A case report of an unrecognized nevoid melanoma in a young woman--clinicopathological diagnostic challenge

Nevoid melanoma is a rare form of melanoma histologically resembling benign melanocytic nevi and may be overlooked in routine histological sections. Authors are presenting a case of a 31-year-old woman who presented with bizarre pigmented skin lesions in the area of the postoperative scar on the back where, 6 years earlier, a "nevus pigmentosus epidermo-dermalis" was excised and hystologically confirmed in outer institution. The lesions were surgically removed and histopathological findings were characteristic for nevoid melanoma. Additionally, specimen of primary removed lesion was reexamined and primary nevoid melanoma was then recognized, therefore indicating that the lesions our patient presented with are nevoid melanoma recidivisms. Extensive diagnostic procedures showed no signs of melanoma dissemination. Three months later, the patient returned for consultation and presented with two new brownish-pigmented papules in the area of the new postoperative scar. The lesions were excised and new nevoid melanoma recidivism was confirmed. The patient remained under the regular follow up and, almost 9 years after the removal of primary nevoid melanoma, followed by two cutaneous recidivisms, remains disease-free. This case aims to highlight the problematic area in the analysis of pigmented skin lesions where nevoid melanoma represents one of the clinical and pathological diagnostic challenges.     

Ultraviolet wavebands and melanoma initiation

In view of claims that ultraviolet radiation-emitting sunbeds are safe, or safe when they emit only longer wavelengths, research findings are reviewed here on the effects of ultraviolet wavebands A and B (UVA, 315-400 nm and UVB, 290-315 nm) on mutagenesis and carcinogenesis in skin, with particular reference to melanocytes and melanoma. Both UVA and UVB radiation have been shown to induce mutations, as well as mutagenic photoproducts such as cyclobutane pyrimidine dimers, in human skin. UVB can induce melanoma in susceptible mice and in xenografted human skin engineered to express melanocyte growth factors. There is evidence for photosensitization of melanocytes by melanin, especially pheomelanin. UVA can induce melanoma in pigmented fish, and melanocytic hyperplasia in pigmented opossums, but has not generally been tested for melanoma induction in pigmented mammals or in human skin. There is no experimental basis for a claim that UVA is safe, and recreational exposure to this known mutagen should be discouraged.     

Black lesions of the skin

Benign melanocytic lesions include lentigo, ephelid (freckle), pigmented nevus, sacral spot, blue nevus, and combined nevus and blue nevus. Malignant melanocytic lesions are melanomas, which arise from melanocytes at the epidermodermal junction, or, rarely, from blue nevi. They usually originate in brown plaques known as lentigo maligna, in pigmented nevi, or in normal skin. Melanoma is diagnosed clinically in less than 50 per cent of instances. Biopsy is therefore of great importance, since practically all melanoma can be cured by adequate early resection.     

Two-photon excitation fluorescence spectrum of the light-harvesting complex LH2 from Chromatium minutissimum within 650-745 nm range is determined by two-photon absorption of bacteriochlorophyll rather than of carotenoids

Two-photon fluorescence excitation spectra of the peripheral light-harvesting complex LH2 from the purple photosynthetic bacterium Chromatium minutissimum were examined within the expected spectral range of the optically forbidden S1 singlet state of carotenoids. LH2 preparations isolated from wild-type and carotenoid-depleted cells were used. 100-fs laser pulses in the range of 1300-1490 nm with an energy of 7-9 mW (corresponding to one-photon absorption between 650 and 745 nm) were used for two-photon fluorescence excitation. It was shown that two-photon fluorescence excitation spectra of LH2 complex from wild and carotenoid-depleted cells are very similar to each other and to the two-photon fluorescence excitation spectrum of bacteriochlorophyll a in acetone. It was concluded that direct two-photon excitation of bacteriochlorophyll a determines the fluorescence of both samples within the 650-745 nm spectral range.     

Discriminating nevus and melanoma on paraffin-embedded skin biopsies using FTIR microspectroscopy

FTIR microspectroscopy, in combination with cluster analysis, has been used to characterise skin tissues, in order to discriminate cancerous from non-cancerous ones. The main objective of this in vitro study was to demonstrate the applicability of infrared spectral imaging to separate, on paraffinised biopsies, pigmented nevi (benign skin lesions) from melanomas (malignant skin lesions). Infrared spectra were collected from paraffin-embedded samples of nevi and melanomas, without deparaffinisation. Despite the important contribution of the paraffin in these spectra, it was possible to find meaningful and discriminating spectral regions. Spectral imaging was first performed to localize different skin layers (dermis and epidermis). Spectra extracted from the images were subjected to hierarchical classification algorithm, which allowed the discrimination of melanomas from the nevi, using selected spectral windows that correspond to vibrations of DNA and melanin content. The diversity of skin lesions and direct accessibility to the skin make this organ an interesting field of investigation using this technique.     

The usefulness of c-Kit in the immunohistochemical assessment of melanocytic lesions

C-Kit (CD117), the receptor for the stem cell factor, a growth factor for melanocyte migration and proliferation, has shown differential immunostaining in various benign and malignant melanocytic lesions. The purpose of this study is to compare c-Kit immunostaining in benign nevi and in primary and metastatic malignant melanomas, to determine whether c-Kit can aid in the differential diagnosis of these lesions. c-Kit immunostaining was performed in 60 cases of pigmented lesions, including 39 benign nevi (5 blue nevi, 5 intradermal nevi, 3 junctional nevi, 15 cases of primary compound nevus, 11 cases of Spitz nevus), 18 cases of primary malignant melanoma and 3 cases of metastatic melanoma. The vast majority of nevi and melanomas examined in this study were positive for c-Kit, with minimal differences between benign and malignant lesions. C-Kit cytoplasmatic immunoreactivity in the intraepidermal proliferating nevus cells, was detected in benign pigmented lesions as well as in malignant melanoma, increasing with the age of patients (P=0.007) in both groups. The patient's age at presentation appeared to be the variable able to cluster benign and malignant pigmented lesions. The percentage of c-Kit positive intraepidermal nevus cells was better associated with age despite other variables (P=0.014). The intensity and percentage of c-Kit positivity in the proliferating nevus cells in the dermis was significantly increased in malignant melanocytic lesions (P=0.015 and P=0.008) compared to benign lesions (compound melanocytic nevi, Spitz nevi, intradermal nevi, blue nevi). Immunostaning for c-Kit in metastatic melanomas was negative. Interestingly in two cases of melanoma occurring on a pre-existent nevus, the melanoma tumor cells showed strong cytoplasmatic and membranous positivity for c-kit, in contrast with the absence of any immunoreactivity in pre-existent intradermal nevus cells. C-Kit does not appear to be a strong immunohistochemical marker for distinguishing melanoma from melanocytic nevi, if we consider c-Kit expression in intraepidermal proliferating cells. The c-Kit expression in proliferating melanocytes in the dermis could help in the differential diagnosis between a superficial spreading melanoma (with dermis invasion) and a compound nevus or an intradermal nevus. Finally, c-Kit could be a good diagnostic tool for distinguishing benign compound nevi from malignant melanocytic lesions with dermis invasion and to differentiate metastatic melanoma from primary melanoma.     

Nucleolar organizer regions in pigmented skin lesions. Value in the differential diagnosis of Spitz nevi.

Forty-one cases of typical melanocytic skin lesions (15 intradermal nevi, 14 Spitz nevi and 12 malignant melanomas) were used to investigate the value of staining of nucleolar organizer regions (NORs) in the differential diagnosis of such pigmented lesions. Histologic sections were stained by the silver colloid (Ag) method, with and without the prior use of a melanin blocking agent. There were statistically significant differences in the mean numbers of AgNORs per nucleus between the groups of lesions studied (1.658 for intradermal nevi, 3.0042 for Spitz nevi and 6.669 for malignant melanomas). Sections treated with potassium permanganate (melanin blocking agent) prior to staining showed an obvious increase in the AgNOR scores in all groups; this increase was highest for Spitz nevi. Although AgNOR staining allows a distinction to be made between intradermal nevi and malignant melanomas, the striking overlap between the counts for Spitz nevi and malignant melanomas precludes the use of this technique as the sole method for establishing the diagnosis of malignancy. Other clinical and morphologic data are especially required to make the diagnosis of Spitz nevi.     

Comparison of image analysis with flow cytometry for DNA content analysis in pigmented lesions of the skin.

The DNA ploidy of 85 melanocytic skin lesions was determined by flow cytometry (FCM) and interactive image analysis (IA) using nuclear extracts of paraffin-embedded tissue. Of the 85 lesions analyzed, 43 were malignant melanomas in different stages of evolution, 15 were dysplastic nevi, 11 were Spitz nevi, and 16 were other types of nevi. Some of the last had features of congenital nevi. Within the melanoma category, there was 42% aneuploidy by FCM versus 56% by IA. Of those melanomas aneuploid by FCM, all but one were aneuploid by IA. All dysplastic nevi, 10/11 Spitz nevi and 15/16 other nevi were diploid by both methods. One of the 16 nevi from the "other types" category was tetraploid by IA but diploid by FCM. A single Spitz nevus was tetraploid by FCM but diploid by image analysis. While our results suggest that interactive IA is potentially a more sensitive method than FCM for detecting aneuploidy in cutaneous pigmented lesions, it remains to be shown whether this will translate into better prognostic assessment of the biologic behavior of melanocytic neoplasms than provided by flow cytometric ploidy analysis.     

Tissue Factor Expression and Serum Level in Patients with Melanoma does not Correlate with Disease Progression

Not only does tissue factor (TF) play a crucial role in hemostasis and thrombosis, but it is also involved in tumor progression and metastatic potency in some malignant tumors. We evaluated the clinical relevance of TF expression in melanocytic tumors and TF serum level in patients with malignant melanoma. TF expression in benign and malignant melanocytic lesions was examined by immunoperoxidase staining in 20 nevi, 41 primary, and 24 metastatic melanoma lesions. TF was detected in 94, 95, and 100% of these lesions, respectively. The staining pattern was membranous and cytoplasmic both in nevi and melanoma cells. This finding was confirmed by western blot analysis using cultured human melanocytes, nevi cells, and melanoma cell lines. TF was also expressed on blood vessels in benign and malignant melanocytic lesions. Expression of TF in primary melanoma lesions was not associated with any clinicopathological variables. In addition, the serum level of TF was elevated in 14% of patients with melanoma; however, it was not correlated with disease progression. These results suggest that TF was ubiquitously expressed in melanocytic cells and its expression was not correlated with disease progression and/or metastatic potency of melanoma cells.     

Evaluation of texture features in spatial and frequency domain for automatic discrimination of histologic tissue

OBJECTIVE: To investigate the applicability of different texture features in automatic discrimination of microscopic views from benign common nevi and malignant melanoma lesions. STUDY DESIGN: In tissue counter analysis (TCA) the images are dissected into square elements used for feature calculation. The first class of features is based on the histogram, the co-occurrence matrix and the texture moments. The second class is derived from spectral properties of the wavelet Daubechie 4 and the Fourier transform. Square elements from images of a training set are classified by Classification and Regression Trees analysis. RESULTS: Features from the histogram and the co-occurrence matrix enable correct classification of 94.7% of nevi elements and 92.6% of melanoma elements in the training set. Classification results are applied to individual test set cases. Discriminant analysis based on the percentage of "malignant elements" showed correct classification of all nevi cases and 95% of melanoma cases. Features derived from the wavelet and Fourier spectrum showed correct results for 88.8% and 79.3% of nevi and 85.6% and 81.5% of melanoma elements, respectively. CONCLUSION: TCA is a potential diagnostic tool in automatic analysis of melanocytic skin tumors. Histogram and co-occurrence matrix features are superior to the wavelet and the Fourier features.     

Apaf‐1 expression in human cutaneous melanoma progression and in pigmented nevi

Malignant melanoma is notoriously refractive to therapy and resistant to apoptosis. This may reflect the downregulation of Apaf‐1, an important mediator of mitochondrial‐dependent apoptosis, observed in vitro in melanoma cell lines and by immunohistochemistry for Apaf‐1 protein in histological samples of primary and metastatic melanomas. Although it has been suggested that loss of Apaf‐1 expression may be an indicator of malignant transformation in melanoma, previous studies on Apaf‐1 expression in benign pigmented nevi were performed without reference to their histologic type. Here we have evaluated the expression of Apaf‐1 mRNA by fluorescence in situ hybridization and of Apaf‐1 protein by immunohistochemistry in a large panel of human melanomas and in eight types of pigmented nevi, considered potential precursors for cutaneous melanoma. We observe a strong negative correlation between melanoma progression assessed according to Clark classification and the expression of Apaf‐1. A significant decrease in Apaf‐1 expression was observed between Clark II and Clark III lesions, the stages usually associated with a transition from horizontal to vertical growth phase of melanoma. There was also statistically significant difference in Apaf‐1 mRNA expression between melanomas of Breslow thickness <1 mm and >4 mm. No Apaf‐1 expression could be detected in lymph node melanoma metastases. These results suggest that Apaf‐1 expression may become a prognostic marker for progress of human cutaneous melanoma and further support the notion that loss of Apaf‐1 may be an important contributory factor in the development of the disease.     

DNA ploidy and nuclear morphometry for the classification of dysplastic nevi

OBJECTIVE: To examine the diagnostic value of DNA ploidy and nuclear morphometric features in sporadic dysplastic nevi as compared to those in compound nevi and melanoma. STUDY DESIGN: DNA ploidy profiles plus seven direct and three derived nuclear features were obtained in a series of 120 melanocytic skin neoplasms (30 dysplastic nevi [DN], 30 melanomas [MM], 60 compound nevi [CN]) and the results compared. RESULTS: DNA ploidy separated melanomas from benign melanocytic skin neoplasms with 96.5% accuracy in classifying the grouped cases. The derived nuclear shape factor Form PE and nuclear axis ratio were the most successful discriminants separating DN from MM but allowed only 73.3% correct classification of cases. Separation of DN from CN was best achieved using Form PE and mean nuclear area (74.4% correctly classified). Results from compound nevi in subjects < 25 years of age fell between those for DN and MM. CONCLUSION: Quantitative nuclear cytologic characteristics in sporadic dysplastic nevi span a range seen in common nevi through to those in thin melanomas. Cytologic changes in sporadic dysplastic nevi overlap those seen in other melanocytic skin neoplasms. Therefore, other reproducible morphometric features need to be assessed in order to further refine the histopathologic diagnosis of this entity.     

Dermatoscopy and High Frequency Sonography: Two Useful Non-Invasive Methods to Increase Preoperative Diagnostic Accuracy in Pigmented Skin Lesions

Dermatoscopy and high frequency sonography have recently been combined to increase diagnostic preoperative accuracy in the treatment of pigmented skin lesions. In this monocentric study 80 patients with pigmented skin lesions were evaluated clinically, by dermatoscopy, and 20 MHz-sonography followed by dermatohistopathological evaluation; 39 malignant melanomas, 37 common nevi, 3 dysplastic nevi, and 1 nevus Spitz were diagnosed histologically. In 72 of the 80 cases (91.3%) dermatoscopical diagnoses were confirmed by histopathology, compared to only 79% correct clinical diagnoses. For the mere clinical diagnosis of melanoma sensitivity was 79%, specificity was 78% and diagnostic accuracy was 65%. All diagnostic values increased by dermatoscopy: sensitivity reached 90%, specificity was 93%, and diagnostic accuracy was 83%. In order to determine tumor thickness preoperatively tumor thickness was measured by 20 MHz sonography. The correlation of tumor thickness between histometric and sonographic results was determined for nevi (r = 0.93) and melanoma (r = 0.95); 74.3% of melanomas were diagnosed correctly within an 0.2 mm range. Regarding the clinical important limit of 1 mm tumor thickness, 87.2% were diagnosed in accordance with histometric evaluation. An increase of 18% in diagnostic accuracy by dermatoscopy and 87.2% of correctly diagnosed cases of tumor thickness of malignant melanoma by high frequency sonography clearly demonstrate that these methods should be considered standard procedures in the diagnosis of pigmented skin lesions and will facilitate the decision on necessary surgical treatment.     

Two-photon excitation spectrum of fluorescence of the light-harvesting complex B800–850 from Allochromatium minutissimum within 1200–1500 (600–750) nm spectral range is not carotenoid mediated

Two types of peripheral light-harvesting complexes LH2 (B800–850) from photosynthetic purple bacterium Allochromatium minutissimum were studied. First type containing carotenoids was prepared from wild type cells. The other one was obtained from carotenoid depleted cells grown with diphenylamine. We have shown that under laser femtosecond excitation within absorption 1200–1500 nm wavelength range the two-photon excitation of LH2 complexes takes place. This can be observed as fluorescence of bacteriochlorophyll (BChl) spectral form B850 (BChl molecules of circular aggregate with strong exciton interaction in 850 nm spectral domain). LH2 fluorescence excitation spectra under two-photon excitation are the same for carotenoid-containing and carotenoidless preparations. In both cases the broad band with peak near 1350 (675) nm (FWHM ～ 240 (120) nm) was found. It is concluded that the broad band with peak near 1350 (675) nm in two-photon excitation spectra of LH2 complexes from Allochromatium minutissimum cannot be interpreted as two-photon excitation band of the optically forbidden S₀ → S₁ transition of carotenoids (rhodopin). Possible nature of this band is discussed.     

Towards in vivo melanin radicals detection in melanomas by electron paramagnetic resonance (EPR) spectroscopy: a proof-of-concept study

Melanoma is the most aggressive skin tumour type. Although complete cure can be achieved when the whole tumour is resected, prognostic dramatically drops when melanoma cells reach deeper tissues and lymph nodes. Hence, there is an urgent need to develop accurate tools allowing (i) discriminating benign naevi from malignant tumours and (ii) being able to characterise melanoma infiltration. For that purpose, we exploited the paramagnetic properties of melanin by using electron paramagnetic resonance (EPR) spectroscopy to measure the melanin content in pigmented (B16F10 cancer cells) and non-pigmented melanomas (WM2664 cancer cells) inoculated intradermally in nude mice. Specifically, we took advantage of a new clinical EPR device (1?GHz), which provides sensitive measurements of radical species in vivo. Results showed that the melanin-specific EPR signal increased with tumour growth in pigmented tumours, whereas no EPR signal could be detected in achromic melanomas. These data plead for the development of new EPR spectrometers/imagers with an improved in-depth resolution for the detection of invasive melanomas.     

Melanin quantification by in vitro and in vivo analysis of near‐infrared fluorescence

Objective measurements of melanin can provide important information for differentiating melanoma from benign pigmented lesions and in assessing pigmentary diseases. Herein, we evaluate near‐infrared (NIR) fluorescence as a possible tool to quantify melanin. Various concentrations of in vitro Sepia melanin in tissue phantoms were measured with NIR fluorescence and diffuse reflectance spectroscopy. Similar optic measurements were conducted in vivo on 161 normal human skin sites. Diffuse reflectance spectroscopy was used to quantify the melanin content via Stamatas–Kollias algorithm. At physiologic concentrations, increasing in vitro melanin concentrations demonstrated higher fluorescence that was linearly correlated (R² = 0.99, p < .001). At higher concentrations, the fluorescence signal plateaued. A linear relationship was also observed with melanin content in human skin (R² = 0.59, p < .001). Comparing the fluorescence and reflectance signals with in vitro and in vivo samples, the estimated melanin concentration in human skin ranged between 0 and 1.25 mg/ml, consistent with previous quantitative studies involving invasive methods.     

Molecular pathogenesis of malignant melanoma: a different perspective from the studies of melanocytic nevus and acral melanoma

The Clark model for melanoma progression emphasizes a series of histopathological changes beginning from benign melanocytic nevus to melanoma via dysplastic nevus. Several models of the genetic basis of melanoma development and progression are based on this Clark’s multi-step model, and predict that the acquisition of a BRAF mutation can be a founder event in melanocytic neoplasia. However, our recent investigations have challenged this view, showing the polyclonality of BRAF mutations in melanocytic nevi. Furthermore, it is suggested that many melanomas, including acral and mucosal melanomas, arise de novo, not from melanocytic nevus. While mutations of the BRAF gene are frequent in melanomas on non-chronic sun damaged skin which are prevalent in Caucasians, acral and mucosal melanomas harbor mutations of the KIT gene as well as the amplifications of cyclin D1 or cyclin-dependent kinase 4 gene. Amplifications of the cyclin D1 gene are detected in normal-looking ‘field melanocytes’, which represent a latent progression phase of acral melanoma that precedes the stage of atypical melanocyte proliferation in the epidermis. Based on these observations, we propose an alternative genetic progression model for melanoma.     

One- and two-photon induced fluorescence of Pacific Blue-labeled human serum albumin deposited on different core size silver colloids

We studied one- and two-photon induced fluorescence of Pacific Blue (PB)-labeled human serum albumin (HSA) in the presence of different size silver colloids. The PB fluorescence emission intensity was observed with small (30-40 nm) and large (about 120 nm) colloids and compared with PB emission in absence of colloids. For the system with a small core size colloids we did not detect any fluorescence enhancement with one-photon excitation and the enhancement observed with two-photon excitation was about 2.5-fold. In contrast, for large silver colloids we observed about a 2-fold increase in PB fluorescence brightness for one-photon excitation, and the enhancement with two-photon excitation excided 13-folds. Much stronger increases in brightness observed with two-photon excitation, compared to one-photon excitation, indicate a dominant role of enhanced local field in fluorescence enhancement on silver colloids in solutions.     

Two-photon excitation and photoconversion of EosFP in dual-color 4Pi confocal microscopy

Recent years have witnessed enormous advances in fluorescence microscopy instrumentation and fluorescent marker development. 4Pi confocal microscopy with two-photon excitation features excellent optical sectioning in the axial direction, with a resolution in the 100 nm range. Here we apply this technique to cellular imaging with EosFP, a photoactivatable autofluorescent protein whose fluorescence emission wavelength can be switched from green (516 nm) to red (581 nm) by irradiation with 400-nm light. We have measured the two-photon excitation spectra and cross sections of the green and the red species as well as the spectral dependence of two-photon conversion. The data reveal that two-photon excitation and photoactivation of the green form of EosFP can be selectively performed by choosing the proper wavelengths. Optical highlighting of small subcellular compartments was shown on HeLa cells expressing EosFP fused to a mitochondrial targeting signal. After three-dimensionally confined two-photon conversion of EosFP within the mitochondrial networks of the cells, the converted regions could be resolved in a 3D reconstruction from a dual-color 4Pi image stack.     

Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin

Multiphoton excitation microscopy at 730 nm and 960 nm was used to image in vivo human skin autofluorescence from the surface to a depth of approximately 200 microm. The emission spectra and fluorescence lifetime images were obtained at selected locations near the surface (0-50 microm) and at deeper depths (100-150 microm) for both excitation wavelengths. Cell borders and cell nuclei were the prominent structures observed. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence at 730 nm excitation. With 960 nm excitation, a two-photon fluorescence emission at 520 nm indicates the presence of a variable, position-dependent intensity component of flavoprotein. A second fluorescence emission component, which starts at 425 nm, is observed with 960-nm excitation. Such fluorescence emission at wavelengths less than half the excitation wavelength suggests an excitation process involving three or more photons. This conjecture is further confirmed by the observation of the super-quadratic dependence of the fluorescence intensity on the excitation power. Further work is required to spectroscopically identify these emitting species. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells.