Ablative fractional laser-assisted photodynamic therapy for lentigo maligna: a prospective pilot study.

BACKGROUND: Lentigo maligna (LM) is an in situ form of melanoma carrying a risk of progression to invasive lentigo maligna melanoma (LMM). LM poses a clinical challenge, with subclinical extension and high recurrence rates after incomplete surgery. Alternative treatment methods have been investigated with varying results. Photodynamic therapy (PDT) with methylaminolaevulinate (MAL) has already proved promising in this respect. OBJECTIVES: To investigate the efficacy of ablative fractional laser (AFL)-assisted PDT with 5-aminolaevulinic acid nanoemulsion (BF-200 ALA) for treating LM. METHODS: In this non-sponsored prospective pilot study, ten histologically verified LMs were treated with AFL-assisted PDT three times at 2-week intervals using a light dose of 90 J/cm2 per treatment session. Local anaesthesia with ropivacaine was used. Four weeks after the last PDT treatment the lesions were treated surgically with a wide excision and sent for histopathological examination. The primary outcome was complete histopathological clearance of the LM from the surgical specimen. Patient-reported pain during illumination and the severity of the skin reaction after the PDT treatments were monitored as secondary outcomes. RESULTS: The complete histopathological clearance rate was 7 out of 10 LMs (70%). The pain during illumination was tolerable, with the mean pain scores for the PDT sessions on a visual assessment scale ranging from 2.9 to 3.8. Some severe skin reactions occurred during the treatment period, however. CONCLUSIONS: Ablative fractional laser-assisted PDT showed moderate efficacy in terms of histological clearance. It could constitute an alternative treatment for LM but due to the side effects it should only be considered in inoperable cases.

Hyperspectral imaging system in the delineation of Ill-defined basal cell carcinomas: a pilot study.

BACKGROUND: Basal cell carcinoma (BCC) is the most common skin cancer in the Caucasian population. Eighty per cent of BCCs are located on the head and neck area. Clinically ill-defined BCCs often represent histologically aggressive subtypes, and they can have subtle subclinical extensions leading to recurrence and the need for re-excisions. OBJECTIVES: The aim of this pilot study was to test the feasibility of a hyperspectral imaging system (HIS) in vivo in delineating the preoperatively lateral margins of ill-defined BCCs on the head and neck area. METHODS: Ill-defined BCCs were assessed clinically with a dermatoscope, photographed and imaged with HIS. This was followed by surgical procedures where the BCCs were excised at the clinical border and the marginal strip separately. HIS, with a 12-cm2 field of view and fast data processing, records a hyperspectral graph for every pixel in the imaged area, thus creating a data cube. With automated computational modelling, the spectral data are converted into localization maps showing the tumour borders. Interpretation of these maps was compared to the histologically verified tumour borders. RESULTS: Sixteen BCCs were included. Of these cases, 10 of 16 were the aggressive subtype of BCC and 6 of 16 were nodular, superficial or a mixed type. HIS delineated the lesions more accurately in 12 of 16 of the BCCs compared to the clinical evaluation (4 of 16 wider and 8 of 16 smaller by HIS). In 2 of 16 cases, the HIS-delineated lesion was wider without histopathological confirmation. In 2 of 16 cases, HIS did not detect the histopathologically confirmed subclinical extension. CONCLUSIONS: HIS has the potential to be an easy and fast aid in the preoperative delineation of ill-defined BCCs, but further adjustment and larger studies are warranted for an optimal outcome.

Matrix metalloproteinases as mediators of tissue injury in different forms of cutaneous lupus erythematosus.

BACKGROUND: Matrix metalloproteinases (MMPs) contribute to tissue destruction, regeneration, inflammation and apoptosis and several of them are upregulated by ultraviolet (UV) radiation in skin. Although some MMPs associate with organ manifestations of systemic lupus erythematosus (SLE), their role in cutaneous lupus erythematosus (LE) is elusive. OBJECTIVES: Our aim was to evaluate the expression of MMPs in SLE, subacute cutaneous LE (SCLE) and discoid LE (DLE) skin lesions and their relation to apoptosis and epidermal changes. METHODS: Lesional skin biopsies from 20 patients with SLE, 20 with DLE and 17 with SCLE, and from UVA/UVB-photoprovoked skin of healthy volunteers were immunostained using antibodies to multiple MMPs and tissue inhibitors of metalloproteinases (TIMPs). The TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling) method was used for detection of apoptosis. RESULTS: MMP-3, -10, -19 and -26 were abundantly expressed by keratinocytes in SLE, DLE and SCLE skin samples. MMP-7 was detected in keratinocytes in regions of oedema and vacuolization especially in SLE and SCLE, while MMP-14 was only occasionally observed in keratinocytes. Photoprovocation did not induce MMP-10 or -26 expression in skin of healthy volunteers. Epithelial TIMP-1 expression was low while occasional positive fibroblasts were seen in the dermis. TIMP-3 was abundantly expressed in the epidermis, endothelial cells and macrophages. CONCLUSIONS: Different subtypes of cutaneous LE are fairly similar in their MMP expression profile. MMP-3 and -10 mediate both epidermal changes and dermal tissue remodelling but are not present in lymphocytes. Low expression of TIMP-1 suggests that lupus skin is characterized by proteolytic events, and targeted action using selective MMP inhibitors may reduce lupus-induced damage in inflamed tissues.

Expression of matrix metalloproteinase (MMP)-7 and MMP-13 and loss of MMP-19 and p16 are associated with malignant progression in chronic wounds.

BACKGROUND: The risk of squamous cell carcinoma (SCC) is significantly increased in chronic leg ulcers. Very little is known about the molecular pathogenesis of these tumours, which are often undiagnosed for a long time. As matrix metalloproteinases (MMPs) are implicated at all stages of tumorigenesis, we investigated whether the pattern of epithelial MMP expression can predict development of SCC from pseudoepitheliomatous hyperplasia of chronic wounds. METHODS: Samples from nine patients with SCCs that had arisen in chronic wounds and 31 with venous leg ulcers were studied using immunohistochemistry for MMP-7, MMP-8, MMP-9, MMP-13, MMP-19 and the tumour suppressor p16. In situ hybridization was performed for MMP-1, MMP-3, MMP-7, MMP-12 and MMP-13. RESULTS: MMP-7 was expressed by malignantly transformed epithelium, while it was absent from chronic wounds. MMP-9 was detected in the epithelium in both SCCs and chronic wounds. Epithelial MMP-13 expression was strong in SCC, but was absent in chronic wounds. MMP-12 was expressed in the epithelium in two SCCs, while macrophages were positive in chronic wounds. MMP-19 was induced in proliferating epithelium of wounds, but was absent from invasive areas of SCC. p16 was expressed by keratinocytes in half of the chronic wounds and at superficial margins of SCCs, while invasive areas were negative. CONCLUSIONS: Our results suggest that epithelial expression of MMP-7, MMP-12 and MMP-13, but not that of MMP-1, MMP-3, MMP-8, MMP-9 and MMP-10, in chronic wounds provides a diagnostic clue for distinguishing SCCs from nonmalignant wounds. The loss of MMP-19 and p16 from the epithelium could aid in making the differential diagnosis between well-differentiated SCCs and nonmalignant chronic wounds.

Overexpression of tissue inhibitor of metalloproteinases-3 in intestinal and cutaneous lesions of graft-versus-host disease.

Matrix metalloproteinases (MMPs) have been implicated in the pathobiology of various T-cell-mediated inflammatory disorders of the intestine and skin. Their synthetic inhibitor has been shown to prevent lethal acute graft-versus-host disease in animal models. We intended to determine the expression of MMPs 1, 3, 7, 9, 10, 12, and 19 and tissue inhibitors of metalloproteinases (TIMPs) 1 and 3 in intestinal and cutaneous lesions of patients suffering from graft-versus-host disease after bone marrow transplantation. In situ hybridizations for MMPs 1, 3, 7, 10, and 12 as well as TIMPs 1 and 3 were performed using (35)S-labeled cRNA probes on intestinal (n = 13) and cutaneous specimens (n = 9) from patients with graft-versus-host disease. Immunohistochemical stainings were carried out to localize MMP-9, MMP-19, TIMP-3, and TGF-beta1 proteins, and TUNEL staining, to detect apoptotic cells. TIMP-3 mRNA and protein were detected in cutaneous lesions in areas with vacuolar degeneration of the basal epidermal layer in all skin samples, and they colocalized with apoptotic keratinocytes and partly with staining for TGF-beta. None of the MMPs examined were overexpressed in skin lesions. Signals for MMP-1 and MMP-3 mRNA was found in 10/13 and 5/13 intestinal biopsies, respectively. In the gut, MMP-19-positive epithelial cells, particularly in the crypts, were found in 10/13 samples. Expression of MMPs 7, 9, 10, and 12 was absent or very low. TIMPs 1 and 3 were expressed by stromal cells in 12/13 and 10/13 gut samples, respectively. Whereas TIMP-1 was expressed particularly by subepithelial cells where epithelium had shed away, TIMP-3 was detected in deeper areas. We conclude that MMPs are differentially regulated in the skin and gut lesions of graft-versus-host disease. In agreement with previous data on cancer cells, TIMP-3, induced by TGF-beta1, may contribute to the apoptosis of keratinocytes in cutaneous graft-versus-host disease lesions, leading to typical histopathological changes. We also conclude that MMPs play a less important role as effector molecules in intestinal graft-versus-host disease than in celiac or inflammatory bowel disease.

Prognosis of primary melanoma.

MATERIALS AND METHODS: To investigate the prognosis of primary melanoma, we studied a Finnish population of 298 primary melanoma patients, the majority with stage I or II tumours. The median clinical follow-up (4.8 years) was acquired from the patients' records, and the overall survival thereafter was collected from patient registries. The median follow-up for overall survival was 9.5 years. RESULTS: The overall survival rate was 66.8%. 24.5% developed metastasis, 17.8% died of melanoma, and 15.4% died of some other cause. Surgical margins had no effect on survival. In univariate analysis the most significant prognostic factors for disease-free and overall survival were stage of tumour (p < 0.0001), thickness of tumour (p < 0.0001), depth of tumour invasion (p < 0.0001) and tumour ulceration (p = 0.0005, p < 0.0002). Ulceration was an unfavorable prognostic marker. Younger patients had better survival outcomes than older ones (p = 0.04). Accordingly, in the multivariate Cox model the independent prognostic factors for both disease-free and overall survival were stage of tumour and thickness of tumour. Tumour location on trunk was an independent adverse prognosticator for overall survival. CONCLUSION: We conclude that the prognosis of primary melanoma has improved in Finland in the last decades being in line with a global tendency.

Expression of human macrophage metalloelastase (MMP-12) by tumor cells in skin cancer.

Matrix metalloproteinases play an essential role in tumor growth and invasion. Different matrix metalloproteinases are often expressed in cancers with distinct patterns. To investigate the role of human macrophage metalloelastase (MMP-12) in epidermal tumors, we studied human macrophage metalloelastase mRNA and protein expression in malignant squamous cell and basal cell carcinomas, and in premalignant Bowen's disease. Human macrophage metalloelastase was detected in 11 of 17 squamous cell carcinomas in epithelial cancer cells, whereas macrophages were positive in 15 of 17 samples. In basal cell carcinomas, human macrophage metalloelastase was more often found in macrophages (seven of 19) than in cancer cells (four of 19). Human macrophage metalloelastase mRNA was also detected in three cell lines derived from squamous cell carcinomas of the head and neck and in transformed HaCaT cells, whereas premalignant tumors and primary keratinocytes were negative for human macrophage metalloelastase mRNA. Western analysis revealed human macrophage metalloelastase protein in squamous cell carcinoma cells. Our results show that human macrophage metalloelastase can be expressed in vivo and in vitro by transformed epithelial cells and indicate that the level of human macrophage metalloelastase expression correlates with epithelial dedifferentiation and histologic aggressiveness.

Cryptococcosis during systemic glucocorticosteroid treatment.

Cryptococcosis is an opportunistic infection caused by a fungus, Cryptococcus neoformans. It is usually seen in immunocompromised patients with AIDS, leukaemia, lymphoma, sarcoidosis or immunosuppressive treatments. We describe a patient who was treated with systemic glucocorticosteroids for 4 years because of lung sarcoidosis. During the last year of treatment, a papular eruption developed which later became ulcerative. In a histopathological examination of a skin biopsy, there was granulomatous inflammation, and the disease was treated as sarcoidosis without success. After 1 year's unsuccessful treatment, another skin biopsy and skin fungal culture revealed C. neoformans. Cryptococcal antigen was found in blood and cerebrospinal fluid, too. The patient was successfully treated first with an amphotericin-B-flucytosine combination and later with fluconazole.

Accumulation of matrilysin (MMP-7) and macrophage metalloelastase (MMP-12) in actinic damage.

Photodamage is characterized by degradation of collagen and accumulation of abnormal elastin in the superficial dermis and several matrix metalloproteinases have previously been implicated in this process. Using immunohistochemistry and in situ hybridization, we have studied the localization of two elastolytic matrix metalloproteinases, matrilysin (matrix metalloproteinase-7) and human macrophage metalloelastase (matrix metalloproteinase-12) in solar damage. Human macrophage metalloelastase protein was detected in the superficial dermis in areas of elastotic material. Matrix metalloproteinase-7 was seen in the mid-dermis in regions with less damaged elastic fibers and morphologically better preserved collagen as well as in a band-like pattern below basal keratinocytes in eight of 18 solar elastosis. In samples taken from healthy volunteers 3 d after repeated ultraviolet A or ultraviolet B photoprovocation, occasional immunopositive cells for human macrophage metalloelastase (stromal) or matrix metalloproteinase-7 (sweat gland epithelium) were detected. In samples taken 1 d after ultraviolet B exposure, however, basal keratinocytes were matrix metalloproteinase-7 immunopositive, explaining the linear immunostaining below basal keratinocytes noted particularly in ultraviolet B treated 3 d specimens. Upregulation of metalloelastase was also demonstrated in the skin of hairless mice after repeated ultraviolet exposure. In normal skin, no staining for human macrophage metalloelastase or matrix metalloproteinase-7 was observed in association with elastin. The amount of immunoreactivity for the substrates of matrix metalloproteinase-7, versican, and tenascin, was clearly increased in solar elastosis and photoprovocated skin; versican but not tenascin was detected in the same areas as matrix metalloproteinase-7. Our results suggest that both matrix metalloproteinase-7 and -12 may contribute to remodeling of elastotic areas in sun-damaged skin.

Transforming growth factor beta 1 and its latent form binding protein-1 associate with elastic fibres in human dermis: accumulation in actinic damage and absence in anetoderma.

Latent transforming growth factor-beta 1 (TGF-beta 1) and its binding protein-1 (LTBP-1) are components of the extracellular matrix microfibrils of cultured human fibroblasts. Using immunohistochemistry we have studied the localization of TGF-beta 1 and LTBP-1 and compared their distribution with that of elastic fibres in the interstitial connective tissue matrix of the human dermis. Prominent LTBP-1 specific fibrillar staining co-localized with the elastic fibres in normal human skin. Co-distribution was also observed in a number of pathological states of the elastic fibres such as solar elastosis, solar keratosis and pseudoxanthoma elasticum. TGF-beta 1 had a staining pattern similar to that of LTBP-1 in solar elastosis and solar keratosis. No staining for LTBP-1 or TGF-beta 1 was found in dermis devoid of elastic fibres, as in anetoderma. LTBP-1 is released from the extracellular matrix of cultured human fibroblasts, epithelial and endothelial cells by proteases. Analogously, the immunoreactivity for LTBP-1 and TGF-beta 1 were also lost from the skin sections by elastase, and by trypsin, a protease pretreatment commonly used in immunohistochemistry. These results indicate that LTBP-1 is a component of the elastin-associated microfibrils of the interstitial connective tissue matrix of human skin. Furthermore, the small latent form of TGF-beta 1 is likely to associate with the extracellular matrix of human dermis via LTBP-1. The release of latent TGF-beta 1 from the matrix, as a consequence of proteolytic cleavage of LTBP-1, is a plausible extracellular mechanism for the regulation of TGF-beta 1 activation.

Expression of Mad, an antagonist of Myc oncoprotein function, in differentiating keratinocytes during tumorigenesis of the skin.

The Myc oncoprotein is associated with cell proliferation and is often down-regulated during cell differentiation. The related Mad transcription factor, which antagonises Myc activity, is highly expressed in epidermal keratinocytes. Mad also inhibits cell proliferation in vitro. To study Mad expression in keratinocyte proliferation and differentiation, we have analysed Mad RNA expression in regenerating and hyperproliferative epidermal lesions and epidermal tumours of varying degrees of differentiation using the RNA in situ hybridisation and RNAase protection techniques. Mad was strongly expressed in differentiating suprabasal keratinocytes in healing dermal wounds and in benign hyperproliferative conditions, but also in squamous cell carcinomas, in which the keratinocytes retain their differentiation potential. However, Mad expression was lost in palisading basal carcinoma cells and poorly differentiated squamous cell carcinomas, which lacked the epithelial differentiation marker syndecan-1. We therefore suggest that Mad expression is closely associated with epithelial cell differentiation, and that this association is retained in epithelial tumours of the skin.

Imedeen for the treatment of degenerated skin in females.

Imedeen, a new compound for oral administration consisting of special protein fractions and some glucosaminoglycans extracted from marine fish, has been shown in previous pilot studies to have a repairing effect on sun-damaged skin. In an open study, 10 females with sun-damaged skin, aged 39-61 years, were treated with 0.5 g/day Imedeen for 90 days. At baseline and after 30, 60 and 90 days, the following parameters were clinically evaluated: wrinkles; mottles; dryness of skin; and brittleness of hair and nails. After 90 days' treatment all signs of sun-damage had improved and brittleness of hair and nails was normalized in all cases. These clinical observations were confirmed by changes in skin thickness and elasticity. In a second double-blind study, 30 females in the same age range and with similar signs of sun-damage were treated with 0.5 g/day Imedeen or placebo for 90 days. The results in the Imedeen-treated group corresponded to those in the first study whereas no response to treatment was observed in the placebo treatment group.