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Actinic keratoses (AK) may occur in all sun-exposed skin areas. Those occurring outside the head area are generally more resistant to treatment than those on the face.To determine efficacy and safety of BF-200 ALA versus vehicle in the treatment of mild-to-severe AK located on extremities, trunk, and neck with red light photodynamic therapy (PDT).This phase III study had an intra-individual design with 50 patients in 6 centers in Germany. Each patient received a maximum of 2 field-directed PDTs. Clinical end points and 1-year follow-up results were recorded.BF-200 ALA was superior to the vehicle with respect to total lesion clearance rates (86.0% vs 32.9%; P .0001) and patient complete clearance per patient's side (67.3% vs 12.2%, P .0001). One-year overall lesion recurrence rate was 14.1% versus 27.4% (BF-200 ALA vs vehicle; P = .0068). Patients were more satisfied by the cosmetic outcome of BF-200 ALA/PDT than the vehicle/PDT. Adverse events were consistent with the known safety profile of BF-200 ALA/PDT.Small number of severe lesions; limited sample size; unbalanced but representative distribution of AK.BF-200 ALA showed significantly higher AK clearance rates on extremities, trunk, and neck than the vehicle and was well tolerated.

Background: Dynamic optical coherence tomography (D-OCT) allows in vivo visualization of blood vessels in the skin and in malignant tumours. Vessel patterns in malignant melanoma may be associated with tumour stage. Objective: The aim of this study was to describe blood vessel patterns in melanomas and to correlate them with stage. Methods: One hundred fifty-nine malignant melanomas were assessed in a multicentre study. Every tumour was imaged using D-OCT prior to surgery and histologic evaluation. The tumour data such as thickness and ulceration as well as the staging at primary diagnosis and a follow-up of at least 40 months resulted in a stage classification. The vessel patterns were assessed according to predefined categories, compared with healthy adjacent skin, and correlated to stage. Results: Melanomas contained more blood vessels in different patterns compared with healthy adjacent skin. In particular, irregular vascular shapes such as blobs, coils, curves and serpiginous vessels were more common in melanomas. In addition, these patterns were significantly more often found in high-risk and metastatic melanomas than in low-risk lesions. Conclusion: In melanomas, the density of the blood vessels is increased, and irregular vascular patterns are more frequent. At higher stages, especially in metastatic melanomas, these atypical vessels are significantly more common.

Simple Summary The assessment of actinic keratoses (AKs) in prevention and therapeutic trials, as well as clinical practice, could significantly benefit from the incorporation of non-invasive imaging technology. Such technology has the potential to enhance the objective evaluation of clinical and subclinical AKs with the added advantage of sequential monitoring. In vivo reflectance confocal microscopy (RCM) allows for the non-invasive imaging of AKs at a cellular level. We aimed to establish an in in vivo RCM protocol for AK response monitoring, ultimately leading to more reliable characterization of longitudinal responses and therapy optimization. Abstract Reflectance confocal microscopy (RCM) presents a non-invasive method to image actinic keratosis (AK) at a cellular level. However, RCM criteria for AK response monitoring vary across studies and a universal, standardized approach is lacking. We aimed to identify reliable AK response criteria and to compare the clinical and RCM evaluation of responses across AK severity grades. Twenty patients were included and randomized to receive either cryotherapy (n = 10) or PDT (n = 10). Clinical assessment and RCM evaluation of 12 criteria were performed in AK lesions and photodamaged skin at baseline, 3 and 6 months. We identified the RCM criteria that reliably characterize AK at baseline and display significant reduction following treatment. Those with the highest baseline odds ratio (OR), good interobserver agreement, and most significant change over time were atypical honeycomb pattern (OR: 12.7, CI: 5.7–28.1), hyperkeratosis (OR: 13.6, CI: 5.3–34.9), stratum corneum disruption (OR: 7.8, CI: 3.5–17.3), and disarranged epidermal pattern (OR: 6.5, CI: 2.9–14.8). Clinical evaluation demonstrated a significant treatment response without relapse. However, in grade 2 AK, 10/12 RCM parameters increased from 3 to 6 months, which suggested early subclinical recurrence detection by RCM. Incorporating standardized RCM protocols for the assessment of AK may enable a more meaningful comparison across clinical trials, while allowing for the early detection of relapses and evaluation of biological responses to therapy over time.

Background and objectives Energy-based devices have been widely applied for skin ablation. A novel ablation technique based on thermomechanical principles (Tixel© ) has been recently developed. The aim of this study was to examine the wound-healing process and clinical aspects after thermomechanical skin ablation. Study design/materials and methods Six female participants were treated with Tixel© on healthy skin of the dorsal side of the right forearm in a single session with a 600 µm protrusion and 12 milliseconds pulse. The treated area was examined with confocal laser scanning microscopy on day 1, 2, 7, and 14 after treatment. Clinical symptoms were evaluated at the same time-points. Results All patients developed erythema and mild edema on the treated areas, which completely disappeared within 14 days. No post-inflammatory hyperpigmentation or scarring was observed. Thermomechanical skin ablation resulted in the formation of homogeneous micro-ablation zones. Two weeks after ablation, the honeycomb patterns of the epidermis in all examined layers was thoroughly restored. Thus, wound-healing was completed. Conclusions Wound healing after thermomechanical skin ablation is much faster compared with other fractionated ablation methods. Treatment intervals of 2-4 weeks could be recommended. Lasers Surg. Med. © 2020 The Authors. Lasers in Surgery and Medicine published by Wiley Periodicals, Inc.

Particle separation from gases is an important unit operation in manifold industrial applications, such as those conducted in environmental protection. For analysis of particle penetration and separation in fiber filters, standard dust particles (Al2O3) were loaded in the gas flow of a filter test facility and deposited within new and uncharged fiber filters. The loaded filters were analyzed by micro-computer tomography and scanning electron microscopy. Three-dimensional tomograms of the samples show an exponential decay of the penetration depth of the particles. This dependency is confirmed by simulations conducted using the discrete element method coupled with computational fluid dynamics within unloaded and loaded fiber structures. Microscale processes of particle separation at the fibers as well as the filtration efficiency and time-dependent filtering process are derived from the simulations. Local particle clustering in the filter medium and partial filter clogging are thus identified.

Introduction: Fractionated radiofrequency (RF) tissue tightening is an alternative method to fractionated laser treatment of skin wrinkling, laxity and acne scars, with reduced risk of scarring or persistent pigmentation. The aim of this study was to evaluate and quantify the wound healing process after RF treatment. Materials and methods: 12 patients were treated with a 64-pin fractional bipolar RF device with 60 mJ/pin applied energy. Confocal laser scanning microscopy (CLSM) examination was performed on day 1, day 2, day 7 and day 14 after treatment. Clinical wound healing process was measured and expressed as a percentage. Results: All patients developed erythema, mild edema and crusts at the treated areas. Two weeks after treatment clinical symptoms resolved. During ablation patients reported moderate pain. Directly after ablation microscopic ablation zones could be detected in CLSM. Measurement of MAZ at epidermis, dermo–epidermal junction and papilary dermis showed a constant diameter until two weeks after treatment. Re-epithelization of the MAZ could be detected already 1 week after treatment. However, 2 weeks after ablation the honeycomb pattern of the epidermis was not yet completely restored. Discussion: Bipolar fractionated RF treatment demonstrates clinically a rapid wound healing response. The subepidermal remodelling process still ongoing after 14 days, showing new granulation tissue. Therefore, treatment intervals of at least 14 days should be recommended to allow completion of the remodelling process.

Background Dynamic optical coherence tomography (D-OCT) has recently been introduced in dermatology. In contrast to 'Standard' OCT imaging, which exclusively relies on the morphological analysis of the tissue, D-OCT allows the in vivo visualization of blood flow. Preliminary D-OCT data showed differences in the vascularization of nevus to melanoma transition, suggesting that this technology may help to differentiate between benign and malignant lesions. Objective Several factors may influence the quality of D-OCT imaging. Therefore, standard operating procedures as well as a common terminology are required for better validation and comparison of the images. Methods Here, we present practical guidelines for optimal image acquisition and a proposed terminology on vascular patterns observed by D-OCT. Results Dynamic OCT allows the morphologic distinction of different vascular shapes (e.g. dots, blobs, curves, lines), their distribution and organization within skin lesions. Conclusion D-OCT adds functional information on skin microvasculature and the vascular networks within lesions.

BACKGROUND Basal cell carcinomas (BCCs) have previously been treated off-label with ingenol mebutate (IM). Ablative fractional laser (AFL) may improve efficacy of IM by increasing drug uptake in the tumour. Optical coherence tomography (OCT) and reflectance confocal microscopy (RCM) detect BCC non-invasively. Our aim was to investigate BCC response and tolerability after combined AFL and IM treatment of low-risk BCCs. METHODS Twenty patients with histologically verified superficial (n = 7) and nodular (n = 13) BCCs were treated with combined fractional CO2 -laser (10 600 nm) and IM 0.015% or 0.05%, the concentration depending on anatomical location. BCC response was evaluated clinically, by OCT and RCM at day 29 and 90 after first treatment, and histologically at day 90. Treatment was repeated at day 29 if BCC persisted. Local skin reactions (LSRs) were assessed using LSR scale at all visits. RESULTS At day 29, 18/20 patients received a second treatment due to residual BCC detected clinically, by OCT or RCM. OCT and RCM presented subclinical BCCs in five of 20 cases (25%). At day 90, overall histological cure rate was 70%, corresponding to clinical (65%) and OCT/RCM (60%) cure rates, and agreement between evaluation methods was substantial (kappa ≥ 0.796, P

Background Drug-induced immunosuppression is necessary to prevent rejection of the foreign organ in transplanted patients, but neoplastic and virus-associated skin diseases are frequent complications. Reflectance confocal microscopy (RCM) recently emerged as a promising tool for the early diagnosis of skin lesions. Materials and methods A total of 61 skin lesions, among them 20 basal cell carcinomas, six Bowen's diseases, 23 actinic keratoses, and 12 verrucae, were analyzed. All lesions were clinically evaluated followed by RCM evaluation by two independent dermatologists and histological examination. Results For the diagnosis of basal cell carcinoma, a sensitivity of 100% by both investigators (INV I + II) and a specificity of 100% by INV I and 80% by INV II were achieved. The sensitivity average rate for RCM features reached by both investigators ranged between 60% and 100%, and the specificity between 55% and 90%. For the diagnosis of actinic keratosis, a concordant sensitivity of 94.4% and a specificity of 80% (INV I) and 60% (INV II) were detected. The sensitivity average rate of specific RCM criteria ranged between 72.3% and 97.2%, whereas specificity ranged between 20% and 90%. Regarding verrucae, RCM confirmed the histological diagnosis with a sensitivity of 85.7% (INV I) and 100% (INV II), while specificity was 100% and 80%, respectively. Conclusion Reflectance confocal microscopy resulted to be a reliable tool for the noninvasive diagnosis of neoplastic and virus-associated skin changes in organ transplant recipients. Nevertheless, given the frequency and diagnostic complexity of the hyperkeratotic lesions occurring post-transplantation, larger cohorts of patients are required to confirm and consolidate these findings.

The routine diagnostic procedure of actinic keratosis (AK) and invasive squamous cell carcinoma (SCC) is a histological examination after taking a biopsy. In the past decades, non-invasive optical methods for skin examination have been developed. Patients with clinical diagnosis of AK or SCC were examined. The morphological criteria were determined for healthy, AK and SCC skin and compared for statistically significant differences. In this study, the applicability of multiphoton tomography (MPT) as an in vivo diagnostic tool for AK and SCC was evaluated. Changes in the morphology of the keratinocytes such as broadened epidermis, large intercellular spaces, enlarged nucleus and a large variance in cell shape could easily be recognized. The cell nuclei of AK and SCC were significantly larger compared to healthy skin cells in all cell layers. The nucleus-cytoplasm ratio was also significantly higher for AK and SCC than for the healthy skin cells. It was even higher in SCC compared to spinous and basal cell layer of AK. The cell density in AK and SCC was significantly lower than in the basal and spinous cell layers of healthy skin. In SCC, the cell density was significantly lower than in AK. Concerning the intercellular spaces, significant differences were found for AK and healthy skin in spinous and basal cell layer and for SCC compared to AK and healthy skin. In this study, MPT proved to be a valuable non-invasive imaging method for in vivo detection and discrimination of AK and SCC from healthy skin.

Pseudomonas aeruginosa produces increased levels of alginate in response to oxygen-deprived conditions. The regulatory pathway(s) that links oxygen limitation to increased synthesis of alginate has remained elusive. In the present study, using immunofluorescence microscopy, we show that anaerobiosis-induced alginate production by planktonic PAO1 requires the diguanylate cyclase (DGC) SadC, previously identified as a regulator of surface-associated lifestyles. Furthermore, we found that the gene products of PA4330 and PA4331, located in a predicted operon with sadC, have a major impact on alginate production: deletion of PA4330 (odaA, for oxygen-dependent alginate synthesis activator) caused an alginate production defect under anaerobic conditions, whereas a PA4331 (odaI, for oxygen-dependent alginate synthesis inhibitor) deletion mutant produced alginate also in the presence of oxygen, which would normally inhibit alginate synthesis. Based on their sequence, OdaA and OdaI have predicted hydratase and dioxygenase reductase activities, respectively. Enzymatic assays using purified protein showed that unlike OdaA, which did not significantly affect DGC activity of SadC, OdaI inhibited c-di-GMP production by SadC. Our data indicate that SadC, OdaA and OdaI are components of a novel response pathway of P. aeruginosa that regulates alginate synthesis in an oxygen-dependent manner.

Background: Basal cell carcinoma (BCC) represents the most common nonmelanoma skin cancer worldwide, affecting mainly adult, fair-skinned individuals. The World Health Organization distinguishes aggressive and nonaggressive forms, of which prototypical variants of the latter are primary nodular and superficial BCC. Objective: To demonstrate noninferiority of BF-200 ALA (a nanoemulsion gel containing 5-aminolaevulinic acid) compared with MAL (a cream containing methyl aminolaevulinate) in the treatment of nonaggressive BCC with photodynamic therapy (PDT). Noninferiority of the primary efficacy variable (overall patient complete response 12 weeks after last PDT) would be declared if the mean response for BF-200 ALA was no worse than that for MAL, within a statistical margin of = -15%. Methods: The study was a randomized, phase III trial performed in Germany and the U.K. with ongoing 5-year follow-up. Of 281 randomized patients, 138 were treated with BF-200 ALA and 143 with MAL. Patients received two PDT sessions 1 week apart. Remaining lesions 12 weeks after the second PDT were retreated. Illumination was performed with a red light source (635 nm, 37 J cm(-2)). The results shown include clinical end points and patients' reassessment 12 months after the last PDT. The study was registered with EudraCT (number 2013-003241-42). Results: Of the BF-200 ALA-treated patients, 93 4% were complete responders compared with 91 8% in the MAL group. The difference of means was 1 6, with a one-sided 97 5% confidence interval of -6 5, establishing noninferiority (P 0001). The results for secondary efficacy parameters were in line with the primary outcome. Recurrence rates 12 months after the last treatment were 10%. Conclusion: sTreatment of nonaggressive BCC with BF-200 ALA-PDT is highly effective and well tolerated with proven noninferiority to MAL-PDT. It demonstrates low recurrence rates after 1 year of follow-up. What's already known about this topic? Photodynamic therapy (PDT) using BF-200 aminolaevulinic acid (ALA) gel is registered and highly effective in the treatment of mild-to-moderate actinic keratosis and field cancerization. BF-200 ALA gel was recently approved for the treatment of superficial and/or nodular basal cell carcinoma (BCC) unsuitable for surgical treatment. PDT using methyl aminolaevulinate (MAL) cream is approved for the treatment of thin or nonhyperkeratotic and nonpigmented actinic keratoses, Bowen disease, and superficial and nodular BCCs when other therapies are considered less appropriate. What does this study add? BF-200 ALA-PDT is confirmed to be significantly noninferior to MAL-PDT for the treatment of nonaggressive BCC. Treatment-emergent adverse events were comparable between the two patient groups, with similar or slightly lower recurrence rates for BF-200 ALA gel compared with MAL cream after 12 months.

Background: Early detection of various types of nonmelanoma skin cancer has been a challenge in dermatology. Noninvasive examination procedures such as optical coherence tomography (OCT) play an increasingly important role, besides the established gold standard of histological tissue sample analysis. OCT is a noninvasive, cross-sectional, real-time technique that allows conclusions to be drawn with regard to the presence of pathologies. Objective: The objective of this study was to investigate whether it is possible to distinguish between different types of nonmelanoma skin cancer using OCT or not. Methods: A study population of a total of 25 cases, comprising 5 cases, each, of 5 tumor entities (i.e., basal cell carcinoma, superficial basal cell carcinoma, actinic keratosis, squamous cell carcinoma, and Bowen disease) was examined. Relevant lesions were scanned both centrally and peripherally in the multislice mode. All OCT images were blinded, randomized, analyzed, and evaluated by 2 clinicians experienced in OCT. Results: This study demonstrated that it is possible to determine correlations between various types of tumors and recurring tumor characteristics. Conclusion: This study showed that it is possible to distinguish between the different nonmelanoma skin cancers by using OCT, but further prospective studies have to be conducted to validate the sensitivity and specificity of the criteria.

Aging depicts one of the major challenges in pharmacology owing to its complexity and heterogeneity. Thereby, advanced glycated end-products modify extracellular matrix proteins, but the consequences on the skin barrier function remain heavily understudied. Herein, we utilized transmission electron microscopy for the ultrastructural analysis of ribose-induced glycated reconstructed human skin (RHS). Molecular and functional insights substantiated the ultrastructural characterization and proved the relevance of glycated RHS beyond skin aging. In particular, electron microscopy mapped the accumulation and altered spatial orientation of fibrils and filaments in the dermal compartment of glycated RHS. Moreover, the epidermal basement membrane appeared thicker in glycated than in non-glycated RHS, but electron microscopy identified longitudinal clusters of the finest collagen fibrils instead of real thickening. The stratum granulosum contained more cell layers, the morphology of keratohyalin granules decidedly differed, and the stratum corneum lipid order increased in ribose-induced glycated RHS, while the skin barrier function was almost not affected. In conclusion, dermal advanced glycated end-products markedly changed the epidermal morphology, underlining the importance of matrix&ndash, cell interactions. The phenotype of ribose-induced glycated RHS emulated aged skin in the dermis, while the two to three times increased thickness of the stratum granulosum resembled poorer cornification.

BACKGROUND Malignant melanoma is an aggressive skin cancer, which can lead to metastasis development. Vascularization enhancement is fundamental for tumor growth, worsening the prognosis. Dynamic optical coherence tomography (D-OCT) enables the in vivo evaluation of vascular patterns in skin lesions. OBJECTIVE In vivo evaluation of the melanoma vessel morphology by means of D-OCT and correlation with Breslow index. METHODS Retrospective analysis of histologically proven melanomas, evaluated by D-OCT at three different depths (150, 300 and 500 μm), was performed. Vessels were classified according to morphology (dots, blobs, coiled, line, curved, serpiginous), distribution (regular, irregular) and the presence/type of branches. The data were correlated with Breslow thickness. RESULTS A total of 127 melanomas were evaluated. Dotted vessels were recorded at all depths, and their irregular distribution was associated with lesions thicker than 1.0 mm (from 75% to 91%), compared with thin ones (42%) at 150 μm (P = 0.031), and from 33% to 57% vs 18% at 300 μm (P = 0.021). Serpiginous and branching vessels with bulges were predominantly seen in melanomas thicker than 2 mm at 150 μm (from 14% to 27%, P

Background Multi-beam optical coherence tomography (OCT) is a novel method of non-invasive skin imaging allowing the evaluation of tissue at high level of lateral and axial resolution. It permits the horizontal and vertical evaluation of the extent of diseases. Objective Herein, we aimed to validate diagnosing basal cell carcinoma (BCC) by OCT using a newly developed scoring system (‘Berlin Score’-BS). This was based on the predetermined criteria such as dark border underneath the tumour and ovoid structures. Their frequency and distribution in subtypes of BCC were evaluated. Methods The study was conducted in two phases, in which the experience of examiner differed. A total of 127 BCC and 50 other skin diseases were examined. In phase one, students performed the evaluation of skin lesions using the BS, while in phase two an expert performed the scoring in a different subset of patients. Results Application of BS by students revealed sensitivity and specificity of 92.8% (95% CI 85.4–96.8) and 24.1% (95% CI 11.0–43.4) when reaching the lower threshold BS≥8. The most common BCC subtypes were superficial (28.7%) and nodular (22.6%) BCC. Second phase was carried out to verify collected data by a dermatological specialist and expert in using OCT. Increased sensitivity and specificity for OCT amounted to 96.6% (95% CI 80.4–99.8) and 75.2% (95% CI 52.5–90.9). Thereby 88% of all diagnoses were correctly classified confirmed by histopathology. Conclusion Multi-beam optical coherence tomography revealed to be a fast and promising device for assessing lesions by means of BS. Both students, who benefit from practice in handling OCT, and experts are able to perform this procedure. However, experience and training in the interpretation markedly increased sensitivity and specificity of the BS in our study. Moreover, redefinition and refining of the criteria seems necessary and may further increase the diagnostic value of OCT for NMSC.

Actinic keratosis (AK), a frequently diagnosed cutaneous neoplasm in individuals with chronic sun exposure or fair skin, is a risk factor for squamous cell carcinoma. AK presents as clinically visible lesions and/or as subclinical lesions where an entire field of area (field cancerization) contains lesions of various grades. The diagnosis and surveillance of subclinical AK is challenging. We report a new AK management approach, including subclinical AK, with noninvasive in vivo reflectance confocal microscopy (RCM) monitoring of field-directed topical 5-fluorouracil 0.5%/salicylic acid 10.0% (5-FU/SA; currently approved for single lesions). In this case series, eight patients with primarily recurrent, multiple AKs received ≤6 weeks of field-directed 5-FU/SA; complete clearance of clinical/subclinical AKs on various body areas was shown in most patients using RCM. RCM facilitated the detection/characterization of subclinical AKs in the setting of field cancerization. Topical field-directed 5-FU/SA monitored with RCM is a promising management approach for subclinical AKs.

The subtype of basal cell carcinoma (BCC) influences the choice of treatment. Optical coherence tomography (OCT) is a non-invasive imaging tool, and a recent development of an angiographic version of OCT has extended the application of OCT to image the cutaneous microvasculature (so-called dynamic OCT, D-OCT). This study explores D-OCT's ability to differentiate the common BCC subtypes by microvascular and structural imaging. Eighty-one patients with 98 BCC lesions, consisting of three subtypes: 27 superficial BCC (sBCC), 55 nodular BCC (nBCC) and 16 infiltrative BCC (iBCC) were D-OCT scanned at three European dermatology centres. Blinded evaluations of microvascular and structural features were performed, followed by extensive statistical analysis of risk ratio (RR) and multiple correspondence analysis. nBCC lesions displayed most characteristic structural and vascular features. Serpiginous vessels, branching vessels, vessels creating a circumscribed figure and sharply demarcated hyporeflective ovoid structures in the dermis were all associated with a higher risk of the subtype being nBCC. The presence of highly present lines and dark peripheral borders at the margin of ovoid structures was negatively associated with iBCC. Lastly, the finding of hyporeflective ovoid structures protruding from epidermis correlated with sBCC. We identified various microvascular and structural D-OCT features that may aid non-invasive identification of BCC subtypes. This would allow clinicians to individualize and optimize BCC treatment as well as aid follow-up of non-surgical treatment.

Background: In vivo reflectance confocal microscopy (RCM) is a promising non-invasive skin imaging technique that could facilitate early diagnosis of basal cell carcinoma (BCC) instead of routine punch biopsies. However, the clinical value and utility of RCM vs. a punch biopsy in diagnosing and subtyping BCC is unknown. Objective: To assess diagnostic accuracy of RCM vs. punch biopsy for diagnosing and subtyping clinically suspected primary BCC. Methods: A prospective, consecutive cohort of 100 patients with clinically suspected BCC were included at two tertiary hospitals in Amsterdam, the Netherlands, between 3 February 2015 and 2 October 2015. Patients were randomized between two test-treatment pathways: diagnosing and subtyping using RCM imaging followed by direct surgical excision (RCM one-stop-shop) or planned excision based upon the histological diagnosis and subtype of punch biopsy (standard care). The primary outcome was the agreement between the index tests (RCM vs. punch biopsy) and reference standard (excision specimen) in correctly diagnosing BCC. The secondary outcome was the agreement between the index tests and reference standard in correctly identifying the most aggressive BCC subtypes. Results: Sensitivity to detect BCC was similar for RCM and punch biopsy (100% vs. 93.94%), but a punch biopsy was more specific than RCM (79% vs. 38%). RCM expert evaluation for diagnosing BCC had a sensitivity of 100% and a specificity of 75%. The agreement between RCM and excision specimen in identifying the most aggressive BCC subtype ranged from 50% to 85% vs. 77% by a punch biopsy. Conclusion: Reflectance confocal microscopy and punch biopsy have comparable diagnostic accuracy to diagnose and subtype BCC depending on RCM experience. Although experienced RCM users could accurately diagnose BCC at a distance, we found an important difference in subtyping BCC. Future RCM studies need to focus on diagnostic accuracy, reliability and specific criteria to improve BCC subtype differentiation.

Background A clear distinction between actinic keratosis (AK), Bowen's disease (BD) and squamous cell carcinoma (SCC) cannot reliably be made by clinical and dermoscopic evaluation alone. Dynamic optical coherence tomography (D-OCT) is a novel angiographic variant of OCT that allows for non-invasive, in vivo evaluation of the cutaneous microvascular morphology. Objective To investigate the microvascular structures of AK, BD and invasive SCC using D-OCT in order to gain insights into the microvascular morphology of lesions in the spectrum of keratinocyte skin cancers. Methods Forty-seven patients with a total of 54 lesions (18 AK, 12 BD and 24 SCC) were included in the study. D-OCT still images of AK, BD and SCC at three predefined skin depths were prepared and randomized, creating a study set of 162 D-OCT images. Three observers performed blinded evaluations of the randomized study set assessing multiple parameters including the different types of vascular morphology. Non-blinded quantitative measurements of vascular diameter were also performed. Results The blinded observer analysis suggests that D-OCT evaluation of the vascular morphology may aid in distinguishing AK, BD and SCC lesions. We identified two vascular shapes that presented significantly differently across the lesion types, namely 'blobs' and 'curves'. A strong presence of blobs at 300 μm skin depth was characteristically seen in a third of BD cases, while not or only slightly present in AK and SCC lesions. Vascular curves were predominantly present in AK lesions. Conclusion We identified various vascular D-OCT features that may aid in non-invasively differentiating subtypes within the keratinocyte skin cancer spectrum.

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Ingenol mebutate represents a topical treatment for fields with actinic keratosis (AK). The biological effects of ingenol mebutate in AK, subclinical (SC)-AK, and reference-skin were assessed and graded by in vivo reflectance confocal microscopy (RCM) and histology. Patients with AK and SC-AK lesions in one 25 cm2 field on hands or forearms, and with an area of reference skin on the inner upper arm, were included. The two fields were each treated with ingenol mebutate 0.05% gel (n=16), or vehicle (n=8), on 2 consecutive days; clinical and RCM assessments were performed on days 1, 2, 3, 8, and 57, and biopsies on day 3. Local skin responses were more pronounced in AK fields (6.1 (mean) ± 2.6 (SD)) compared with reference skin (3.5 ± 1.5). The clinical AK lesion reduction was 43.8% and 6.3% with ingenol mebutate and vehicle, respectively. RCM and histology evaluations showed that ingenol mebutate induced a significant pronounced cell death and immune response in AK and SC-AK lesions, compared with reference skin. Ingenol mebutate induced RCM-measured reduction in (investigator-1/investigator-2): AK lesions (34/28%), SC-AK lesions (72/56%), and solar elastosis in AK fields (mean, -0.22/-0.25). In conclusion, ingenol mebutate showed selective pronounced biological responses in AK and SC-AK as compared with reference skin.br /br /emJ Drugs Dermatol./em2016;15(10):1181-1189.

SummaryBackground To date, studies with ingenol mebutate gel have used clinical clearance, not histological clearance, as a primary efficacy endpoint. Objectives This phase 1, multicentre, single-arm, open-label study sought to histologically confirm clinical clearance of actinic keratoses (AKs) to support a treatment effect deep in the epidermis. Methods Patients (n = 108) aged ≥18 years with histologically confirmed AK within a 25-cm2 contiguous treatment area on the trunk and extremities received ingenol mebutate 0.05% gel for 2 consecutive days and were followed up on day 3 and week 8. One AK was randomly pre-selected at day 1 for clinical and histological evaluation at week 8; and for reflectance confocal microscopy (RCM) in a subset of patients. Primary endpoint was clinical and histological clearance of AKs at week 8. Results The observed agreement rate between clinical and histological assessments of clearance of a single AK was 81.9% and the positive predictive value of a clinical assessment of clearance was 87%. Agreement between the two pathologists was 88%. The common composite 8-week complete clearance rate was 41% (95% confidence interval 32%, 50%). Observed agreement rates between RCM and pathologist I and II assessments of clearance were 72.9% and 81.4%, respectively. Thirty patients (27.8%) experienced 38 adverse events (AEs). Application-site pain (four patients, 3.7%) was the most common treatment-related AE inside the treatment area. Conclusion Ingenol mebutate achieves histopathological clearance of AKs that correlates with observed clinical clearance. Clinical clearance is a good predictor for histological clearance. This article is protected by copyright. All rights reserved.

Optical coherence tomography (OCT) is a technique that enables real-time in-vivo examination of tissue. This technology provides the clinician with the potential to use a non-invasive tool in the identification and diagnosis of many skin lesions. However, the diagnostic features of basal cell carcinoma have not yet been described with comparison to their histopathology.br /To identify and describe key features of basal cell carcinoma (BCC) and its subtypes as they present in multi-beam Swept Source - OCT (MSS-OCT), and to correlate those against conventional histopathology.br/A total of 40 lesions were assessed by MSS-OCT prior to biopsy. 60-slice OCT images of the lesions were obtained and correlated with histology sections taken in the same plane. OCT scans were assessed retrospectively by a panel to determine the OCT criteria for BCC and its subtypes.br/The following diagnostic criteria were identified: hyporeflective ovoid structures (40/40), dark halo boundaries (38/40), epidermal thinning (28/40), and collagen compression (14/40). Lesional tissue also showed a destruction of layers when compared to the surrounding normal tissue. In addition to the shared criteria, other subtypes showed distinct diagnostic criteria.br/With its higher sensitivity, using MSS-OCT allowed for non-invasive, accurate identification of the key diagnostic features of BCC and its subtypes with high correlation to the histopathologic features found with biopsy.br /br /emJ Drugs Dermatol./em2016;15(5):545-550.

Dynamic optical coherence tomography (D-OCT) is an angiographic variation of OCT that non-invasively provides images of the in vivo microvasculature of the skin by combining conventional OCT images with flow data. The objective of this study was to investigate and report on the D-OCT technique for imaging of the vascular networks in skin as well as to validate the method by comparing the results against already accepted blood flow measuring tools.35 healthy subjects were recruited for the multicentre study, consisting of three experiments set up to examine the vascular blood perfusion during different induced physiologic changes in the blood flow. In order to validate the D-OCT images against existing techniques for blood flow measuring we performed consecutive D-OCT, chromametry and laser speckle contrast imager (LSCI) measurements on identical skin sites in all of the experiments. Blinded observer evaluations were performed in order to evaluate the vascular morphology in the D-OCT images.The results showed a statistically significant positive correlation between the D-OCT measurements and the LCSI flux measurements (rs=0.494; 95% CI [0.357, 0.615]; p0.001), and also the redness a* measurements were positively correlated with the D-OCT measurements (r=0.48; 95% CI [0.406, 0.55]). D-OCT was able to reliably image and identify morphologic changes in the vascular network consistent with the induced physiological changes of blood flow.This study has initiated validation of the use of D-OCT for imaging of skin blood flow. Our results showed that D-OCT was able to reliably image and identify changes in the skin vasculature consistent with the induced physiological blood flow changes. These basic findings support the use of D-OCT imaging for in vivo microcirculation imaging of the skin.

Non-melanoma skin cancer (NMSC) is the most frequent human cancer with continuously rising incidences worldwide. Herein, we investigated the molecular basis for the impaired skin barrier function of organotypic NMSC models. We unraveled disturbed epidermal differentiation by reflectance confocal microscopy and histopathological evaluation. While the presence of claudin-4 and occludin were distinctly reduced, zonula occludens protein-1 was more wide-spread, and claudin-1 was heterogeneously distributed within the NMSC models compared with normal reconstructed human skin. Moreover, the cancer altered stratum corneum lipid packing and profile with decreased cholesterol content, increased phospholipid amount, and altered ceramide subclasses. These alterations contributed to increased surface pH and to 1.5 to 2.6-fold enhanced caffeine permeability of the NMSC models. Three topical applications of ingenol mebutate gel (0.015%) caused abundant epidermal cell necrosis, decreased Ki-67 indices, and increased lactate dehydrogenase activity. Taken together, our study provides new biological insights into the microenvironment of organotypic NMSC models, improves the understanding of the disease model by revealing causes for impaired skin barrier function in NMSC models at the molecular level, and fosters human cell-based approaches in preclinical drug evaluation.

Optical coherence tomography (OCT) represents a non-invasive imaging technology, which may be applied to the diagnosis of non-melanoma skin cancer and which has recently been shown to improve the diagnostic accuracy of basal cell carcinoma. Technical developments of OCT continue to expand the applicability of OCT for different neoplastic and inflammatory skin diseases. Of these, dynamic OCT (D-OCT) based on speckle variance OCT is of special interest as it allows the in vivo evaluation of blood vessels and their distribution within specific lesions, providing additional functional information and consequently greater density of data. In an effort to assess the potential of D-OCT for future scientific and clinical studies, we have therefore reviewed the literature and preliminary unpublished data on the visualization of the microvasculature using D-OCT. Information on D-OCT in skin cancers including melanoma, as well as in a variety of other skin diseases, is presented in an atlas. Possible diagnostic features are suggested, although these require additional validation.

Tyrosine nitration in proteins occurs under physiologic conditions and is increased at disease conditions associated with oxidative stress, such as inflammation and Alzheimer's disease. Identification and quantification of tyrosine-nitrations are crucial for understanding nitration mechanism(s) and their functional consequences. Mass spectrometry (MS) is best suited to identify nitration sites, but is hampered by low stabilities and modification levels and possible structural changes induced by nitration. In this insight, we discuss methods for identifying and quantifying nitration sites by proteolytic affinity extraction using nitrotyrosine (NT)-specific antibodies, in combination with electrospray-MS. The efficiency of this approach is illustrated by identification of specific nitration sites in two proteins in eosinophil granules from several biological samples, eosinophil-cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Affinity extraction combined with Edman sequencing enabled the quantification of nitration levels, which were found to be 8 % and 15 % for ECP and EDN, respectively. Structure modeling utilizing available crystal structures and affinity studies using synthetic NT-peptides suggest a tyrosine nitration sequence motif comprising positively charged residues in the vicinity of the NT- residue, located at specific surface- accessible sites of the protein structure. Affinities of Tyr-nitrated peptides from ECP and EDN to NT-antibodies, determined by online bioaffinity- MS, provided nanomolar K(D) values. In contrast, false-positive identifications of nitrations were obtained in proteins from cystic fibrosis patients upon using NT-specific antibodies, and were shown to be hydroxy-tyrosine modifications. These results demonstrate affinity- mass spectrometry approaches to be essential for unequivocal identification of biological tyrosine nitrations.

Summary Background There are poorly documented variations in the journey a skin cancer patient will follow from diagnosis to treatment in the European Union. Objectives To investigate the possible difficulties or obstacles that a person with a skin malignancy in the European Union may have to overcome in order to receive adequate medical screening and care for his/her condition. In addition, we wished to explore differences in European health systems, which may lead to health inequalities and health inequities within Europe. Methods Ten European countries took part in this investigation (in alphabetical order): Finland, Germany, Greece, Italy, Malta, Poland, Romania, Spain, the Netherlands and the U.K. The individual participants undertook local and national enquiries within their own country and completed a questionnaire. Results This exercise has identified important differences in the management of a skin cancer patient, reflecting major disparities in health care between European countries. Conclusions Further investigation of health disparities and efforts to address health inequalities should lead to improvements in European health care quality and reduction in morbidity from skin cancer.

Background There are limited data regarding the association of actinic keratosis (AK) and other types of nonmelanoma skin cancer (NMSC); studies investigating possible correlation of AK with melanocytic naevi are even scarcer. To our knowledge, there are no data examining the risk of AK in people using specific medications. Objective To investigate constitutional and exposure risk factors leading to AK and the coexistence of AK with NMSC and melanoma. Methods A multicentre hospital-based case-control study was performed in Finland, Germany, Greece, Italy, Malta, Poland, Scotland and Spain, including 343 patients with actinic keratosis (AK), 409 with squamous cell carcinoma (SCC), 602 with basal cell carcinoma (BCC), 360 with invasive melanoma and 119 with in situ melanoma, and 686 control subjects. Exposures were assessed by questionnaires that were partly self-administered and partly filled out by dermatologists. Unconditional logistic regression modelling was used to assess associations including the influence of phenotypic characteristics, presence of naevi, sun-exposure habits and certain drugs on AK risk. Results Differences in hair and eye coloration variably influenced the risk for AK, with red hair signifying a seven times higher risk [odds ratio (OR) 6·9, 95% confidence interval (CI) 4·34-11·00), and brown - compared with blue - eyes, about a 40% reduced risk (OR 0·61, 95% CI 0·13-0·92). The darker the skin phototype, the lower the risk for AK, with phototype IV exhibiting nine times less risk of developing AK. Some and many freckles on the arms were associated with an OR of 1·8 (95% CI 1·08-2·81) and 3·0 (95% CI 1·10-3·54), respectively, while overall number of naevi and high educational level were inversely associated with AK. Sun exposure, thiazide diuretics and cardiac drugs had a higher risk for AK. SCC was the most frequent (58%) skin neoplasm coexisting with AKs, followed by BCC (30%), melanoma in situ (12%) and invasive melanoma (6%). Conclusion In this large case-control study from across Europe the expected associations were confirmed for known risk factors. Some possible new risk factors, including cardiac and diuretic drugs, were identified, creating a new field for further investigation in future studies.

Summary Background During recent years numerous studies have suggested that personal and environmental factors might influence cancer development. Objectives To investigate environmental and personal characteristics associated with skin cancer risk. Methods A multicentre hospital-based case–control study was performed in Finland, Germany, Greece, Italy, Malta, Poland, Scotland and Spain, including 409 patients with squamous cell carcinoma (SCC), 602 with basal cell carcinoma (BCC) and 360 with cutaneous malignant melanoma (CMM) and 1550 control persons. Exposures were assessed by questionnaires that were partly self-administered, partly completed by dermatologists. Unconditional logistic regression modelling was used to assess associations including the influence of certain drugs and food items on skin cancer risk. Results The usual associations were observed for sun exposure and pigmentation characteristics, with chronic sun exposure being most strongly associated with SCC risk, and naevi and atypical naevi with CMM risk. Use of ciprofloxacin was associated with a decreased risk of BCC [odds ratio (OR) 0·33] and use of thiazide diuretics was associated with an increased risk of SCC (OR 1·66). Ciprofloxacin was also associated with SCC (OR 0·34) and thiazines with BCC (OR 2·04), but these associations lost significance after correction for multiple testing. Consumption of pomegranate, rich in antioxidants, was associated with decreased BCC and SCC risk, also after correcting for multiple testing. Recent experience of stressful events was associated with increased risk, particularly of CMM. Conclusions In this large case–control study from across Europe the expected associations were observed for known risk factors. Some new potential protective factors and potential risk factors were identified for consumption of certain food items, medication use and stress, which deserve further investigation in future studies.

Summary Background A wide variety of both surgical and nonsurgical therapies is currently available for patients with skin cancer. Objectives This part of the EPIDERM (European Prevention Initiative for Dermatological Malignancies) project is aimed at the evaluation of the treatment preferences for skin cancer in eight countries of the European Union. Methods A multicentre hospital-based case–control study was carried out at dermatology departments in Finland, Germany, Greece, Italy, Malta, Poland, Scotland and Spain. Patients with skin cancer (basal cell carcinoma, actinic keratosis, squamous cell carcinoma, cutaneous malignant melanoma and Bowen disease) were consecutively enrolled between July 2008 and July 2010. Information on the study variables (sex, age, country, tumour type, anatomical location and treatment) was obtained from questionnaires designed by the EPIDERM project. Results In total, 1708 patients with skin cancer were included. Surgery was the first treatment option in 76·5% of the patients (P = 0·001). Actinic keratosis was the only tumour type in which nonsurgical treatment was more frequent than surgery (91·4%). Tumours on the head were less likely to be surgically excised than those at other locations (odds ratio 0·25, P = 0·001). Simple excision or curettage was the most common surgical procedure (65·4%), followed by graft and flaps (22·4%). Cryotherapy was the most common nonsurgical option (52·4%), followed by imiquimod (18·0%), photodynamic therapy (PDT; 12·0%), 5-fluorouracil (5-FU; 5·7%), and diclofenac with hyaluronic acid (4·0%). Conclusions Surgery remains the first-choice treatment of skin cancer. Regarding nonsurgical treatments, the conservative treatments available (imiquimod, 5-FU, PDT and diclofenac gel) have not yet exceeded the use of ablative options such as cryotherapy despite their accepted benefit of treating field cancerization.

Background Actinic cheilitis (AC) represents the equivalent of actinic keratosis on the lip. Various treatment modalities are available and the efficacy of diclofenac in hyaluronic acid has recently been described. Reflectance confocal microscopy (RCM) is a non-invasive imaging technique which has recently been applied for the diagnosis of actinic keratoses. Herein, we describe the applicability of RCM for the diagnosis of AC and for monitoring of treatment response of AC to diclofenac in hyaluronic acid. Methods Ten Caucasian patients with clinical suspicion for AC were included in this study. To obtain a non-invasive diagnosis, RCM was performed at baseline, followed by biopsy and respective confocal-histopathological correlation. Six patients with a histological diagnosis of AC were treated with diclofenac in hyaluronic acid, whereby monitoring was performed by RCM. Results Reflectance confocal microscopy was able to correctly identify 6/7 cases of AC and 3/3 cases of benign lesions. The most important RCM criteria for diagnosis of AC were cellular atypia at the stratum spinosum and granulosum with atypical honeycomb pattern. One patient with AC was misclassified as inflammatory cheilitis by RCM as it showed marked inflammatory response and lacked clear signs of cellular atypia on RCM imaging. Following topical treatment with diclofenac gel, 5/6 patients (83%) showed a good treatment response with regression of dysplasia on consecutive RCM examination. Conclusions Reflectance confocal microscopy is a promising tool for the non-invasive diagnosis and monitoring of actinic cheilitis. However, marked inflammation represents a potential diagnostic pitfall. In this regard, biopsy should be performed in doubtful cases.

Reflectance confocal microscopy (RCM) is a novel, noninvasive imaging technique based on the collection of in vivo high-resolution images using the differential reflectance of the subcellular structures of the epidermis and dermis when illuminated with a coherent light source. RCM has been used to visualize different skin pathologies, notably actinic keratoses and several types of skin cancer, including squamous and basal cell carcinomas and melanoma. Basal cell carcinoma is particularly amenable to examination by RCM, and its clinical features as visualized by this technique have been described in detail. In addition, RCM allows follow-up of the response of diseased skin to treatment and aids in the determination of the precision of surgical procedures due to the possibility of performing repetitive examinations without further damage of the affected area.

Background: Antibiotic therapy is thought to improve lung function in patients with cystic fibrosis (CF) by decreasing neutrophil-derived inflammation. We investigated the origin and clinical significance of lactate in the chronically inflamed CF lung. Methods: Lactate was measured in sputa of 18 exacerbated and 25 stable CF patients via spectrophotometry and gaschromatography. Lung function was assessed via spirometry. Seven patients with chronic obstructive pulmonary disease (COPD) and three patients with acute lung inflammation served as control groups. Neutrophil and bacterial lactate production was assessed under aerobic and anaerobic conditions. Results: In sputum specimens of patients with respiratory exacerbations lactate concentrations decreased significantly (pb0.005) from 3.4±2.3 mmol/L to 1.4±1.4 mmol/L after 2–3 weeks of intravenous antibiotics. Successful treatment was reflected in 16 patients (88.9%) by FVC increase associated with lactate decrease (pb0.05). In every single sputum lactate was detectable (3.0±3.1 mmol/L, range 0.2–14.1 mmol/L). Lactate was lower (1.6±0.8 mmol/L) in sputa from seven COPD patients, and it was below the detection limit in three patients with acute lung inflammation. Neutrophil lactate production accumulated up to 10.5 mmol/L after 4 days, whereas bacterial lactate production didnot appear to contribute substantiallytosputum lactateconcentrations. Conclusions: Successful antibiotic therapy is reflected by a decrease in lactate concentrations. Neutrophils are the most likely source for lactate in sputum of CF patients. Therefore lactate may be used to monitor responses to antibiotic therapy as an adjunct to lung function measurements.

BACKGROUND Antibiotic combination therapy might be more efficient than single antibiotics to combat Pseudomonas aeruginosa biofilms in the airways of patients with cystic fibrosis. We tested the ability of colistin sulphate-tobramycin combinations and single antibiotics to kill P. aeruginosa biofilms. METHODS P. aeruginosa biofilms were generated in vitro and in rat lungs. In a pilot study, 5 patients with cystic fibrosis inhaled colistin and then tobramycin for 4 weeks. The changes in P. aeruginosa counts and lung function were assessed before and after therapy. RESULTS Antibiotic combination therapy significantly reduced the number of P. aeruginosa cells in P. aeruginosa biofilm models in vitro. When rats were challenged with 1 x 10(7) cfu of P. aeruginosa, which was embedded in alginate beads, mortality rates, lung pathologic findings, and bacterial colony-forming unit counts were significantly lower after 7 days in animals receiving antibiotic combination than in animals receiving single antibiotics. In patients with cystic fibrosis, inhaled colistin-tobramycin was well tolerated and resulted in a mean decrease of 2.52 + /- 2.5 log(10) cfu of P. aeruginosa per milliliter of sputum (P = .027). Measurements of forced expiratory volume in 1 s, obtained both before and after the study, did not differ significantly. CONCLUSION Colistin-tobramycin combinations are more efficient than respective single antibiotics for killing P. aeruginosa in biofilms in vitro, and they significantly reduced P. aeruginosa cell counts in a rat lung infection model and in patients with cystic fibrosis.

Background: Histopathologically, basal cell carcinoma (BCC) is characterized by basaloid tumor nodules of varying size showing peripheral palisading of cells and nuclei, and separation from surrounding stroma by optically empty appearing clefts. These are usually regarded as an artifact, occurring during routine tissue processing. Recently, reflectance confocal microscopy (RCM) has been applied for noninvasive, in vivo evaluation of BCC. Besides other features, small areas of low refractility separating tumor islands from the surrounding tissue can be observed in vivo, suggesting that the presence of amorphous material like mucin might be the causal factor for these clefts. Methods: A total of 13 BCCs were studied by RCM and histopathological techniques. Staining was performed with Alcian blue for the detection of peritumoral mucin. Correlation between RCM images and histopathological samples was studied, and the diameter of hyporefractile areas on RCM as well as the thickness of peritumoral mucin was measured. Results: Good correlation was seen between dark areas on RCM and thickness of peritumoral mucin with a mean diameter of 14 μ m( RCM) and 11.44 μm (histopathology), respectively. Pearson correlation coefficient was 0.605 (p < 0.0001). Conclusions: Our results show that the peritumoral cleft-like spaces seen in BCC on histopathology exist in vivo, and correspond to the peritumoral mucin deposition.

Virulent varicella-zoster virus (VZV) can spread in immunocompetent humans, resulting in symptoms mostly of the skin. In contrast, vaccine Oka (V-Oka), the attenuated VZV vaccine strain, only rarely causes clinical reactions. The mechanisms underlying these pathogenetic differences are unclear. In this study, we comparatively analyzed the ability of virulent VZV and V-Oka to modulate instruction of dendritic cells (DCs) by innate signals. DCs isolated from normal human skin were susceptible to infection with VZV and V-Oka. Moreover, inflammatory DCs, which play a crucial role in the stimulation of Th1 immune responses, accumulated in herpes zoster lesions. Infection of inflammatory DCs generated in vitro with virulent VZV or V-Oka resulted in upregulation of CD1c. Upon coculture with CD1c-restricted innate cells, DCs developed a mature phenotype whether infected with virulent VZV or V-Oka. Intriguingly, a striking difference was detected on the functional level. The release of IFN-γ and IL-12, the signature cytokines of Th1 responses, was enhanced by V-Oka but blocked by virulent VZV. V-Oka and virulent VZV efficiently synergized with CD40L, eliminating the possibility that CD40 signaling was a target of VZV-associated immune evasion. Instead, virulent VZV selectively interfered with signaling through TLR2, which is known to sense VZV. Thus, virulent VZV subverts Th1-promoting instruction of human DCs by blocking TLR2-mediated innate signals that prime IL-12 production by DCs. Taken together, our results demonstrate a novel immune-evasion mechanism of virulent VZV that has been lost during the attenuation process leading to the VZV vaccine strain.