ARTICLE
In recent years several innovative therapies of cancer have been under
investigation. Photodynamic therapy (PDT) is a new, promising treatment
modality for malignancies, with potentially high effectiveness and low
morbidity.
Photodynamic therapy refers to light activation of a tumor-localizing
photosensitizer to generate highly reactive oxygen intermediates, causing
tissue injury and necrosis by oxidizing essential cellular components.
Topical PDT using delta-aminolevulinic acid (ALA) involves photosensitation
with endogenous porphyrins and activation with visible light. ALA is a
small molecule which easily penetrates the abnormal stratum corneum overlaying
many skin tumors, while it hardly penetrates the normal skin. In situ
conversion of ALA to protoporphyrin IX (PpIX), an extremely potent photosensitizer
and fluorescence emitter, is accomplished in normal and neoplastic keratinocytes
to a different degree, by enzymes in the heme pathway, resulting in selective
accumulation in neoplastic tissue and tissue-specific phototoxic effects
[1, 2].
The emission of light following absorption of incident photons by chromophores
is termed fluorescence. PpIX accumulation is characterized by a bright
orange-red fluorescence and can be visualized in the skin when illuminated
with light of appropriate wavelengths. Since the PpIX accumulation is
a gradual process, fluorescence kinetics could be used to determine the
ideal time point post application to initiate the irradiation of the tumor.
In a previous study, we showed that the erythema inspection and quantification
analysis, as a result of tissue-specific phototoxic effect with topical
ALA-PDT, provides a reliable predictor of the therapeutic outcome [3].
The purpose of this study was to assess the clues for optimization of
topical ALA-PDT conditions, to treat epithelial tumors such as basal cell
carcinomas (BCC) and precancerous lesions such as actinic keratoses (AK),
by determination of the accumulation efficiency of the photosensitizer
versus time and the localization efficiency of the photosensitizer
within the tumor versus time.
Materials and methods
Patients
After informed consent, 29 patients (21 men and 17 women, median age
72 years) with 37 histologically proven malignant and precancerous skin
lesions (17 BCC and 20 AK), median diameter 1 cm, were included in this
study. Of the 17 BCC (2 superficial, 7 nodular, 1 nodular-micronodular,
1 micronodular, 1 nodular-cystic, 1 pigmented and 4 metatypical- basal
squamous), median diameter 0.9 cm, 13 were located on the face, 2 on the
neck, 1 on the ear lobe and 1 on the trunk. Of the 20 AK (9 atrophic,
5 acantholytic, 3 hypertrophic and 3 bowenoid), 18 were on the face and
scalp and 2 on the extremities. No lesion had been previously treated.
ALA-PDT procedure
After cleansing the whole area with sterile saline solution, a freshly
prepared w/o cream containing 20% ALA (Sigma Co, St Louis) was applied
with a margin of about 1cm beyond the skin lesion and kept under occlusion
impervious to light for 3-6 hrs, followed by irradiation, without anesthesia
or sedation. The irradiation was carried out with a xenon lamp 300W, fitted
with bandpass filters selecting visible light. The exact moment of starting
the irradiation was deduced according to the findings of peak fluorescence
detection. In 13 BCC and 16 AK a session of PDT with light dose 30 J/cm2
was performed. In a second group of patients, tumors with clinical and
histological factors of poor responsiveness (large size and/or thickness,
pigmentation, marked cellular atypia) were included; four BCC and three
AK received 3 sessions of 60 J/cm2, one monthly (total light
dose 180 J/cm2), while 1 AK of an extremity received a total
light dose of 240 J/cm2.
Studies of fluorescence
The equipment used to image, measure and map the fluorescence intensity
of PpIX in the skin, as well as the color change evaluation by imaging
the light diffuse reflection, consisted of a thermoelectrically cooled
charge coupled device (CCD) color camera, a light source, a system of
optical filters, a frame grabber and a personal computer. The camera had
a wide responsivity, ranging from UV to NIR (320-1,150 nm). The selection
of imaging spectral band was performed with 6 optical filters adapted
on the camera and controlled by specially developed software, which was
also employed for the image calibration processing and analysis. The video
signal obtained from the output of the camera was digitized by a video
grabber, which has been installed on a Pentium personal computer 200 MHz,
with 16 MB RAM and graphics accelerator with 4MB video RAM. The monitor'
s display resolution capacity was 1,024 x 768 pixels, with color depth
24 bit (16.7 million colors). The tissue irradiation was carried out with
a 300 W xenon lamp fitted with bandpass filters to excite PpIX fluorescence
and white light respectively. The light was delivered to tissue via
a ring fiber-optic bundle placed in the front of the camera and surrounding
a zoom macro-lens.
The fluorescence images were recorded in a dark room and between the
fluorescence recordings the area of interest was kept under occlusion.
The power density of the light source was very low (approximately 0.5
mW/cm2) and the exposure time was very short, in order to avoid
possible photobleaching of the photosensitizer. For the study of fluorescence
kinetics, serial in vivo fluorescence images were captured in 9
patients (5BCC, 4AK), every 15 min for 7 hrs and every 2 hrs afterwards
until 24 hrs. For the remaining patients, fluorescence was detected between
2-7 hrs after ALA application. For any image, the medians of the fluorescence
intensity of the pixels which correspond to the lesion area were calculated,
after correcting for background autofluorescence and the sequential integrated
fluorescence signals were then plotted versus time. This correction
was obtained by subtracting the pre-ALA administration medians of autofluorescence
intensity. The maximal fluorescence intensity, as compared with the zero
time baseline intensity, is defined as photosensitizer accumulation efficiency
and the correlation of fluorescence spatial distribution with the area
of the lesion assessed with visual light, is defined as photosensitizer
localization efficiency. In order to better show the spatial and temporal
variation of the PpIX fluorescence (accumulation and localization efficiency)
we used pseudo-colors. The recorded alterations from the baseline are
expressed on a color scale, increasing from black (0%) to white (30%).
The detection of tumor tissue by imaging PpIX fluorescence with pseudo-colors
is based on the fact that each image consists of multiple pixels with
specific coordinates in the RGB color space [3].
The prerequisite for reproducible color measurements is the standardization
of the imaging conditions, such as illumination and capturing conditions.
In the case of diffuse reflection, calibration was performed against a
white perfect diffuser (BaSO4) and for fluorescence emission
imaging against a white fluorescence standard (Spectralon). In both cases
calibration against black was performed with the camera aperture closed.
When the calibration procedure ended, the acquisition was performed with
the stored calibration parameters, thus, all the real time displayed images
were calibrated in the spectral and the intensity domain. The compensation
for any spatial variation of the image intensity caused by the non-uniform
transfer function of the optics or by the non uniform illumination of
the subject was performed with shading correction.
Results
ALA-PDT
Seventeen BCC were treated. The overall cure response rate, without
recurrence was 70.6%. The follow up ranged from 20-35 months (mean 24
months).
Twenty AK were treated with ALA-PDT. The overall cure rate was 85%,
without recurrence and the follow up period was 26-48 months (mean 36
months) (Table I).
Fluorescence kinetics and spatial distribution
In vivo fluorescence kinetics evaluation over time showed that
most of the skin lesions developed maximum fluorescence emission intensity
(photosensitizer accumulation efficiency) between 4-6.5 hrs after ALA
application. There were considerable variations in both the rate of increase
and the maximum intensity of the fluorescence achieved, expressed in arbitrary
units, between tumors of the same type. We, therefore, varied the beginning
of the irradiation, adapting PDT planning to the patient lesion individualities.
Protoporphyrin IX fluorescence kinetics data of BCC and AK are presented
in Figure 1. In principle,
by fluorescence detection, BCC were characterized by high photosensitizer
accumulation efficiency, while AK had much lower photosensitizer accumulation
efficiency. The best localization efficiency of BCC was noted 3.5-5 hrs
after ALA application and thereafter a progressive decrease of fluorescence
intensity became evident on the tumor, as well as diffusion of fluorescence
outside the margins of BCC (Figs.
2 and 3). Despite the persistence
of satisfactory fluorescence intensity values until 14 hrs after ALA application
(Fig. 1), irradiation
starting was chosen to be early, 3.5-5 hrs, when high photosensitizer
accumulation efficiency and maximal localization efficiency on the BCC
was noted, ensuring the selectivity of the procedure (Figs.
1, 2 and 3).
In the case of AK, the fluorescence intensity after achieving the maximal
value (median 5 hrs) declined gradually within the margins of AK (high
photosensitizer localization efficiency) (Fig.
4). Irradiation, therefore was started based on the data of accumulation
efficiency, (approximately 5 hrs after ALA application (Figs.
1 and 4).
High spatial variability of fluorescence intensity was recorded on the
tumor surface, possibly attributed to the unequal distribution of atypical
cells within the tumor or to existing local variations of the tissue optical
properties. Twenty-four hours after topical ALA application hardly any
fluorescence was detectable in tumors, nor in surrounding normal skin.
The fluorescence kinetics study demonstrated that the findings were
reproducible.
Discussion
In the present study a high cure rate was proven in superficial skin
tumors such as BCC and precancerous lesions, such as AK. Some previous
clinical studies yielded high efficacy rates [1, 4, 5]. The perceived
advantages of ALA-PDT, such as localized and short-term photosensitivity
and rapid photo-degradation by light illumination have led this modality
to be very useful for the treatment of superficial non-melanoma skin tumors.
The success of treatment requires an optimal interplay among a number
of several parameters, such as drug and light doses, treatment sessions,
criteria of tumors and patient selection. Limited depth of light and/or
ALA penetration significantly limits PDT for hypertrophic AK and thick
nodular BCC and may offer remarkable risk of tumor recurrence in case
of inadequate photosensitizer concentration. A variety of strategies have
been used to improve the desired PpIX production such as iron chelators
[6] and to enhance the effectiveness of PDT using different wavelengths
and light doses [7, 8].
It becomes, however, clear that there is a need to monitor parameters
other than delivered light dose and irradiance and to correlate these
with ALA-PDT outcome. The determination of the time course of photosensitizer
fluorescence in skin tumors is crucial for effective photodiagnosis and
PDT.
Our imaging procedure is noninvasive, simple, reliable and reproducible
and sets no limits on the size or the shape of the area under examination.
Serial, in vivo and real-time measurements of fluorescence intensity
are of particular advantage when an endogenous photosensitizer is used,
which might be synthesized in various amounts over time in different tumors
of the same type, depending on the metabolic activity of the target cell.
This novel system facilitates further study of skin photobiology and ALA
pharmacokinetics.
Some authors, based on the principle that the intensity of the emitted
fluorescence is a function of the amount of the fluorochrome present,
claimed that the fluorescence of skin lesions after ALA application, using
a Wood's lamp or laser-induced fluorescence, provided an estimate for
prediction of PDT [1, 4, 8]. However, no direct correlation was found
between fluorescence intensity and clinical response by other authors
[6, 9]. On the other hand, the optimal time for PDT could be deduced from
the time-dependent concentration of PpIX, since fluorescence kinetics
is a temporal process. In order to maximize PDT effectiveness after application
of ALA, irradiation of the treatment area should occur at the time of
sufficiently increased concentration of the endogenous photosensitizer
within the lesion.
In our study, fluorescence emission evaluation
was shown to be an accurate diagnostic tool for BCC and AK, since a strong
fluorescence was observed in all lesions under examination. Wennberg et
al. found a good correlation between the fluorescence imaging in
vivo and histological mapping in 50% of BCC and a partial one in other
23%, four hours after ALA application [10]. Fritsch et al. compared
the macroscopical fluorescence intensity after topical ALA-application
by means of Wood's light, with the time-dependent tissue formation of
porphyrins, in epithelial skin tumors and normal skin, and recommended
irradiation 1-6 hrs after ALA-application [11]. However, awareness of
fluorescence time course kinetics is considered critical to the interpretation
of photosensitizer accumulation in skin tumors. Unfortunately, despite
the encouraging results of ALA-induced fluorescence kinetic studies in
animal models [9, 12, 13], little is known concerning the in vivo
porphyrin synthesis and elimination in tumor and normal tissue after topical
application of ALA, in humans. Differences in photosensitizer accumulation
and localization efficiency and transient selectivity may be most likely
attributed to a faster uptake or increased porphyrin production in tumor
versus normal keratinocytes, due to a higher demand for heme biosynthesis,
rather than a reduced activity of the enzyme ferrochelatase [14].
In vivo quantitative determination of the time course of the
porphyrin fluorescence is an essential pre-condition for an effective
PDT, offering valuable information about the optimal time delay for efficacious
and selective PDT. Tope et al. studied fluorescence kinetics in
BCC after oral administration of ALA and showed a full thickness PpIX
accumulation in all BCC histological subtypes and maximal tumor: normal
skin fluorescence ratios 1-3 hrs after ALA ingestion [15].
In our study, excitation of the photosensitizer was performed by light
of 425 ± 10 nm, which penetrates well in depth of about 0.1-0.15
mm, including the normal epidermis and superficial dermis [16]. Therefore,
an estimation of the fluorescence lateral extent in superficial skin lesions
could be achieved only by means of the in vivo kinetic study, whereas
deep situated tumors might not be accurately delineated.
Our finding (Figs. 2
and 3) that in BCC, contrary to AK, fluorescence diffuses
outside the margins of the visible lesion as the time lapses might indicate
a deeper tumor volume [15], or the presence of subclinical deeper seated
tumor nests, or finally be due to neovascularization in and out of the
tumor mass, to support the blood supply required for the increased metabolic
demand of a skin cancer. In conclusion, our results suggest that the optimum
irradiation time for BCC is approximately 3.5-5 hrs after ALA application
and for AK 5 hrs, taking into account both the parameters of photosensitizer
accumulation and localization efficiency (Figs.
1
to 4).
Established methods of treatment for BCC (excisional surgery and cryosurgery)
and for AK (cryotherapy and chemotherapy) are known to yield very high
cure response rates [17-19]. However, PDT is not invasive, has excellent
cosmetic results and is suitable for the treatment of multiple and large
lesions. It is particularly recommended for older patients or with poor
compliance, as well as patients with many other health problems, where
invasive methods are contraindicated.
We also emphasize the importance of further investigation in the field
of ALA-induced fluorescence kinetics, in order to optimize treatment protocols.
Article accepted on 27/3/00
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