Fluorescence Detection (FD)
Tuesday, April 04, 2006
Fluorescence detection (FD) is gaining interest in different
areas of medicine. In dermatology, 5-aminolevulinic acid (ALA) and
methyl 5-aminolevulinate (MAL) are topically applied in the
majority of cases. After penetration into the tumor cell,
photoactive porphyrins (PAP) are formed according to the
physiological haem biosynthesis. PAP accumulate selectively in the
neoplastic cells and emit light in the form of fluorescence when
exposed to light of appropriate wavelength and energy.
This procedure has been developed in parallel to photodynamic
therapy (PDT). In FD, fluorescence of porphyrins is used to detect
tumor tissue. PDT is a relatively new treatment modality for
neoplasms such as epithelial tumors of the skin.
In 1900, Raab demonstrated for the first time that dyes, e.g.
acridine, in combination with light were able to sensitize or to
kill microorganisms, e.g. paramecia. Some years later, the oxygen
dependency of this reaction was postulated and the term
'photodynamic action' was described. Successful treatment of skin
disorders, e.g. condylomata lata, lupus vulgaris, and various skin
tumors, with eosin and white light proved the efficacy of PDT.
Policard used the characteristic brick-red fluorescence of
porphyrins, e.g. hematoporphyrin, for tumor detection (FD) in 1924
for the first time. The predominant porphyrin fluorescence in tumor
tissue was confirmed by several investigators in humans and
animals. A hematoporphyrin derivative (HpD) consisting of porphyrin
derivatives was shown to be even more preferentially stored in
carcinomas. Until 1980, HpD was the most frequently applied
porphyrin compound in PDT. Photofrin (a mixture of
dihematoporphyrinester and ether, DHE; Ipsen Pharma GmbH,
Ettlingen, Germany) and Photosan-3 (Seehof Laboratorium GmbH,
Wesselburen, Germany) are the only approved drugs for systemic PDT.
However, the systemic administration of photosensitizer induced
generalized phototoxicity. Tumor-selective PDT with porphyrins was
obtained in 1990 by applying the porphyrin precursor
δ-aminolevulinic acid (ALA) topically. Exogenous administration of
ALA bypasses the rate-limiting enzyme of heme synthesis, ALA
synthase, which synthesizes ALA from glycine and succinyl CoA.
Thus, ALA treatment induces an increase in tissue porphyrin levels,
especially in neoplastic tissue. However the ALA molecule is
hydrophilic, thus limiting the into the epidermis and accumulation
into tumor tissue.
Therefore, modifications of ALA-molecules were tried
experimentally and, finally, methyl 5-amino-levulinate (MAL), which
contains an esterified carboxyl group that doesn't carry a negative
charge under physiologic conditions, was developed. The higher
lipophilicity of esterified forms permits a more effective
penetration of cutaneous tissue. Studies show evidence of greater
selectivity for neoplastic tissue with MAL.1-3
The efficacy of MAL/ALA-PDT in the treatment of skin tumors was
proven by several studies. Actinic keratoses (AK) in particular
were shown to be highly sensitive to topical MAL/ALA-PDT.
FD with ALA-induced porphyrins was shown to be capable of
differentiating bladder carcinomas from the adjacent normal
We examined the ALA-induced porphyrin fluorescence in various
dermatologic disorders, particularly skin tumors. A correlation was
observed between the clinically detectable fluorescence extension
and the tumor margins examined histopathologically.
Technique of FD
MAL (or ALA 10-20% in an ointment vehicle; 50-200 mg
ALA/cm2) is applied to cutaneous lesions under occlusive
foil to enhance tissue penetration and to avoid photobleaching.
After 4-6 h, intralesional porphyrin formation is evaluated by the
emission of red fluorescence during irradiation with Wood's light
(370-405 nm; Hanau, Fluotest).
Figure 1. Superficial basal cell carcinoma
with ill-defined borders (A) and detection by FD (B)
Fluorescence of Cutaneous Tissues in MAL/ALA-FD
Epidermal neoplasms such as basal cell carcinoma (BCC), squamous
cell carcinoma (SCC), Bowen's disease (BD), AK, and extramammary
Paget's disease show an intensive, uniform red fluorescence. All
other melanotic or amelanotic, benign or malignant tumors, such as
malignant melanoma (MM), lentigo senilis, verruca seborrhoica, and
nevus cell nevus, demonstrate no or only minimal fluorescence. In
all verrucae vulgares, fluorescence is absent. Psoriatic lesions
also revealed bright fluorescence, which is, however, often
inhomogeneous and sometimes absent. Plaques of mycosis fungoides
exhibit intermediate fluorescence intensity (Table 1).
|Tissues Exhibiting Bright Fluorescence After
Application of MAL/ALA
|Basal cell carcinoma
|Squamous cell carcinoma
|Possibilities of FD
|Easy to perform
|Useful modality to detect neoplastic tissue
|Preoperative delineation of ill-defined tumors
|Efficacy control of PDT or other treatment modalities
Intralesional Porphyrin Enrichment
In FD with MAL or ALA, increased ALA-induced porphyrin
biosynthesis was shown in neoplastic, hyperplastic, and also in
inflamed tissues. It is postulated that MAL/ALA treatment induces a
high ALA enrichment and a selective accumulation of porphyrin
metabolites in tumors. The mechanism of preferential intratumoral
uptake of precursors and photosensitizers is still not fully
understood. In the case of MAL/ALA, active transport is the most
likely explanation, but passive diffusion may be operative as well.
Enzymatic differences between normal and neoplastic tissue such as
a lower activity of the ferrochelatase, which in erythropoietic
protoporphyria leads to an accumulation of protoporphyrin, seem to
be less effective in ALA-induced porphyrin sensitization. The level
of synthesized porphyrins depends mainly on the amount of MAL/ALA
penetrating the skin to neoplastic cells. Thus, we assume a reduced
MAL/ALA penetration or uptake in lesions such as verruca vulgaris
and verruca seborrhoica. In the case of psoriatic lesions, the
hyperkeratotic areas may limit the penetration of MAL/ALA and of
MAL/ALA-Induced Porphyrin Fluorescence in Normal Skin
Uninvolved skin shows different fluorescence intensities
depending on the anatomical area and MAL/ALA application time. The
higher fluorescence intensity on the face, axilla, or groin as
compared to the trunk or extremities is probably due to increased
bacterial flora that also produces relatively high porphyrin
levels. In these body areas, fluorescence in normal skin may
interfere with that in neoplastic skin, but differentiation is
still possible due to the higher fluorescence intensity in tumor
tissue. The fluorescence intensity in normal skin is increased by a
longer MAL/ALA exposure time, and preliminary data show that
shorter application times of MAL/ALA, e.g. 3 h, or pretreatment
with an erythromycin-containing cream facilitate and improve the
differentiation between tumor and normal tissue fluorescence.
FD for Preoperative Planning and Control of Tumor Therapy
In anatomically difficult sites, such as the nose or the ear and
especially in pretreated skin, the detection of recurring skin
tumors can be facilitated by FD. In general, all deeply fluorescing
areas probably represent neoplastic tissues, as proven by
histopathology. The clinical fluorescence corresponds well with
histological borders of the tumor. MAL/ALA-FD allows the
delineation of clinically ill-defined tumors and the detection of
tumor relapse or new tumors that were not clinically
- Fritsch C, Homey B, Stahl W, et al. Preferential relative
porphyrin enrichment in solar keratoses upon topical application of
δ-aminolevulinic acid methylester. Photochem Photobiol.
- Gardlo K, Ruzicka T. Metvix (PhotoCure). Curr Opin Investig
Drugs. 2002 Nov;3(11):1672-8.
- Peng Q, Soler AM, Warloe T, et al. Selective distribution of
porphyrins in skin thick basal cell carcinoma after topical
application of methyl 5-aminolevulinate. J Photochem Photobiol
B. 2001Sep 15;62(3):140-5.