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Surgery and Cosmetics

Percy Lehmann, MD

Fluorescence Detection (FD)

Percy Lehmann

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 tissue.

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)

Superficial basal cell carcinoma with ill-defined borders

Superficial basal cell carcinoma with detection by FD

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).

Table 1


Tissues Exhibiting Bright Fluorescence After Application of MAL/ALA
Basal cell carcinoma
Squamous cell carcinoma
Solar keratosis
Paget's disease
Bowen's disease
Psoriatic lesions

Table 2


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 exciting light.

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 detectable.

References

  1. 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. 1998 Aug;68(2):218-21.
  2. Gardlo K, Ruzicka T. Metvix (PhotoCure). Curr Opin Investig Drugs. 2002 Nov;3(11):1672-8.
  3. 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.
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