Biopsy outperforms reflectance confocal microscopy in diagnosing and subtyping basal cell carcinoma: results and experiences from a randomized controlled multicentre trial.

BACKGROUND: Reflectance confocal microscopy (RCM) is a noninvasive method for skin assessment, allowing entire lesion evaluation up to the papillary dermis. RCM is a potentially attractive alternative to punch biopsy (PB) in basal cell carcinoma (BCC). OBJECTIVES: To determine the diagnostic accuracy of RCM vs. PB in diagnosing and subtyping BCC, and to study patient satisfaction and preferences. METHODS: Patients with a clinically suspected primary BCC were randomized between RCM and biopsy. Conventional surgical excision or follow-up were used as reference. Sensitivity and specificity for BCC diagnosis and subtyping were calculated for both methods. BCC subtype was stratified based on clinical relevance: aggressive (infiltrative/micronodular) vs. nonaggressive (superficial/nodular) histopathological subtype and superficial vs. nonsuperficial BCC. Data on patient satisfaction and preferences were collected using a questionnaire and a contingent valuation method. RESULTS: Sensitivity for BCC diagnosis was high and similar for both methods (RCM 99·0% vs. biopsy 99·0%; P = 1·0). Specificity for BCC diagnosis was lower for RCM (59·1% vs. 100·0%; P < 0·001). Sensitivity for aggressive BCC subtypes was lower for RCM (33·3% vs. 77·3%; P = 0·003). Sensitivity for nonsuperficial BCC was not significantly different (RCM 88·9% vs. biopsy 91·0%; P = 0·724). Patient satisfaction and preferences were good and highly comparable for both methods. CONCLUSIONS: Biopsy outperforms RCM in diagnosing and subtyping clinically suspected primary BCC. This outcome does not support routine clinical implementation of RCM, as a replacement for PBs in this patient group.

[Minocycline-induced hyperpigmentation: not uncommon, but nonetheless important to recognise]. / Minocycline-geïnduceerde hyperpigmentatie.

BACKGROUND: Minocycline, a broad-spectrum antibiotic from the group of tetracyclins, is frequently prescribed for acne vulgaris and rosacea. Hyperpigmentation is a relatively common side effect of this drug and can lead to multiple unsightly skin lesions, which are not always reversible. It can take a long period, from a few months to several years, before the lesions have completely vanished. CASE DESCRIPTION: A 24-year-old male was seen in our outpatient clinic because of slowly progressive pigmented lesions on his shins. His medical history included chronic use of minocycline for the treatment of acne vulgaris. Based on the morphology of the lesions, the patient's medical history and analysis of a skin biopsy, the diagnosis 'minocycline-induced hyperpigmentation type 2'was made. CONCLUSION: Hyperpigmentation is a relatively common and undesirable side-effect of minocycline use. Long-term prescription of this drug for more than one year should therefore be considered with caution, especially if the dose exceeds 100 milligrams per day.

A 'waterfall' transfer-based workflow for improved quality of tissue microarray construction and processing in breast cancer research.

A major focus in cancer research is the identification of biomarkers for early diagnosis, therapy prediction and prognosis. Hereby, validation of target proteins on clinical samples is of high importance. Tissue microarrays (TMAs) represent an essential advancement for high-throughput analysis by assembling large numbers of tissue cores with high efficacy and comparability. However, limitations along TMA construction and processing exist. In our presented study, we had to overcome several obstacles in the construction and processing of high-density breast cancer TMAs to ensure good quality sections for further research. Exemplarily, 406 breast tissue cores from formalin-fixed and paraffin embedded samples of 245 patients were placed onto three recipient paraffin blocks. Sectioning was performed using a rotary microtome with a "waterfall" automated transfer system. Sections were stained by immunohistochemistry and immunofluorescence for nine proteins. The number and quality of cores after sectioning and staining was counted manually for each marker. In total, 97.1 % of all cores were available after sectioning, while further 96 % of the remaining cores were evaluable after staining. Thereby, normal tissue cores were more often lost compared to tumor tissue cores. Our workflow provides a robust method for manufacturing high-density breast cancer TMAs for subsequent IHC or IF staining without significant sample loss.

Unilateral lentiginosis with nevus depigmentosus on the other side.

A transmittance of unilateral hypomelanosis in 4 generations and the co-occurrence of unilateral lentigenosis in the youngest family member are reported. It can be explained by paradominant inheritance and the twin-spot theory.

Erythrosis pigmentosa mediofacialis (Brocq) and erythromelanosis follicularis faciei et colli in the same patient.

Erythrosis pigmentosa peribuccalis (Brocq) (or erythrosis pigmentosa mediofacialis) and erythromelanosis follicularis faciei et colli, have been regarded as different disorders, mainly because the first occurs on the mediofacial area and is common in women and the second mostly occurs pre-auricularly in men. Both conditions show histological signs of abnormal follicular keratinization with teleangiectasia and round cell infiltrate. An increase in the level of melanin has been seen in some patients. We describe here a woman in whom lesions started in the middle of the face and later became evident in the pre-auricular area. This suggests that the two conditions are in fact the same disease. As a neutral term for this not uncommon disorder we propose erythrosis pigmentosa faciei et colli.

Skin-derived antileukoproteinase (SKALP) is decreased in pustular forms of psoriasis. A clue to the pathogenesis of pustule formation?

Skin-derived antileukoproteinase (SKALP, also known as elafin) is an inducible epidermal serine proteinase inhibitor, that we have recently characterized at the protein and DNA levels. SKALP is a strong and specific inhibitor of PMN elastase, and is putatively involved in the regulation of cutaneous inflammatory processes. In order to investigate the role of SKALP in the control of elastase in psoriatic epidermis, we compared SKALP expression in normal skin, and in skin from patients with chronic plaque psoriasis and pustular forms of psoriasis. Epidermal scales and biopsies were collected and SKALP expression was studied at the mRNA level and at the protein level both functionally and immunochemically. In epidermal scales, we found that the levels of both free and total SKALP activity in pustular psoriasis were far lower than in plaque psoriasis. A significant number of pustular psoriasis patients showed latent SKALP activity, which represents the amount of SKALP putatively complexed to elastase. In addition, we found free elastase activity in 25% of the pustular psoriasis patients, indicating a total saturation of epidermal SKALP activity. In epidermal biopsies from pustular psoriasis patients, SKALP activity was significantly decreased compared with those from plaque psoriasis patients. Northern blot analysis did not reveal differences in epidermal mRNA levels between chronic plaque psoriasis and pustular psoriasis. We hypothesize that a reduced amount of epidermal SKALP contributes to an imbalance between elastase and its inhibitor, thereby promoting the formation of epidermal pustules. We suggest that these findings could provide a rationale for the treatment of pustular psoriasis with inhibitors of PMN-derived proteinases, as a new therapeutic modality.

Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.

Skin-derived antileukoproteinase (SKALP), also known as elafin, is a serine proteinase inhibitor first discovered in keratinocytes from hyperproliferative human epidermis. In addition to the proteinase inhibiting domain which is directed against polymorphonuclear leukocyte (PMN) derived enzymes such as elastase and proteinase 3, SKALP contains multiple transglutaminase (TGase) substrate domains which enable crosslinking to extracellular and cell envelope proteins. Here we show that SKALP is constitutively expressed in several epithelia that are continuously subjected to inflammatory stimuli, such as the oral cavity and the vagina where it co-localizes with type 1 TGase. All epithelia from sterile body cavities are negative for SKALP. In general, stratified squamous epithelia are positive, whereas pseudostratified epithelia, simple/glandular epithelia and normal epidermis are negative. SKALP was found in fetal tissues of the oral cavity from 17 wk gestation onwards where it continued to be expressed up to adult life. Remarkably, in fetal epidermis SKALP was found from week 28 onwards, but was downregulated to undetectable levels in neonatal skin within three months, suggesting a role during pregnancy in feto-maternal interactions or in the early maturation phase of the epidermis. Immunoelectron microscopy revealed the presence of SKALP in secretory vesicles including the lamellar granules. In culture models for epidermal keratinocytes we found that expression of the endogenous SKALP gene provided protection against cell detachment caused by purified elastase or activated PMNs. Addition of exogenous recombinant SKALP fully protected the keratinocytes against PMN-dependent detachment whereas superoxide dismutase and catalase were only marginally effective. These findings strongly suggest that the constitutive expression of SKALP in squamous epithelia, and the inducible expression in epidermis participate in the control of epithelial integrity, by inhibiting PMN derived proteinases.

Topographic relation between skin-derived antileukoproteinase (SKALP) and leukocyte elastase in a case of annular pustular psoriasis.

We have assessed the distribution pattern of a new epidermal elastase inhibitor, skin-derived antileukoproteinase (SKALP) and polymorphonuclear leukocytes (PMN) in a patient with annular pustular psoriasis, using immunohistochemical methods. In clinically uninvolved skin SKALP was not expressed and only occasionally a few PMN could be identified. In the erythematous distal margin of the lesion, expression of SKALP was shown in suprabasal keratinocytes. Although PMN were present in the dermis, no transepidermal PMN migration had occurred at this stage. In the pustular region a dense SKALP-positive zone was demonstrated in the suprabasal compartment. In the centrally healed area neither SKALP expression nor PMN accumulation was shown. The present case report suggests that the induction of SKALP expression does not result from the passage of PMN through the epidermis. The offswitch of SKALP expression coincides with the disappearance of the PMN infiltration and not with the resolution of the mononuclear infiltrate.

Levels of skin-derived antileukoproteinase (SKALP)/elafin in serum correlate with disease activity during treatment of severe psoriasis with cyclosporin A.

The epidermal serine proteinase inhibitor SKALP (also known as elafin), directed against human leukocyte elastase and proteinase 3, is strongly induced in suprabasal keratinocytes during inflammation. The presence of SKALP/elafin in urine has been demonstrated for several inflammatory skin disorders, such as psoriasis, erythroderma, and erysipelas. In this study we investigated whether SKALP/elafin levels in serum and urine of psoriatic patients can be used as a marker for disease activity during treatment. Patients with severe chronic disabling psoriasis were treated for 16 weeks with cyclosporin A, which resulted in a marked clinical improvement as measured with the PASI score. SKALP/elafin levels both in serum and urine were determined with an enzyme-linked immunosorbent assay (ELISA). Measurements were performed at the start of the cyclosporin A treatment, and after regular intervals up to 16 weeks. The results indicate that 1) SKALP/elafin determination in serum rather than in urine is the preferred method, because the decrease in serum SKALP levels during therapy is more pronounced and correlated better with the clinical course of the patients; 2) SKALP/elafin levels in serum decreased during cyclosporin A treatment (p < 0.05); and 3) SKALP/elafin levels in serum correlate with the PASI score (p < 0.01). We conclude that SKALP/elafin measurement in serum of patients with severe psoriasis provides a tool for monitoring disease activity.

Demonstration of skin-derived antileukoproteinase (SKALP) and its target enzyme human leukocyte elastase in squamous cell carcinoma.

Skin-derived antileukoproteinase (SKALP), also known as elafin, is a strong and specific inhibitor of elastase and proteinase 3. SKALP is not present in normal epidermis, but is expressed by epidermal keratinocytes under hyperproliferative conditions such as psoriasis, wound healing, and in cell culture. In human epidermal tumours, SKALP is differentially expressed and restricted to tumours with distinct squamous differentiation. We have studied the presence of both SKALP and one of its known target enzymes, leukocyte elastase, in 41 squamous cell carcinomas of the skin. SKALP expression correlated with the degree of differentiation: strong expression was seen in well-differentiated cells and expression was absent in poorly differentiated tumour cells. Most of the squamous cell carcinomas showed elastase-positive cells in the tumour stroma and also within the tumour cell nests. SKALP may interfere with the proteolytic activity of infiltrating inflammatory cells or with hitherto unknown proteinases from the tumour cells. We hypothesize that in squamous cell carcinoma progressive loss of SKALP expression could facilitate tumour spread.

Differential expression of SKALP/Elafin in human epidermal tumors.

Recently we described a new epidermal serine proteinase inhibitor, skin-derived antileukoproteinase (SKALP), also known as elafin. SKALP/elafin was found to be absent in normal human epidermis, but can be induced in vitro and in vivo under hyperproliferative conditions. Here we studied the expression of SKALP/elafin in several types of epidermal tumors (basal cell carcinoma, squamous cell carcinoma, Bowen's disease, actinic keratosis, and keratoacanthoma). Using immunohistochemical staining SKALP/elafin appeared to be differentially expressed in these tumors. Functional measurements of anti-proteinase activity, and Western blotting of tumor extracts confirmed our findings at the histological level. In well differentiated squamous cell carcinoma, SKALP/elafin messenger RNA was demonstrated by non-radioactive in situ hybridization. We conclude that SKALP/elafin is a marker for abnormal or disturbed squamous differentiation. A possible role of SKALP/elafin in the control of tumor cell invasion is discussed.

SKALP/elafin: an elastase inhibitor from cultured human keratinocytes. Purification, cDNA sequence, and evidence for transglutaminase cross-linking.

SKALP/Elafin is a proteinase inhibitor found in psoriatic epidermis as a short polypeptide of 6 kDa. Here we present evidence that this protein is synthesized as a larger precursor molecule with distinct biological features. Purification and NH2-terminal sequencing of SKALP/elafin from cultured human keratinocytes and the cloning of its cDNA revealed the existence of a mature protein, which upon cleavage of a hydrophobic signal sequence of 22 amino acids has a calculated molecular mass of 9.9 kDa (95 amino acids). In addition to the known proteinase inhibitor domain, the mature protein contains a domain with 4 repeats which are homologous to putative transglutaminase substrate motifs. We were able to demonstrate on Western blots that immunoreactive SKALP is present in high molecular weight proteins extracted from psoriatic skin. This suggests that SKALP is covalently attached to epidermal proteins. In addition it was found that both the complete SKALP molecule and a synthetic peptide of the NH2-terminal portion of SKALP could be used as a transglutaminase substrate. We therefore speculate that SKALP/elafin, secreted by epidermal keratinocytes in inflamed skin, exists both as a free 6-kDa form and as an immobilized 9.9-kDa form covalently attached to the cornified envelopes by transglutaminase cross-linking.

Demonstration of skin-derived antileukoproteinase (SKALP) in urine of psoriatic patients.

Recently we described a new elastase inhibitor (skin-derived antileukoproteinase, SKALP) that is expressed in psoriatic epidermis and cultured keratinocytes, but is virtually absent in normal skin. In this study we investigated whether SKALP activity could be measured in urine of psoriatic patients and healthy controls. We found that urine of psoriatic patients contained considerable amounts of anti-elastase activity, whereas this activity in urine from normals was significantly lower. The properties of the urinary anti-elastase activity in psoriatic patients were indistinguishable from that of epidermal SKALP. It was found to be a cationic, heat-stable protein with an apparent molecular weight of 11 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and a K(i) of approximately 2 x 10(-11) M. In addition, in Western blotting partially purified inhibitor from urine was found to react with a polyclonal anti-SKALP serum. SKALP in urine was either present in a free form or in a latent form, most likely complexed with elastase. We speculate that SKALP in urine of psoriatic patients is derived from the epidermis, and that it might serve as a marker for disease activity.

Dithranol modulates the leukotriene B4-induced intraepidermal accumulation of polymorphonuclear leukocytes.

Dithranol, with and without the addition of salicylic acid, was applied daily on normal skin according to a short contact protocol as used in the treatment of psoriasis. Sellotape stripping and epicutaneous application of leukotriene B4 (LTB4) were carried out within these pretreated areas. The challenged skin was subsequently biopsied and the intraepidermal accumulation of polymorphonuclear leukocytes (PMN) was quantified using the marker enzyme elastase. Dithranol pretreatment yielded a significant reduction of the LTB4-induced accumulation of PMN, whereas the tape stripping-induced accumulation of PMN was not affected by dithranol pretreatment. The addition of salicylic acid did not significantly enhance the effect of dithranol.