Original Paper Dermatology 2013;227:214–225 DOI: 10.1159/000353775

Received: March 20, 2013 Accepted after revision: June 17, 2013 Published online: October 15, 2013

Correlation between Protoporphyrin IX Fluorescence Intensity, Photobleaching, Pain and Clinical Outcome of Actinic Keratosis Treated by Photodynamic Therapy Filippo Piffaretti a Matthieu Zellweger a Behrooz Kasraee b Jérôme Barge c Denis Salomon b Hubert van den Bergh a Georges Wagnières a a

Medical Photonics Group, Swiss Federal Institute of Technology, SB-ISIC, Lausanne, b Faculté de Médecine Genève, Geneva, and c Photoderma SA, Froideville, Switzerland

Abstract Background: Photodynamic therapy (PDT) with Metvix® is a good therapeutic option to treat actinic keratosis, but it presents drawbacks (pain, lesion recurrences, heterogeneous outcome), emphasizing the possible need to individualize treatment. Objective: We assessed whether PDT clinical outcome and pain during treatment were correlated with protoporphyrin IX fluorescence intensity and photobleaching. Methods: 25 patients were treated by Metvix PDT. The outcome was evaluated after 1.3 (±0.4), 7.6 (±1.8), 13.2 (±1.2) and 33.6 (±3.0) months. After administration of Metvix, red light (632 ± 10 nm) was delivered with a light-emitting diode panel device. The outcome was assessed on a cosmetoclinical scale. Results: All patients who showed a fluorescence level before PDT treatment above a certain threshold had a complete recovery at 33.6 (±3.0) months. Conclusion: Our approach could be used to individualize PDT treatment based on the pretreatment fluorescence level, and to predict its long-term outcome. © 2013 S. Karger AG, Basel

© 2013 S. Karger AG, Basel 1018–8665/13/2273–0214$38.00/0 E-Mail [email protected] www.karger.com/drm

Introduction

Actinic keratosis (AK) is a hyperproliferation of the keratinocytes, usually caused or stimulated by solar radiation. AK is the most common skin condition treated by dermatologists. The skin lesions are clinically identified as rough, scaly, crusted, pink to reddish brown papules. They are found on sun-exposed skin, especially on the head, neck, forearms, and hands. The patients at greatest risk are middle-aged and elderly men, with fair skin and blue eyes, especially those with occupations involving prolonged exposure to UV light, such as farmers, sailors and construction workers [1]. Considerable efforts and means are provided for AK treatment, and it has been estimated that the annual costs generated by this condition were of the order of USD 1 billion in the USA (2004 data) [2]. Three main reasons justify the treatment of AK. The most important one is the difficulty to distinguish the AK lesions that will eventually evolve into the dangerous squamous cell carcinoma from the ones that will spontaneously disappear. Secondly, AK lesions are treated to provide relief for symptoms such as tenderness or itching. Finally, treatment may be desirable for cosmetic reasons. Georges Wagnières Medical Photonics Group, Swiss Federal Institute of Technology Ecole polytechnique fédérale de Lausanne SB-ISIC, Station 6, CH–1015 Lausanne (Switzerland) E-Mail georges.wagnieres @ epfl.ch

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Key Words Fluorescence · Imaging · Protoporphyrin IX · Actinic keratosis · Photodynamic therapy · Metvix® · Pain · Bleaching

Curettage, cryosurgery and topical treatments such as application of 5-fluorouracil and photodynamic therapy (PDT) are the most common AK treatments [3, 4]. Curettage is effective for almost all clinical types of AK but has potential complications such as scarring and infection, and local anaesthesia is frequently required before the procedure. Cryosurgery is cheap and generally well tolerated but may result in transitory hypopigmentation and is limited to patients with only a small number of tiny, localized lesions. The topical application of 5-fluorouracil has a high success rate but is generally not well tolerated. It is also characterized by prolonged erythema in the treated zones. Finally, the topical treatment by PDT is well tolerated and presents excellent cosmetic outcomes. It is particularly well suited for areas of field AK [4, 5], and can be associated with only 2 main drawbacks, such as a relatively high cost and the pain experienced during skin irradiation, which needs to be properly managed by the clinician. A PDT treatment requires a photosensitizer (PS). In dermatology, the PS of choice is the group of molecules called photo-activable porphyrins (PaPs), the most widely studied member of this group being protoporphyrin IX (PpIX). For the sake of clarity, we will use PpIX as the abbreviation for ‘PaPs, including PpIX’ throughout. In particular, the PDT procedure applied along the present study relies on the administration of a precursor of PpIX, Metvix® (approved for AK and basal cell carcinoma in Europe) or Levulan® (approved for AK in the USA). This topical administration is very convenient for AK treatment, as the precursor molecule has to diffuse to a depth of approximately 100 μm from the skin surface [6] to reach the hyperplastic and dysplastic cells, where it induces the biosynthesis of PpIX. To improve the homogeneity of the distribution of PpIX, it is common to carefully scrape out the lesion’s scales and crust typical of this condition. Some groups report much longer preparation procedures [7]. The PpIX is then activated by illuminating the skin with non-coherent lamps or light-emitting diode (LED) arrays. Red light illumination allows treatment of skin lesions to a depth of 2 mm [8–10]. AK lesions are also commonly treated with blue light (405 nm), which penetrates efficiently to 200–300 μm in the skin [4, 11–14]. Metvix-based PDT is an efficient treatment modality for AK and is also characterized by excellent cosmetic outcome. Several studies report clearance rates varying from 68 to 89% after a single treatment, with follow-ups of up to 12 months [10, 12, 13, 15–17], or 2 courses of treatment separated by a 3-month interval [18]. Despite these results, lesion recurrences and heterogeneous clini-

cal outcome are common. This may be due to the fact that PDT efficacy depends on several parameters, including the PS’s microscopic and macroscopic distribution at treatment time, the light dose, the tissular oxygen distribution, the irradiance and the tissue’s optical properties at the treatment wavelength. Therefore, it is difficult to ensure that optimal treatment parameters are applied during each individual treatment [19]. Different strategies were developed to study and monitor the light dosimetry of PDT [20–25] or to monitor other AK treatment modalities [26], but none of them is currently in routine or systematic use. The parameters involved in the cascade of reactions leading to the PDT effect may vary dynamically and interdependently during the treatment. Furthermore, sophisticated technologies and challenging measurement modalities, with efficacies/ reliabilities that have yet to be proven in many cases, are not well accepted in the clinical field, which explains the limited use of these strategies (e.g. PDT dose monitoring by measuring the 1O2 luminescence intensity [23]). Finally, there is a possibility that measuring the PS’s photobleaching (i.e. the reduction in the fluorescence intensity of PpIX, caused by the PDT irradiation) may be a useful way to evaluate – and adjust – the administration of the PDT light dose [27–31]. If demonstrated, this simple way to individualize treatment parameters would render more complicated approaches unnecessary. The objective of this work was to determine if the PDT clinical outcome and the pain induced during the treatment of AK were correlated with PpIX fluorescence intensity and bleaching, measured before and after irradiation. As a consequence, our report could also provide useful information to determine whether the PDT light dose used in the treatment of AK might be individually adjusted with a simple, optical monitoring strategy, which could possibly also help to predict the treatment outcome. To reach these goals, an imaging device was designed and adapted to the measurement of skin lesion fluorescence in a clinical environment.

Outcome of Actinic Keratosis Photodynamic Therapy

Dermatology 2013;227:214–225 DOI: 10.1159/000353775

This work was designed in collaboration with the Hôpitaux Universitaires de Genève in order to study the PDT treatment of AK lesions and its follow-up. This work aimed at quantitatively recording and correlating PS fluorescence and the PDT-induced PS photobleaching with patient age, roughness of the lesion’s surface, incubation period, pain experienced during the treatment, and eventually the follow-up of the clinical outcome. The PS was PpIX induced by topical Metvix administration, according to the standard guidelines (drug-light interval = 180 min). All subjects

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Materials and Methods

Lesion Identification Each AK lesion was identified by feeling the roughness of the lesion surface with the index finger. The lesions were then graded in a scale from 1 to 3, where 1 implied a barely detectable difference with respect to normal skin and 3 meant a rough surface. Subsequently the borders of the lesions were demarcated by following the roughness threshold grade 1, with a green permanent marker (Steadler® Lumocolor-M). Therefore, the determination of the roughness and the border of each lesion allowed us to study the influence of the latter over the intensity of the PpIX fluorescence measured before PDT illumination. Lesion Preparation Prior to Metvix administration, all the lesions were gently scraped clean with a curette (Bruns® 17-003-0) to efficiently remove the stratum corneum and the hyperkeratotic tissue. This lesion preparation is a standard procedure [4, 32–35] to facilitate the diffusion of the precursor into the lesion, and ensure homogenous light distribution in the tissues. This procedure was not intended to be a therapeutic curettage, and generally no bleeding was observed. Successively, all AK lesions were precisely indexed in order to localize them several months after the treatment. Additionally, the fluorescence imaging device’s orientation (device described below) and angle with respect to the lesion were precisely defined and kept constant for each fluorescence measurement. These rules of conduct had to be strictly defined since we aimed at comparing fluorescence intensities of the lesions at 2 different times during the procedure, namely just before and just after the PDT treatment. Due to the limited field of view (diameter: 40 mm, see ‘Monitoring Imaging Apparatus’ section) a precise positioning of the monitoring apparatus by the operator was required in order to allow a repeatable observation of the lesion. After this preliminary preparation procedure, a 1-mm layer of 16% Metvix cream (Galderma SA, Paris, France) was administered over the entire surface of the lesion. The lesion was then covered with aluminium foil and affixed with a bandage (Micropore®, 3M)

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to avoid interaction between parasitic daylight and the PpIX accumulated within the lesion. The patients were asked to wait for 180 min before illumination. All the information gathered during this preparation phase (number of lesions, surface roughness, delimitation and the drug-light interval) was reported and documented on the patient’s anatomical sketch. PDT Treatment and Pain Assessment The PDT light dose was delivered with a standard red LED panel delivery device (Aktilite® CL128, 632 ± 10 nm, Galderma [36]). The irradiation time (540 s, 36 J/cm2) and the distance between the lesion and the LED panel (approx. 5 cm) were kept constant. To further homogenize the illumination, each lesion was irradiated perpendicularly. Consequently, for patients presenting multiple lesions, each of them was illuminated separately. PDT illumination sessions are frequently associated with moderate to severe pain. Because pain is the main adverse effect for this treatment modality, a tablet of anti-inflammatory drug (Méfénacide®, 500 mg) was given to each patient 1 h before treatment [35, 37–40]. Additionally the lesions were sprayed with cold water (approx. 5 ° C), during illumination, to further reduce the burning sensation. In addition to this appropriate management of the pain sensation, the protocol provided the possibility to interrupt the PDT illumination for breaks of 1–2 min, to give the patient time to recover. This happened in about two thirds of cases. The pain experienced by the patient was quantified with a visual analogue pain scale [41, 42]. For each lesion, the patients were asked to rank their pain sensation during illumination from 1 to 10.  

 

Evaluation of the Clinical Outcome The clinical outcome was assessed by ranking the lesions’ evolution on a 6-level scale derived from the scale proposed by Szeimies et al. [43] and described in detail in table 1. It should be noted that ranks from 2a and above are complete responses, and that the difference between ranks from 2a to 2d is cosmetic only. The clinically relevant outcomes are 0, 1 and 2a-d, expressing a recurrence, a response in less than 75% of the lesion surface, and a complete response, respectively. Monitoring Imaging Apparatus The monitoring system was designed to be simple, ergonomic, and as much as possible made of commercially available parts to be accepted in the clinical field. Figure 1 depicts the main parts of this monitoring system. A standard clinical photo camera (Nikon®-D90 coupled with the objective Nikon® AF D 50 mm f/1.4) was used as fluorescence imaging sensor. A custom illumination apparatus was developed to excite the inspected lesions with a constant and homogeneous illumination. A stable and spatially homogeneous excitation is important to record quantitative fluorescence images. The custom illumination apparatus was mainly composed of 4 functional components: the emission filter, the excitation diodes, the excitation homogenizing tube, and the fluorescence reference ring. Of all parts of our set-up, only the homogenizing tube and the filter support are not commercially available and had to be prepared in house, using simple parts. The emission filter stage involves a long pass filter (Kodak® Wratten filter No. 9) mounted on a sliding support to easily switch from conventional white light reflectance imaging to fluorescence imaging. The diode excitation stage was designed with 2 sets of 5 diodes placed radially along the circumference of the excitation

Piffaretti /Zellweger /Kasraee /Barge / Salomon /van den Bergh /Wagnières  

 

 

 

 

 

 

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were undergoing AK treatment by PDT as part of their normal dermatological treatment, and the only impact that our work had on their otherwise routine treatment was that 2 images of their lesions were taken (one before and one after treatment). The work was compliant with the Hôpitaux Universitaires de Genève guidelines valid at the time the images were taken. From September 2009 to January 2010, 25 patients (mean age = 72 ± 11 years, 77 AK lesions) scheduled for PDT treatment of their AK lesions accepted to be included in this work. The treatment outcome was evaluated at 3 different times, scheduled at 1.3 months (±0.4), 7.6 months (±1.8), 13.2 months (±1.2), and 33.6 months (±3.0) after the PDT session. Patients included in this work were specifically informed about the clinical procedure (both the routine clinical procedure warranted to treat their AK lesions and the additional 2 images) and gave their consent. During the entire set of PDT treatments, a single and experienced physician was in charge of the patient management, lesion evaluation and PDT treatment. Therefore, all the gathered measurements were less affected by subjective differences in lesion preparation, in lesion roughness judgement, and differences in the handling of the specific PDT and monitoring devices. The clinical outcome at each time point was also assessed by a single physician.

Color version available online

a

b

Fig. 1. Overall schematics of the monitoring system used to detect the fluorescence and bleaching of AK lesions by fluorescence imaging. a Fluorescence monitoring apparatus: 1 = standard photo camera Nikon®-D90; 2 = emission filter; 3 = excitation diodes; 4 = excitation homogenizing tube; 5 = reference ring. b Zoom picture over the reference ring (5): the fluorescence image of the reference spot is shown in the upper right corner of the right image; λex. = 405 nm, filtered by a Kodak® Wratten filter No. 9, LP460.

Level

Description

0

Recurrence on the same spot

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Response in less than 75% of the lesions/lesion surface

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Complete response in at least 75% of the lesions/lesion surface and poor cosmetic outcome (extensive scarring, atrophy or induration)

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Complete response in at least 75% of the lesions/lesion surface and fair cosmetic outcome (slight to moderate scarring, atrophy or induration)

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Complete response in at least 75% of the lesions/lesion surface and good cosmetic outcome (moderate occurrence of redness or change in pigmentation)

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Complete response in at least 75% of the lesions/lesion surface and excellent cosmetic outcome (only slight occurrence of redness or change in pigmentation)

tube. The first set emits a broad white light emission (superbrightled® RL5-W6030, 400–660 nm) to record conventional white light reflectance images. The second emits a narrow blue band light (superbrightled® RL5-UV2030) to efficiently excite the PpIX accumulated within the lesion and thus record fluorescence images. This stage was powered by a commercially available power supply (24 V DC, 20 mA). With the help of a switch, it was possible to select between the two illumination modes. An excitation filter (Edmund® NT52–530) had to be placed in front of the blue diodes to reject the parasitic fluorescence of the printed circuit board where the diodes where mounted. The homogenizing tube is an aluminium tube having a highly diffusing layer (Delrin®) on its internal surface. The length was

optimized to obtain a compact and lightweight (total weight approx. 1.7 kg) system. This system delivers a flat excitation profile over the entire focal plane. The fluorescence reference ring was fastened to the distal end of the homogenizing tube (fig. 1b). These stable fluorescent spots localized in the image focal plane allow the normalization of the fluorescence of the inspected lesions. These spots contain stable fluorescing pigments (Lentalux®) embedded in epoxy resin. Pits located around the ring were filled with increasing concentrations of this mixture.

Outcome of Actinic Keratosis Photodynamic Therapy

Dermatology 2013;227:214–225 DOI: 10.1159/000353775

Image Processing The fluorescence images were analyzed with an image-processing software (ImageJ® version 1.41). Figure 2 depicts the analysis

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Table 1. Assessment scale of the outcome of the PDT treatment for AK lesions

Color version available online

a

b

areas. A region of interest was selected by following the lesion borders. The mean fluorescence intensity of the lesion was computed in the region of interest, and simultaneously the total lesion surface was evaluated. Two circular areas were also chosen: the first area contained the reference fluorescing spot, measured at the beginning of each session to assess the status of the measurement device on a given day; the second circular area contained a lesion-free portion of the field, and gave us a measure of the intensity of the tissue autofluorescence before (TAutoB) and after (TAutoA) PDT. To compare the results from different images, the fluorescence intensities of the lesion and of the control zone were first normalized by the mean fluorescence intensity of the reference spot (FRef). The lesion’s fluorescence bleaching (BPS) was calculated by subtracting the normalized fluorescence calculated after PDT (FA) from the corresponding value measured before PDT (FB), see equations 1–3 below. The PpIX contribution to the recorded fluorescence signal before and after irradiation is symbolized respectively by FPSB and FPSA and the stable fluorescence intensity of the reference spot by FRef. FB = (FPSB + TAutoB)/FRef

(1)

FA = (FPSA + TAutoA)/FRef

(2)

BPS = FB − FA

(3)

Results

The normalized fluorescence intensity measured before PDT illumination FB, the fluorescence photobleaching BPS, and other clinical parameters were monitored before and after the PDT of 77 AK lesions in 25 patients. The drug-light interval was not always exactly 180 min due to the constraints of the clinical settings. It was on average 170 min (±21). One patient (with 6 lesions) was excluded from the interpretation of results at 33.6 months ± 3.0 because his skin bore such a large number of AK lesions (‘field cancerization’) that it proved impossible to assess 218

Dermatology 2013;227:214–225 DOI: 10.1159/000353775

if a lesion was a recurrence or a new lesion. He was included in the early follow-ups, however, as the initial treatment outcome could be assessed. During the first follow-up consultations at 1.3 months (±0.4), 12 patients (44 lesions) were controlled. During the second (7.6 ± 1.8 months) 12 patients (46 lesions), during the third (13.2 ± 1.2 months) 5 patients (17 lesions) and during the last scheduled follow-up consultation (33.6 ± 3.0 months) 10 patients (36 lesions) were controlled. Lesion Roughness and Patient Age versus Fluorescence Intensity We studied the influence of the lesion roughness and of patient age on the PpIX fluorescence signal FB. To emphasize the influence of lesion roughness we subdivided them into 3 subgroups depending on their roughness grade (see ‘Lesion Identification’ section for details). Sixteen lesions were characterized by a roughness grade of 1, 16 by a grade of 2, and 14 by a grade of 3. The influence of the patient age was studied by analyzing the fluorescence signal FB as a function of the age of each patient in the year 2010. No correlation was observed (fig.  3) between fluorescence intensity and lesion surface roughness (fig. 3a) or patient age (fig. 3b), suggesting that those two parameters are not relevant for the tissular production of PpIX. Pain versus Fluorescence Intensity To study the relationship between the pain experienced by patients and the fluorescence signal FB, all the treated lesions were considered independently from other clinical parameters (n = 77). Figure 4a shows the pain experienced during the PDT illumination as a function of the fluoresPiffaretti /Zellweger /Kasraee /Barge / Salomon /van den Bergh /Wagnières  

 

 

 

 

 

 

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Fig. 2. Fluorescence images of the PpIX accumulated within AK lesions recorded before (a) and after (b) the PDT session (λex. = 400 nm). Metvix was administered 180 min before illumination. Ctrl = Control zone; Ref = fluorescence reference spot; L = lesion.

0.8

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60 70 80 Patient age (years)

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Fig. 3. Normalized fluorescence intensity measured before treatment as a function of lesion surface roughness (a) and patient age (b).

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cence signal measured just before illumination (FB). Figure 4b plots the pain as a function of the fluorescence signal multiplied by the lesion surface (see ‘Image Processing’ section). The number of lesions characterized by the same pain rank is mentioned in round parentheses on the right side of the plots. Despite the small number of lesions, likely insufficient to draw firm conclusions, the results

show an absence of correlation (fig. 4). This is probably linked to the strong patient subjectivity in pain evaluation and likely to other uncontrolled parameters, e.g. differences in the physiology or lesion innervation, heterogeneous PAP build-up, bias due to treatment interruptions, etc. The distribution of the experienced pain confirms that PDT is associated with moderate to severe pain. In fact,

Outcome of Actinic Keratosis Photodynamic Therapy

Dermatology 2013;227:214–225 DOI: 10.1159/000353775

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Fig. 4. Pain experienced by the patient during PDT irradiation as a function of the fluorescence intensity measured before treatment. a Pain as a function of the normalized fluorescence. b Pain as a function of the normalized fluorescence × lesion surface.

Clinical Outcome The patients were controlled as follows: during the first follow-up consultation at 1.3 months (±0.4) 12 patients (44 lesions), during the second (7.6 ± 1.8 months) 12 patients (46 lesions), during the third (13.2 ± 1.2 months) 5 patients (17 lesions) and during the last followup consultation (33.6 ± 3.0 months) 10 patients (36 lesions). In figures 6 and 7, the clinical outcome of all the lesions controlled during the 4 follow-up consultations is plotted as a function of PpIX fluorescence before treatment (FB, fig. 6), and of PpIX photobleaching (BPS, fig. 7). The symbols refer to the clinical outcomes assessed during the first consultation (1.3 ± 0.4 months), the second (7.6 ± 1.8 months), the third (13.2 ± 1.2 months) and the fourth (33.6 ± 3.0 months) after PDT treatment. Apart from 1 outlying point, all patients showing a fluorescence level before PDT treatment above a certain threshold (0.4 for fluorescence intensity; 0.2 for fluorescence bleaching with our set-up) had a complete recovery during the last follow-up visit. If this is confirmed in larger cohorts of patients, this suggests that one could predict long-term treatment outcome by measuring the pretreatment fluorescence level. 220

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0.4 0.3 0.2 0.1 R2 = 0.91

0 0.1

0.2

0.3 0.4 0.5 0.6 Normalized fluorescence (AU)

0.7

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Fig. 5. PpIX fluorescence photobleaching as a function of the nor-

malized fluorescence measured before treatment.

It is noteworthy that figures 6a and 7a show points spread between level 1 and 2d, likely showing that whilst most lesions are treated, some take time to fully heal. Figures 6b and 7b show mainly points on the two highest levels (2c and 2d), showing a very good treatment efficacy. Figures 6c and 7c show points spread over the entire scale, showing either some incomplete responses, or some recurrences, and figures 6d and 7d show no points on level 2d (best cosmetic outcome with full recovery). A different subset of the group of patients has been controlled at each point in time, explaining the differential distribution of points on the various graphs of figures 6 and 7.

Discussion

Our results show that the use of either the normalized fluorescence before treatment, or the magnitude of photobleaching during treatment are valuable parameters to provide information on the clinical outcome of PDT for AK indications including, to a certain extent, that in the long term. Studying the correlation between the PpIX fluorescence intensity and/or its photobleaching during PDT of AKs with the pain perceived by the patient during the treatment and the clinical outcome is an interesting and important topic. Our report has shown that the 4 parameters cannot be easily correlated, although PpIX fluorescence intensity, photobleaching and the clinical outcome can be. Surprisingly, to our knowledge [29, 44–46], Piffaretti /Zellweger /Kasraee /Barge / Salomon /van den Bergh /Wagnières  

 

 

 

 

 

 

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Fluorescence Photobleaching versus Fluorescence Intensity The measured normalized fluorescence FB was correlated with the calculated photobleaching BPS following PDT (see equation 3 in the ‘Materials and Methods’ section). All the treated lesions (n = 77) were considered for this analysis. The results show a strong linear correlation (fig. 5), suggesting that the rate of PpIX photobleaching measured with our system is approximately constant across all the PDT sessions. The PpIX molecules accumulated in the epidermis may indeed interact efficiently with the illumination light and the tissue oxygen, which in part diffuses from the atmosphere, so that an efficient fluorescence photobleaching occurs. Detailed analysis of the results presented in figure 5 demonstrates that the linear regression has a slope of approximately 1, but that the regression line is affected by an offset of approximately 0.1. This is likely to be due to photobleaching of the skin autofluorescence or could possibly suggest the existence of an unbleachable part of the accumulated PpIX or, alternatively, the presence of unbleachable photoproducts.

PpIX photobleaching (AU)

60% of the treated lesions were ranked with a pain feeling on the scale smaller than 5 (mild to moderate pain), and almost all lesions (85%) were ranked below 7.

2d Clinical outcome (AU)

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tion of the normalized fluorescence measured before illumination of AK lesions treated by Metvix-based PDT and controlled at 1.3 ± 0.4 months (a), 7.6 ± 1.8 months (b), 13.2 ± 1.2 months (c) and at 33.6 ± 3.0 months (d) after the PDT session.

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Fig. 6. Clinical outcome as a func-

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only a very limited number of publications have addressed this issue in detail. Our report demonstrates that this correlation, although not perfect, does exist, including in the medium to long term (>33 months), and that it can be measured with a set-up mainly made of commercially available parts. The PpIX fluorescence intensity was measured before and after PDT with an imaging device developed in our laboratory, mainly with standard parts. Although this device provides, in our conditions, reproducible, background-free and linear (data not shown) values of PpIX fluorescence, some of its features can still be improved for a more convenient and routine use in the clinic. It is worth noting that should this method gain wide clinical acceptance, one could expect the set-up to be made simpler and even more ergonomic than our research device. The irradiance of the excitation light should and will be increased, typically by one order of magnitude, to reduce the time necessary to record the fluorescence images with an acceptable signal-to-noise ratio. Such a modification

will not significantly increase the PpIX photobleaching since the fluorescence acquisition time will be reduced accordingly. By analyzing the recorded images and the plots reporting the fluorescence intensity, we show that important intra- and interpatient variations affect these measurements. These variations are probably due to parameters influencing the PpIX accumulation as reported by Dögnitz et al. [8] or Wiegell et al. [35], such as the precursor diffusion rate, the lesion’s temperature, temporal and spatial variations of the metabolic activity, and differences in the density of active cells and local tissue optical properties. Unfortunately, the limited number of patients involved in the present report does not enable us to subdivide the treated lesions according to their anatomical subgroups, or any other relevant subgroup. We studied the influence of the patient’s age and of the AK lesion’s roughness on the PpIX fluorescence intensity. As reported in the literature, we observed that the patient age is not a parameter that significantly influences the

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Clinical outcome (AU)

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biosynthesis of PpIX [47, 48]. This conclusion is somewhat surprising since we were expecting an impact of skin aging on the precursor penetration and on the metabolic activities that play a role in the PpIX build-up. Similarly, no correlation was observed between the roughness of the lesion surface and the PpIX fluorescence intensity. To our knowledge the correlation between these two parameters has not been studied before. However, the work of Wiegell et al. [49] demonstrated a positive correlation between the measured AK-PpIX fluorescence and the lesion’s redness and inflammation. The rationale that brought Wiegell et al. [49] to study this correlation is somewhat similar to ours, but the results cannot be compared. Indeed the concepts of redness and inflammation are at best very poorly correlated with the concept of ‘roughness’ (data not shown). Similarly, Smits et al. [50] reported that there was a positive correlation between the PpIX fluorescence intensity and the ‘severity’ of AKs. According to the classification proposed by Anwar et al. [51], Cockerell and Wharton [52], and Yantsos et al. [1], the grade of AKs 222

2a

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tion of PpIX photobleaching of AK lesions treated by Metvix-based PDT and controlled at 1.3 ± 0.4 months (a), 7.6 ± 1.8 months (b), 13.2 ± 1.2 months (c) and at 33.6 ± 3.0 months (d) after the PDT session.

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Fig. 7. Clinical outcome as a func-

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PpIX bleaching (AU)

is closely related to the proliferation of atypical keratinocytes involving different percentages of the epidermis (i.e. the grading is tightly related to the amount of dysplastic and hyperplastic cells within the lesion). As a consequence, the absence of correlation we have observed between the PpIX fluorescence and the lesion roughness indicates that this feature is not correlated with the ‘severity’ grading. In the present piece of work, we could not demonstrate a correlation between the pain experienced by the patient during the PDT and the normalized fluorescence intensity. Although several groups have extensively studied the pain sensation during the PDT illumination, no clear agreement has been found up to now regarding this correlation. Some studies demonstrated a statistically significant correlation [35, 39, 53], whereas others, as in our case, did not observe a clear correlation [49, 54]. This discrepancy is probably due, at least in part, to the difficulty of measuring and documenting the sensation of pain. The fact that we gave pain relievers to the patients also introPiffaretti /Zellweger /Kasraee /Barge / Salomon /van den Bergh /Wagnières  

 

 

 

 

 

 

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Clinical outcome (AU)

2d

When the clinical outcome is evaluated at 33.6 months following PDT, high levels of fluorescence intensity or PpIX photobleaching correspond to a more potent therapeutic effect. These encouraging results are supported by other publications that report correlations between the photobleaching and clinical outcome [27, 29, 31, 44–46], albeit for shorter follow-up periods in most cases. It should be noted that most of these studies reported that the fluorescence photobleaching, and not the fluorescence intensity, is well correlated with the clinical outcome. This discrepancy could be explained by the fact that these groups used either ‘point’ measurements, instead of imaging measurements and/or because they measured the fluorescence qualitatively instead of using a scientific camera presenting a linear response, as was the case in our work. With our imaging system, it appears that a PpIX fluorescence intensity of 0.4, and a PpIX fluorescence photobleaching above 0.2, will lead to a positive clinical outcome at 33.6 months. Only a subset of the group of treated patients attended each of the follow-up consultations. The scanty participation was due to the fact that the studied cohort was composed by an elderly population (mean age = 72 ± 11 years): several patients declined to attend some of the scheduled follow-up visits, sometimes for health reasons, sometimes considering that it was not necessary. Moreover, at least 3 of the treated patients passed away (due to nondermatological causes) in the period between the PDT treatment and the last follow-up visit. When we measured the fluorescence of the PaP accumulated in the lesions just before treatment, we assessed the selectivity of the PaP for the lesions. This allowed us to assess our protocol to prepare the lesions, and the delineation of the lesions to be treated. We assigned a selectivity level based on a 3-point scale, namely: presence of fluorescence in the lesion and in its full periphery (no selectivity); presence of fluorescence in the lesion and in parts of its periphery (partial selectivity); presence of fluorescence only in the lesion and not in its periphery (full selectivity). Measured on 76 lesions, we found that 41 (54%) had a partial selectivity, 35 (46%) had full selectivity, and no single lesion scored on the lowest point of no selectivity at all. This shows that the preparation protocol we chose is efficient whilst remaining simple, and that validates this part of the treatment methodology, also in the case of private practices. In conclusion, we can claim that, in the case of AK treated with Metvix, the fluorescence measured before PDT and the PpIX fluorescence photobleaching are both correlated with clinical outcome. These prelimi-

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duced an element of discrepancy that cannot be evaluated. More generally, pain is a difficult parameter to study because it is subject to large intra- and interpatient variations. In fact, pain assessment is strongly affected by subjective and emotional aspects that depend on the patient’s earlier experiences of pain, and on the degree of anxiety of the patient during treatment [41, 54–56]. It should be noted that the heterogeneity of the AK lesions is very likely to be the most important factor playing a role in this discrepancy. It should also be noted that we offered the possibility to patients to either cool the lesion with water spraying during treatment, or to interrupt the treatment to ease the pain. It is likely that both elements introduce an unquantifiable bias in our measurements. One could also imagine that a patient who asked for the treatment to be interrupted because of the pain would expect the pain to be high again when the treatment resumes. In general, the PpIX fluorescence and the pain seem to be correlated in normal tissue and early AKs, whereas this correlation no longer exists for more advanced lesions [39, 40]. Considering the limited number of lesions in our report, the lesions could not be adequately subdivided in different anatomical zones. This may also degrade the correlation between the PpIX fluorescence and the pain since it has been reported that anatomical innervation differences may play an important role [37, 57]. The strong linear correlation presented in figure 5 between PpIX photobleaching and the normalized fluorescence before PDT is an expected, but interesting result. The correlation, with a slope of 1, is in agreement with several studies reporting that, in well-oxygenated conditions, the PpIX and its photoproducts can be efficiently and completely bleached during the illumination [19, 28, 31, 58, 59]. It is also worth noting that this correlation is observed for all the treated lesions, independently of the anatomical place and independently of all other possible tissue parameters. In addition, the limited deviation from the regression line (R2 = 0.91) further supports the possibility to use the fluorescence intensity, measured before treatment, as a surrogate of the PpIX photobleaching following PDT. Finally it should be noted that the intercept of this regression line with the y-axis appears not to be exactly at (0, 0), but near (0.1, 0). This is likely to be due to photobleaching of the skin autofluorescence [60] or could possibly suggest the existence of an unbleachable part of the accumulated PpIX or alternatively, the presence of unbleachable photoproducts. This interesting conclusion deserves further studies to clearly determine the exact origin of this effect.

nary clinical results support the point that a satisfactory clinical outcome can only be reached if the PpIX fluorescence intensity or the PpIX fluorescence photobleaching is above a certain threshold (0.4 or 0.2 in our specific case). However, surprisingly no correlation was observed in the present report between the pain and the PpIX fluorescence measured before treatment, as well as between this last parameter and the lesion roughness and the patient’s age.

Acknowledgements This work was supported by the Swiss National Science Foundation (grant No. 205320-130518) and funded in part by the J. Jacobi Trust. The authors also wish to acknowledge the contributions of Thomas Glanzmann and Eddy Forte.

Disclosure Statement The authors declare that they have no conflict of interest.

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Correlation between protoporphyrin IX fluorescence intensity, photobleaching, pain and clinical outcome of actinic keratosis treated by photodynamic therapy.

Photodynamic therapy (PDT) with Metvix® is a good therapeutic option to treat actinic keratosis, but it presents drawbacks (pain, lesion recurrences, ...
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