nursing support services, but also to the study of efficacy in relation to optimising PDT for various types of skin cancer. The combination of science and clinical skills is seen as a key aspect of the multidisciplinary team. By January 2004, three years after the Centre opened, 437 (1900 treatments) patients with skin cancer and 113 patients (191 treatments) with internal malignancies (Table 2) had been treated/investigated. The importance of cooperation and constructive development, with contributions by clinicians and scientists, not only within the Centre but also within different parts of Scotland and England (particularly with Professor Keyvan Moghissi and Professor Hugh Barr), had

Number of patients/treatments over period January 2001—January 2004.

Skin SYSTEMIC Brain Hepatobiliary Gastrointestinal Oral Bronchial

No. of patients

No. of treatments

Comment

437

1900



38 8 10 19 38

62 11 10 38 70

Of which 18 (patients and treatments) were PD — — Of which 12 patients/17 treatments were lichen planus —

222 enabled the Centre to develop PDT and PD for a wide range of internal and cutaneous malignancies.

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Photodynamic therapy in dermatology: Dundee clinical and research experience.

Topical photodynamic therapy (PDT) is increasingly accepted and used as a highly effective treatment for superficial non-melanoma skin cancer and dysp...
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