The figure shows a Jablonski diagram. The length of the arrow indicates the energy of the photon (wavelength is written next to the arrow). The solid arrows indicate laser photons, and the dashed arrows indicate fluorescence photons. A, Conventional 1-photon excitation; B, simultaneous 2-photon excitation; C, stepwise 2-photon excitation of melanin fluorescence. A and B illustrate excitation suitable for measuring the conventional autofluorescence of skin tissue. The corresponding fluorophores show no absorption of 800-nm photons; therefore, the latter can be used for fluorescence excitation only in a simultaneous 2-photon absorption process. However, melanin has sufficient absorption at 800 nm and therefore can absorb 2 such photons in a stepwise absorption process (mechanism illustrated in panel C), which is substantially more effective than the mechanism shown in panel B. This way, the ultraweak fluorescence of melanin in skin, which otherwise is completely masked by the autofluorescence, is detectable. (For details, see Eichhorn et al.3)
A-C, Paraffin-embedded skin tissue samples (measured with 800-nm/2.5-ns pulses). A, Normally pigmented skin; B, nevus; and C, malignant melanocytic melanoma. Each tissue sample was measured at histologically proven reference samples, especially in panel B, a compound nevus, and in panel C, a superficially spreading melanoma. D and E, Ascending cells in the upper epidermis. D, In all of the 8 paraffin-embedded samples of the present study (E) in a histologically proven, paraffin-embedded sample of a nodular melanoma with transepidermal melanocytic migration (measured with 800 nm/2.5 ns pulses). The measurement points are integral intensity values via 16 nm (error bars indicate means [SDs] of measurements in 10 areas of the tissue type). The diameter of the measured skin area is 50 µm. The solid lines are for guidance only. An additional signal appears at 400 nm, if the measuring area is in the dermis, resulting from collagen. Under the present conditions of excitation, there is no contribution of paraffin to the fluorescence.
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Scholz M, Buder S, Kerl K, Dummer R, Garbe C. New Diagnostic Method for Lesions With Transepidermal Melanocytic Migration. JAMA Dermatol. 2014;150(6):654–656. doi:10.1001/jamadermatol.2013.8119
Transepidermal melanocytic migration (TEM) is a histological feature that is frequently observed in malignant melanoma but may also occur in nevi (eg, Spitz nevi, acral nevi). Pagetoid TEM is considered a key indicator of malignant disease. We refer to an unusual case report by Kerl et al1 in a young female patient. Eleven melanomas were originally diagnosed within 10 years, and in a comprehensive retrospective diagnosis, these could be reclassified as nevi. (See Kerl et al1 for details of the patient’s medical history and the context of this case.) Herein, we report on the application of a new diagnostic method for melanocytic lesions to the issue of TEM as melanoma indicator.
Recently, a new diagnostic method has been described for malignant melanocytic melanoma,2,3 which is based on the stepwise 2-photon excited melanin fluorescence (STPMF) of melanosomes. The mechanism of STPMF is illustrated in Figure 1; for a description of the method used see Eichhorn et al.3 This fluorescence shows a high information content. The STPMF spectra of the fluorescence from melanocytes (Figure 2A), from nevus cell nevi (Figure 2B), and from malignant melanocytic melanoma (Figure 2C) are characteristically different and diagnostically useful . The mechanism of STPMF is illustrated in Figure 1C; for a description of the method used (briefly, to measure melanin fluorescence in the skin, separated from the other fluorophores) see Eichhorn et al.3 Furthermore, nonaltered dermis is characterized by a specific signal from collagen, which occurs at the 400-nm second harmonic of the excitation radiation. With this collagen signal, both nevus components in the dermis (eg, the compound nevus) and melanoma invasion through the basal membrane can be characterized. This finding applies to the tissue in vivo4 and ex vivo2,4 and in histological preparations.3 The parents of the patients were informed about the study and provided written informed consent to a molecular workup of all tissues and to the publication of the case report. Institutional review board approval was not necessary in this setting.
In this this study, 8 histological preparations of specimens from the patient described by Kerl et al1 were examined with STPMF. According to the original diagnosis, the samples included 1 melanoma in situ, 2 melanomas in situ in conjunction with a compound nevus, 3 atypical compound nevi, 1 atypical nevus, and 1 compound nevus. For the STPMF measurement of fluorescence, the preparations were each covered with a measuring grid with 50-μm increments. In this way, the entire epidermis and the dermis, to a depth of up to 900 μm, were detected. The result per sample—depending on its size—was 250 to 1000 fluorescence spectra, each associated with a tissue region of about 30 μm in diameter.
No signs of melanocytic malignant degeneration were found in any of the 8 samples, including those from the lesions originally classified as melanoma in situ. The fluorescence spectra indicate uniformly benign compound nevi. These results correspond to the revised diagnostic findings of these samples described in the article by Kerl et al.1
It should be noted that the histological observation of TEM in the 8 samples is equivalent to detection of the occurrence of fluorescence of melanosomes in the upper layers of the epidermis using the method described herein. But, interestingly, the spectral properties of these melanosomes correspond to those from nevus cells, not to those from melanocytes (Figure 2). This finding agrees with the fact that the absence of melanosome transfer to the keratinocytes is characteristic of nevus cells. However, the latter usually form nests, which are missing in this case.
It is worth mentioning that in the case of TEM in histologically proven malignant melanomas, the fluorescence of the ascending cells corresponds to that of melanoma cells (Figure 2C). An example is given in Figure 2E, which shows the resulting fluorescence from TEM in a nodular malignant melanoma.
The results show that STPMF-based diagnosis in the histologically complex case of TEM gives clear results. This new method shows an early malignant melanocytic degeneration with high sensitivity, even before it is histologically detectable2; therefore, the complete absence of such melanoma spectra among the total of 5650 measured tissue areas of the 8 samples is a sure indication of their uniform benignity.
Corresponding Author: Claus Garbe, MD, Universitätsklinikum Tübingen, Zentrum für Dermatoonkologie, Liebermeisterstraße 25, 72076 Tübingen (firstname.lastname@example.org).
Accepted for Publication: September 5, 2013.
Published Online: April 23, 2014. doi:10.1001/jamadermatol.2013.8119.
Author Contributions: Dr Scholz had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Dummer.
Acquisition of data: Scholz, Kerl, Dummer.
Analysis and interpretation of data: Scholz, Buder, Garbe.
Drafting of the manuscript: Scholz.
Critical revision of the manuscript for important intellectual content: All authors.
Administrative, technical, and material support: Scholz, Dummer, Garbe.
Study supervision: Scholz, Buder, Dummer, Garbe.
Conflict of Interest Disclosures: None reported.
Funding/Support: The study was supported in part by the European Union and the Land Berlin (ProFit grant No. 10 152 586).
Role of the Sponsors: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Additional Contributions: Dr Scholz thanks Dieter Leupold, DSc, and Goran Stankovic, Dip-Ing (FH), LTB Lasertechnik Berlin, for initiating this work and for valuable contributions.