Metformin shows anti-neoplastic properties by inhibition of oxidative phosphorylation and glycolysis in epidermolysis bullosa-associated aggressive cutaneous squamous cell carcinoma.

BACKGROUND: While most cutaneous squamous cell carcinomas (cSCCs) are treatable, certain high-risk cSCCs, such as those in recessive dystrophic epidermolysis bullosa (RDEB) patients, are particularly aggressive. Owing to repeated wounding, inflammation and unproductive healing, RDEB patients have a 68% cumulative risk of developing life-threatening cSCCs by the age of 35, and a 70% risk of death by the age of 45. Despite aggressive treatment, cSCC represents the leading cause of premature mortality in these patients, highlighting an unmet clinical need. Increasing evidence points to a role of altered metabolism in the initiation and maintenance of cSCC, making metabolism a potential therapeutic target. OBJECTIVES: We sought to determine the feasibility of targeting tumour cell energetics as a strategy to selectively hinder the growth advantage of aggressive cSCC. METHODS: We evaluated the cell energetics profiles of RDEB-SCC cells by analysing available gene expression data against multiple gene signatures and single-gene targets linked to metabolic reprogramming. Additionally, we employed real-time metabolic profiling to measure glycolysis and respiration in these cells. Furthermore, we investigated the anti-neoplastic properties of the metformin against human and murine high-risk cSCCs in vitro and in vivo. RESULTS: Gene expression analyses highlighted a divergence in cell energetics profiles between RDEB-SCC and non-malignant RDEB keratinocytes, with tumour cells demonstrating enhanced respiration and glycolysis scores. Real-time metabolic profiling supported these data and additionally highlighted a metabolic plasticity of RDEB-SCC cells. Against this background, metformin exerted an anti-neoplastic potential by hampering both respiration and glycolysis, and by inhibiting proliferation in vitro. Metformin treatment in an analogous model of fast-growing murine cSCC resulted in delayed tumour onset and slower tumour growth, translating to a 29% increase in median overall survival. CONCLUSIONS: Our data indicate that metformin exerts anti-neoplastic properties in aggressive cSCCs that exhibit high-risk features by interfering with respiration and glycolytic processes.

Consensus reclassification of inherited epidermolysis bullosa and other disorders with skin fragility.

BACKGROUND: Several new genes and clinical subtypes have been identified since the publication in 2014 of the report of the last International Consensus Meeting on Epidermolysis Bullosa (EB). OBJECTIVES: We sought to reclassify disorders with skin fragility, with a focus on EB, based on new clinical and molecular data. METHODS: This was a consensus expert review. RESULTS: In this latest consensus report, we introduce the concept of genetic disorders with skin fragility, of which classical EB represents the prototype. Other disorders with skin fragility, where blisters are a minor part of the clinical picture or are not seen because skin cleavage is very superficial, are classified as separate categories. These include peeling skin disorders, erosive disorders, hyperkeratotic disorders, and connective tissue disorders with skin fragility. Because of the common manifestation of skin fragility, these 'EB-related' disorders should be considered under the EB umbrella in terms of medical and socioeconomic provision of care. CONCLUSIONS: The proposed classification scheme should be of value both to clinicians and researchers, emphasizing both clinical and genetic features of EB. What is already known about this topic? Epidermolysis bullosa (EB) is a group of genetic disorders with skin blistering. The last updated recommendations on diagnosis and classification were published in 2014. What does this study add? We introduce the concept of genetic disorders with skin fragility, of which classical EB represents the prototype. Clinical and genetic aspects, genotype-phenotype correlations, disease-modifying factors and natural history of EB are reviewed. Other disorders with skin fragility, e.g. peeling skin disorders, erosive disorders, hyperkeratotic disorders, and connective tissue disorders with skin fragility are classified as separate categories; these 'EB-related' disorders should be considered under the EB umbrella in terms of medical and socioeconomic provision of care. Linked Comment: Pope. Br J Dermatol 2020; 183:603.

Increased tumour cell PD-L1 expression, macrophage and dendritic cell infiltration characterise the tumour microenvironment of ulcerated primary melanomas.

BACKGROUND: Primary melanoma ulceration is an unfavourable prognostic factor included in current staging systems. Yet, the immunological and molecular alterations responsible for this adverse outcome have not been fully elucidated. OBJECTIVES: We aimed to identify immunological differences between ulcerated and non-ulcerated primary melanomas concerning both innate and adaptive immunity and to correlate these with clinical outcome. METHODS: Formalin-fixed paraffin-embedded primary melanomas from 112 patients (pts) were analysed by immunohistochemistry. The expression of various markers identifying tumour-infiltrating lymphocytes, macrophages and dendritic cells was evaluated semi-quantitatively by three independent investigators. Tumour cell expression of programmed death-ligand 1 (PD-L1), transporter of antigen processing 1 and the MxA protein was also analysed. RESULTS: Recurrence occurred in 21/56 pts (37.5%) with ulcerated vs. 14/56 pts (25.0%) with non-ulcerated tumours (P = 0.15). Tumour ulceration was associated with more frequent development of brain metastasis (17.6 vs. 3.6% of pts, P = 0.015). Immunohistochemistry showed an association of ulceration with the presence of intratumoural CD68+ macrophages (P = 0.028) as well as with increased numbers of intratumoural CD11c+ dendritic cells (P = 0.014) and CD163+ macrophages (P = 0.001). PD-L1 positivity (expression in >1% of tumour cells) was more frequent in ulcerated than non-ulcerated tumours [40 (72.7%) vs. 25 (44.6%), P = 0.003]. A positive correlation between intratumoural CD11c+ (Spearman's correlation coefficient ρ: 0.42) and CD163+ (ρ: 0.31) cell count and frequency of tumour cell PD-L1 expression was detected. CONCLUSIONS: Our results confirm the adverse clinical outcome associated with primary melanoma ulceration, particularly concerning the risk of recurrence and subsequent development of brain metastases. The observed immunological differences suggest a conceivable role of increased intratumoural macrophage and dendritic cell counts associated with enhanced tumour cell PD-L1 expression potentially contributing to the immunosuppressive, growth-promoting microenvironment of ulcerated primary melanomas.

An ex vivo RNA trans-splicing strategy to correct human generalized severe epidermolysis bullosa simplex.

BACKGROUND: Generalized severe epidermolysis bullosa simplex (EBS-gen sev) is a genetic blistering skin disease in which autosomal dominant mutations in either the keratin KRT5 or KRT14 genes lead to impaired function of the intermediate filament cytoskeleton in the basal epidermis. Here we present an ex vivo RNA trans-splicing-based therapeutic approach to correct the phenotype. OBJECTIVES: To correct a mutation within exon 1 of the KRT14 gene, using a 5'-trans-splicing approach, where any mutation within the first seven exons could be replaced by a single therapeutic molecule. METHODS: A therapeutic RNA trans-splicing molecule containing wild-type exons 1-7 was stably transduced into an EBS patient-derived keratinocyte line. Trans-splicing was confirmed via reverse-transcriptase polymerase chain reaction, Western blotting and immunofluorescence microscopy. Skin equivalents generated from corrected keratinocytes were grafted onto nude mice and analysed about 8 weeks post-transplantation for regular epidermal stratification, trans-splicing-induced green fluorescent protein expression and blistering. RESULTS: Transplanted skin equivalents generated from trans-splicing-corrected patient keratinocytes showed a stable and blister-free epidermis. KRT14 correction disrupted EBS-gen sev-associated proinflammatory signalling, as shown at the mRNA and protein levels. Disruption of the pathogenic feedback loop in addition to overall downregulation of KRT14 expression highlighted the effect of KRT14 correction on the EBS pathomechanism. CONCLUSIONS: Our data demonstrate that trans-splicing-mediated mRNA therapy is an effective method for the correction of dominantly inherited KRT14 mutations at the transcriptional level. This results in the rescue of the EBS-gen sev phenotype and stabilization of the epidermis in a xenograft mouse model.

[Mucosal manifestations of epidermolysis bullosa : Clinical presentation and management]. / Schleimhautbeteiligung bei Epidermolysis bullosa : Klinik und Therapiemaßnahmen.

Mucosal lesions occur with different prevalence and severity in all subtypes of hereditary epidermolysis bullosa (EB), a group of rare genodermatoses. They are associated with increased morbidity and mortality, especially in severe junctional and dystrophic subtypes. Despite progress in clinical approaches to curative therapy, the management of these patients is still primarily symptom-oriented. Current recommendations mainly rely on expert opinion and experience from health care professionals of specialized centers, since the rarity of this disease largely limits the availability and feasibility of randomized controlled trials. Accurate preventive and supportive care measures, however, can significantly lessen symptoms, avoid/ameliorate complications, and enhance the quality of life of these patients.

Construction and validation of an RNA trans-splicing molecule suitable to repair a large number of COL7A1 mutations.

RNA trans-splicing has become a versatile tool in the gene therapy of monogenetic diseases. This technique is especially valuable for the correction of mutations in large genes such as COL7A1, which underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa. Over 800 mutations spanning the entire length of the COL7A1 gene have been associated with defects in type VII collagen, leading to excessive fragility of epithelial tissues, the hallmark of dystrophic epidermolysis bullosa (DEB). In the present study, we designed an RNA trans-splicing molecule (RTM) that is capable of repairing any given mutation within a 4200 nucleotide region spanning the 3' half of COL7A1. The selected RTM, RTM28, was able to induce accurate trans-splicing into endogenous COL7A1 pre-mRNA transcripts in a type VII collagen-deficient DEB patient-derived cell line. Correct trans-splicing was detected at the RNA level by semiquantitative RT-PCR and correction of full-length type VII collagen was confirmed at the protein level by immunofluorescence and western blot analyses. Our results demonstrate that RTM28, which covers >60% of all mutations reported in DEB and is thus the longest RTM described so far for the repair of COL7A1, represents a promising candidate for therapeutic applications.

[Molecular diagnosis of methicillin-resistent Staphylococcus aureus: Methods and efficacy]. / Molekulare Diagnostik von Methicillin-resistentem Staphylococcus aureus : Methoden und Effektivität.

Methicillin-resistant Staphylococcus aureus (MRSA) isolates are a serious public health problem whose ever-increasing rate is commensurate with the pressure it is exerting on the healthcare system. At present, more than 20% of clinical S. aureus isolates in German hospitals are methicillin-resistant, in Austria less than 10%. Strategies from low-prevalence countries show that this development is not necessarily inevitable. In the Scandinavian countries and the Netherlands, thanks to a rigorous prevention programme, MRSA prevalence has been kept at an acceptably low level (< 1-3%). Central to these search-and-destroy control strategies is an admission screening using several MRSA swabs taken from mucocutaneous colonisation sites of high-risk patients (MRSA surveillance). It has also been reported that the speed with which MRSA carriage is detected has an important role, as it is a key component of any effective strategy to prevent the pathogen from spreading. Since MRSA culturing involves a 2-3 day delay before the final results are available, rapid detection techniques (commonly referred to as MRSA rapid tests) using polymerase chain reaction (PCR) methods and, most recently, rapid culturing methods have been developed. The implementation of rapid tests reduces the time of detection of MRSA carriers from 48-72 to 2-5 h. Clinical evaluation data have shown that MRSA can thus be detected with very high sensitivity. Specificity, however, is sometimes impaired due to false-positive PCR signals occurring in mixed flora specimens. In order to rule out false-positive PCR results, a culture screen must always be carried out simultaneously. The data provide preliminary evidence that a PCR assay can reduce nosocomial MRSA transmission in high-risk patients or high-risk areas, whereas an approach that screens all patients admitted to the hospital is probably not effective. Information concerning the cost effectiveness of rapid MRSA tests is still sparse and thus the issue remains debated.

[Molecular diagnostics in melanoma]. / Molekulare Diagnostik beim Melanom.

The molecular landscape of melanoma is changing more rapidly than ever since new molecular technology approaches have made it possible to examine human melanoma for genetic alterations underlying the disease. In recent years, these approaches have identified new familial melanoma susceptibility genes, most of them also conferring risk to other cancers. This has implications for clinical testing and surveillance. Furthermore, molecular testing of melanoma to determine therapeutic eligibility for targeted therapies is now standard of care and should be familiar to the dermatologist.

[Darier disease]. / Morbus Darier.

Darier disease (Darier-White disease, dyskeratosis follicularis) is a rare autosomal dominant genodermatosis with regional differences in prevalence. The responsible mutations have been identified on chromosome 12q23-24.1. The gene encodes a calcium-ATPase type 2 in the sarco-/endoplasmic reticulum (SERCA2), which belongs to the large family of P-type cation pumps. This pump couples ATP hydrolysis to the transport of cations across membranes and thus plays a significant role in intracellular calcium signaling. Neuropsychiatric disorders are often associated with Darier disease. However, these diseases are not due to mutations in the gene ATP2A2 but to a susceptibility locus in a 6.5 Mb region near this gene. Currently, the treatment is strictly limited to the relief of symptoms. In severe cases, oral retinoids (acitretin: initial 10-20 mg/Tag and isotretinoin: 0.5-1 mg/kg/day) lead to a response in 90% of cases. However, side effects often prevent long-term use of vitamin A derivatives.

Autoimmune bullous diseases in Austria.

Autoimmune bullous diseases (AIBD) are chronic disorders associated with significant morbidity and even mortality, for which the 19 dermatologic departments in Austria apply standard modalities to provide state-of-the-art diagnosis and treatment. Most of the affected individuals are initially treated on an inpatient basis, with follow-up done in specialized outpatient clinics or in private practices. A well-established system of care for AIBD patients is thus available nationwide. Considering the significant morbidity and mortality but also rareness of AIBD, national and international standardization of AIBD administration in registries is a major requirement of further improvement in patient care.

Cytokeratin 8 interacts with clumping factor B: a new possible virulence factor target.

Staphylococcus aureus is a human pathogen of growing clinical significance, owing to its increasing levels of resistance to most antibiotics. Infections range from mild wound infections to severe infections such as endocarditis, osteomyelitis and septic shock. Adherence of S. aureus to human host cells is an important step, leading to colonization and infection. Adherence is mediated by a multiplicity of proteins expressed on the bacterial surface, including clumping factor B. In this study, we aimed to identify new targets of clumping factor B in human keratinocytes by undertaking a genome-wide yeast two-hybrid screen of a human keratinocyte cDNA library. We show that clumping factor B is capable of binding cytokeratin 8 (CK8), a type II cytokeratin. Using a domain-mapping strategy we identified amino acids 437-464 as necessary for this interaction. Recombinantly expressed fragments of both proteins were used in pull-down experiments and confirmed the yeast two-hybrid studies. Analysis with S. aureus strain Newman deficient in clumping factor B showed the clumping factor B-dependence of the interaction with CK8. We postulate that the clumping factor B-CK8 interaction is a novel factor in S. aureus infections.