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.

Slug Is A Surrogate Marker of Epithelial to Mesenchymal Transition (EMT) in Head and Neck Cancer.

BACKGROUND: Epithelial to mesenchymal transition (EMT) promotes therapy resistance in head and neck cancer (HNC) cells. In this study, EMT was quantified in HNC tumor samples by the cellular co-localization of cytokeratin/vimentin, E­cadherin/ß­catenin and by Slug expression. METHODS: Tissue samples from HNC patients were stained with antibody pairs against cytokeratin/vimentin and E-cadherin/ß-catenin. Epithelial-mesenchymal co-localization was quantified using immunofluorescence multichannel image cytometry. Double positivity was confirmed using confocal microscopy. Slug was semi-quantified by 2 specialists and quantified by bright field image cytometry. RESULTS: Tumor samples of 102 patients were investigated. A loss of E-cadherin positive cells (56.9 ± 2.6% vs. 97.9 ± 1.0%; p < 0.0001) and E-cadherin/ß-catenin double positive cells (15.4 ± 5.7% vs. 85.4 ± 1.2%; p < 0.0001) was observed in tumor samples. The percentage of Slug positive cells was increased in tumor samples (12.1 ± 3.6% vs. 3.2 ± 2.6%; p = 0.001). Ordinal Slug scores judged by two specialists closely correlated with percentage of Slug-positive cells (Spearman's rho = 0.81; p < 0.001). Slug score correlated negatively with the percentage of E-cadherin positive cells (r = 0.4; p = 0.006), the percentage of E-cadherin/ß-catenin positive cells (r = 0.5; p = 0.001) and positively with cytokeratin/vimentin positive cells (r = 0.4, p = 0.003). CONCLUSION: EMT can be assessed in HNC tumor probes by cytokeratin/vimentin co-expression and loss of E-cadherin/ß-catenin co-expression. Slug score provides a convenient surrogate marker for EMT.

Surgical rescue for persistent head and neck cancer after first-line treatment.

PURPOSE: Surgical rescue is a treatment option for persistent disease after first-line treatment treatment of head and neck cancer (HNC). METHODS: Patients with persistent HNC treated with rescue surgery between 2008 and 2016 were included. Patients who received a rescue neck dissection (ND only) and who received primary site surgery ± ND were analysed separately (primary site surgery ± ND). RESULTS: During the observation period, 35 patients received ND only and 17 primary site surgery ± ND. No perioperative mortality was observed. In nine patients with ND only and 12 patients with primary site surgery ± ND at least one complication was encountered. 41/52 (79%) patients had a complete response. Median overall survival of patients receiving rescue surgery was 56 months (95% CI 44-69 months). Median overall survival was best for patients with initial laryngeal and oropharyngeal cancer and worst for patients with hypopharyngeal cancer (p = 0.02). Functional deficits following rescue surgery were mainly observed in the domains speech, nutrition, and shoulder/arm mobility. The risk of functional impairment was higher for patients with rescue surgery at the primary tumor site (OR 2.5 ± 2; p = 0.07). CONCLUSION: Rescue surgery offers patients with resectable, persistent disease a realistic chance to achieve long-term survival. Especially patients with laryngeal and oropharyngeal cancer profited from rescue surgery. Rescue neck dissection is an effective and safe procedure. Patients with rescue surgery at the primary tumor site ± ND should expect complications and permanent functional impairment.

Pleiotropic Effects of Epithelial Mesenchymal Crosstalk on Head and Neck Cancer: EMT and beyond.

Epithelial mesenchymal crosstalk (EMC) describes the interaction of the tumor stroma and associated fibroblasts with epithelial cancer cells. In this study we analysed the effects of EMC on head and neck cancer cells. In tumor cell lines EMC was induced using media conditioned from a mix-culture of cancer cells and fibroblasts. Cell proliferation and chemotherapy response were assessed using direct cell counting. Flow cytometry, immunohistochemistry of markers of epithelial-mesenchymal transition (EMT) and subsequent TissueFaxs™ acquisition and quantification and western blot analysis were performed. Holotomographic microscopy imaging was used to visualize the effects of EMC on Cisplatin response of SCC-25 cells. EMC induced a hybrid epithelial-mesenchymal phenotype in SCC-25 cells with co-expression of vimentin and cytokeratin. This hybrid phenotype was associated with chemotherapy resistance and increased proliferation of the cells. The EMC conditioned medium led to an activation of the IL-6/STAT3 pathway with subsequent phosphorylation of STAT3. EMC induced a hybrid epithelial-mesenchymal phenotype in HNSCC cells accompanied by increased therapy resistance and cell proliferation. The IL-6/STAT3 pathway might be one of the major pathways involved in these EMC-related effects.

Galanin modulates human and murine neutrophil activation in vitro.

AIMS: Polymorphonuclear neutrophils are key players in innate immunity. The innate immune system needs to be tightly controlled to ensure proper activation but also no overactivation. Galanin has been shown to regulate inflammatory reactions, and therefore, we aimed to elucidate the expression of galanin and its three receptors (GAL1 -GAL3 ) in polymorphonuclear neutrophils and to evaluate whether galanin exerts direct or indirect effects on human and murine polymorphonuclear neutrophils. METHODS: Human peripheral polymorphonuclear neutrophils were isolated from fresh blood of healthy donors, and murine polymorphonuclear neutrophils were isolated from bone marrow of C57BL/6N mice. Gene expression was evaluated by qRT-PCR. As a marker for polymorphonuclear neutrophil activation, CD11b integrin surface expression was measured by FACS analysis. Furthermore, a label-free technology measuring ligand-induced dynamic mass redistribution was used to evaluate the response of polymorphonuclear neutrophils to galanin. RESULTS: GAL2 receptor expression was found in both human and murine polymorphonuclear neutrophils, galanin and GAL3 receptor were exclusively expressed in murine bone marrow polymorphonuclear neutrophils, and GAL1 receptor was not detectable in polymorphonuclear neutrophils of either species. Galanin treatment was not able to induce CD11b integrin surface expression or dynamic mass redistribution in human polymorphonuclear neutrophils and murine bone marrow polymorphonuclear neutrophils. However, galanin treatment significantly enhanced the response of polymorphonuclear neutrophils of both species to interleukin-8. CONCLUSION: Galanin can be regarded as an immunomodulatory peptide as it can sensitize polymorphonuclear neutrophils towards pro-inflammatory cytokines in humans and mice.

New treatment strategies for HPV-positive head and neck cancer.

Epidemiological studies show an increasing incidence of human papilloma virus-associated oropharyngeal cancer. HPV-positive head and neck squamous cell carcinoma (HNSCC) is recognized as a special subgroup of HNSCC. Because HPV-positive patients are often younger and have an outstanding prognosis, long-term toxicities of therapy have become an important issue. Current clinical trials focus on a reduction of treatment-related toxicity and the development of HPV-specific therapies. New treatment strategies include a dose reduction of radiotherapy, the use of cetuximab instead of cisplatin for chemoradiation and transoral robotic surgery (TORS). Increasing comprehension of the molecular background of HPV-associated HNSCC has also lead to more specific treatment attempts including immunotherapeutic strategies. Whereas recently published data shed light on immune mechanisms resulting in a tolerogenic niche for HPV and HPV-associated HNSCC, other studies focus on specific vaccination of HPV-positive HNSCC. This study will summarize current therapy approaches and illustrate ongoing clinical trials in the field of HPV-positive HNSCC.

[New aspects of HPV-positive head and neck cancer. Highlights from the 2011 ASCO Congress]. / Neue Aspekte HPV-positiver Kopf-Hals-Karzinome. Highlights vom ASCO-Kongress 2011.

HPV-positive head and neck carcinoma is significantly different than tobacco- and alcohol-induced cancer. Between 30% and 50% of oropharyngeal cancers are associated with human papillomavirus (HPV). Studies still show an increasing incidence. HPV-positive head and neck cancer patients have a better prognosis due to a better response to therapy. Especially patients with gene overexpression of immunological proteins in the antigen presentation are suggested to benefit from radiotherapy. A current retrospective study shows better outcomes for patients treated with radiotherapy in combination with biological targets compared to radiochemotherapy.

The neuropeptide galanin is a novel inhibitor of human hair growth.

BACKGROUND: Galanin is a trophic factor of the central and peripheral nervous system that shows widespread distribution in human skin. However, the exact localization and the role of galanin in the hair follicle (HF) remain to be clarified. OBJECTIVES: To characterize galanin expression in human scalp HFs and to examine the effects of galanin on normal human scalp HF growth in organ culture. METHODS: Immunohistochemistry was performed on cryosections of human female scalp skin. Anagen HFs were microdissected and cultured up to 9 days and treated with 100 nmol L(-1) galanin. Staining for Ki-67, TUNEL and Masson-Fontana were used to analyse proliferation, apoptosis and hair cycle staging of the HFs. Functional effects of galanin were tested in serum-free HF organ culture. RESULTS: Galanin-like immunoreactivity was detected in the outer root sheath (ORS) and inner root sheath. Additionally, galanin mRNA was detected in ORS keratinocytes and all HF samples tested. Galanin receptor transcripts (GalR2, GalR3) were also detected in selected samples. Galanin reduced proliferation of hair matrix keratinocytes in situ compared with vehicle-treated controls, shortened the hair growth phase (anagen) in vitro and reduced hair shaft elongation. This was accompanied by the premature development of a catagen-like morphology of galanin-treated HFs. CONCLUSIONS: We present the first evidence that human HFs are both a source and a functionally relevant target of galanin. Due to its hair growth-inhibitory properties in vitro, galanin application deserves further exploration as a potential new treatment strategy for unwanted hair growth (hirsutism, hypertrichosis).

Postoperative course and clinical significance of biochemical blood tests following hepatic resection.

BACKGROUND: Hepatic resection continues to be associated with substantial morbidity. Although biochemical tests are important for the early diagnosis of complications, there is limited information on their postoperative changes in relation to outcome in patients with surgery-related morbidity. METHODS: A total of 835 consecutive patients underwent hepatic resection between January 2002 and January 2008. Biochemical blood tests were assessed before, and 1, 3, 5 and 7 days after surgery. Analyses were stratified according to the extent of resection (3 or fewer versus more than 3 segments). RESULTS: A total of 451 patients (54·0 per cent) underwent resection of three or fewer anatomical segments; resection of more than three segments was performed in 384 (46·0 per cent). Surgery-related morbidity was documented in 258 patients (30·9 per cent) and occurred more frequently in patients who had a major resection (P = 0·001). Serum bilirubin and international normalized ratio as measures of serial hepatic function differed significantly depending on the extent of resection. Furthermore, they were significantly affected in patients with complications, irrespective of the extent of resection. The extent of resection had, however, little impact on renal function and haemoglobin levels. Surgery-related morbidity caused an increase in C-reactive protein levels only after a minor resection. CONCLUSION: Biochemical data may help to recognize surgery-related complications early during the postoperative course, and serve as the basis for the definition of complications after hepatic resection.

Benefit, risks and cost-effectiveness of screening for abdominal aortic aneurysm.

PURPOSE: Abdominal aortic aneurysms (AAA) cause a considerable number of deaths. A ruptured AAA is associated with a mortality rate of 80%. The purpose of this study was to summarize the current evidence from published health economic models for the long-term effectiveness and cost-effectiveness of screening programs for AAA. MATERIALS AND METHODS: Medical, economic and health technology assessment (HTA) databases were systematically searched for cost-effectiveness models up to October 2007. Only models with a lifetime time horizon of evaluating AAA screening in men over 65 years were included in the review. Study data were extracted, standardized and summarized in evidence tables and cost-effectiveness plots. RESULTS: We reviewed 8 cost-effectiveness models published between 1993 and 2007 comparing AAA screening and lack of screening in men over 60. One model yielded a loss of life-years at additional costs. The remaining seven models yielded gains in life expectancy ranging from 0.02 to 0.28LYs. Gains in quality-adjusted life expectancy reported by six of the seven models ranged from 0.015 to 0.059 QALYs. Incremental costs ranged from 96 to 721 Euros. Incremental cost-effectiveness ratios (ICER) ranged from 1443 to 13 299 Euros per LY or QALY gained. CONCLUSION: Based on our analysis, the introduction of a screening program to identify AAA will probably gain additional life years and quality of life at acceptable extra costs. The target population for a screening program should be men 65 years and older.

Galanin family of peptides in skin function.

The skin, the largest organ of the body, functions as a barrier between the body proper and the external environment, as it is constantly exposed to noxious stressors. During the last few years, the concept of an interactive network involving cutaneous nerves, the neuroendocrine axis, and the immune system has emerged. The neuroendocrine system of the skin is composed of locally produced neuroendocrine mediators that interact with specific receptors. Among these mediators are neuropeptides, including members of the galanin peptide family--galanin, galanin-message associated peptide, galanin-like peptide, and alarin--which are produced in neuronal as well as nonneuronal cells in the skin. Here, we review the expression of the galanin peptides and their receptors in the skin, and the known functions of galanin peptides in different compartments of the skin. We discuss these data in light of the role of the galanin peptide family in inflammation and cell proliferation.

Lack of complex I is associated with oncocytic thyroid tumours.

Oncocytic tumours are characterised by hyperproliferation of mitochondria. We immunohistochemically analysed all enzymes of the oxidative phosphorylation system in 19 oncocytic thyroid tumours. A specific lack of complex I was detected, which was expressed at <5% of the level determined in surrounding non-cancerous tissue.

Galanin family of peptides in skin function.

The skin, the largest organ of the body, functions as a barrier between the body proper and the external environment, as it is constantly exposed to noxious stressors. During the last few years, the concept of an interactive network involving cutaneous nerves, the neuroendocrine axis, and the immune system has emerged. The neuroendocrine system of the skin is composed of locally produced neuroendocrine mediators that interact with specific receptors. Among these mediators are neuropeptides, including members of the galanin peptide family--galanin, galanin-message-associated peptide, galanin-like peptide, and alarin--which are produced in neuronal as well as nonneuronal cells in the skin. Here we review the expression of the galanin peptides and their receptors in the skin, and the known functions of galanin peptides in different compartments of the skin. We discuss these data in light of the role of the galanin peptide family in inflammation and cell proliferation.

Effect of a whole-body vibration session on knee stability.

The aim of the study was to investigate the effect of WBV on stretch reflexes involved in knee joint control. We evoked stretch reflexes of the hamstring muscles by inducing an anterior tibial translation during standing in 23 healthy subjects which were divided into a control and an intervention group. WBV with a frequency of 30 Hz and a vertical amplitude of 4 mm was induced by an uniformly oscillating platform. The WBV session lasted 60 seconds and was repeated twice. Short (SLR) and medium latency responses (MLR) of the hamstring muscles and maximum tibia translation were assessed using surface EMG and linear potentiometers. While there were no significant changes in latency, the size of the lateral and medial hamstring SLR was significantly increased after WBV (p = 0.039 and p = 0.043, respectively). No significant differences were found for the hamstring MLR size after WBV. Maximum tibial translation was significantly decreased after WBV (p = 0.031). Our results suggest that single WBV exposure has a positive effect on knee joint stability as a short-term adaptation on neuromuscular level. This appears to be directly associated with an increase of hamstring SLR size in response to the anterior tibial movement which may cause the decrease in anterior tibial translation.

Introducing a fast and simple PCR-RFLP analysis for the detection of mutant thiopurine S-methyltransferase alleles TPMT*3A and TPMT*3C.

BACKGROUND: Azathioprine, in combination with corticosteroids, is the first-line therapy of severe forms of pemphigus vulgaris. Patients with an impaired thiopurine S-methyltransferase (TPMT) activity are at risk of developing severe myelo-suppression upon treatment with thiopurines such as azathioprine. Analysis of the TPMT status prior to drug administration is therefore highly recommended. However, because of the limited availability of TPMT testing outside of specialized centres, pre-emptive TPMT testing is not widespread. To avoid laborious biochemical and sequencing assays, we evaluated a new restriction fragment length polymorphism (RFLP) analysis. METHODS: We designed a rapid genetic polymerase chain reaction (PCR)-RFLP screen for the most prevalent mutant TPMT*3A and TPMT*3C alleles that are known to result in reduced TPMT enzyme activity. RESULTS: Validating our fast system on 871 Caucasian DNA samples, we observed that 8.61% of our probands carried the TPMT*3A allele and 0.23% were heterozygous for the TPMT*3C allele, which is in concordance with previously reported allele frequencies. CONCLUSION: This simple and low-cost PCR-RFLP TPMT polymorphism testing approach can be performed in a standard laboratory. It should be applied to all patients prior to receiving thiopurine drug therapy to avoid the severe, but predictable, haematopoietic side-effects of thiopurine drug administration.

Mitochondrial DNA mutations in renal cell carcinomas revealed no general impact on energy metabolism.

Previously, renal cell carcinoma tissues were reported to display a marked reduction of components of the respiratory chain. To elucidate a possible relationship between tumourigenesis and alterations of oxidative phosphorylation, we screened for mutations of the mitochondrial DNA (mtDNA) in renal carcinoma tissues and patient-matched normal kidney cortex. Seven of the 15 samples investigated revealed at least one somatic heteroplasmic mutation as determined by denaturating HPLC analysis (DHPLC). No homoplasmic somatic mutations were observed. Actually, half of the mutations presented a level of heteroplasmy below 25%, which could be easily overlooked by automated sequence analysis. The somatic mutations included four known D-loop mutations, four so far unreported mutations in ribosomal genes, one synonymous change in the ND4 gene and four nonsynonymous base changes in the ND2, COI, ND5 and ND4L genes. One renal cell carcinoma tissue showed a somatic A3243G mutation, which is a known frequent cause of MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, stroke-like episode) and specific compensatory alterations of enzyme activities of the respiratory chain in the tumour tissue. No difference between histopathology and clinical progression compared to the other tumour tissues was observed. In conclusion, the low abundance as well as the frequently observed low level of heteroplasmy of somatic mtDNA mutations indicates that the decreased aerobic energy capacity in tumour tissue seems to be mediated by a general nuclear regulated mechanism.

Pedal artery imaging--a comparison of selective digital subtraction angiography, contrast enhanced magnetic resonance angiography and duplex ultrasound.

OBJECTIVE: to evaluate selective digital subtraction angiography (DSA), contrast-enhanced magnetic resonance angiography (CE-MRA) and duplex ultrasound (duplex) in preoperative pedal artery imaging. MATERIAL AND METHODS: DSA, CE-MRA and duplex were studied prospectively in 37 patients suffering from critical leg ischaemia. Two radiologists independently reviewed both the CE-MRA and DSA images. The pedal vessels were scored on a scale from 0 to III (0=vessel not visualised, I=vessel faintly visualised, II=stenosis >50%, III=vessel without relevant stenosis). Duplex ultrasound was performed by an angiologist blind to both the DSA and MRA findings and the pedal arteries were scored 0-III according to their diameter. Each examiner named the pedal artery best suitable for bypass surgery. Agreement in artery assessment was expressed as kappa values. Patency of the bypass at 30 days was used as validation of the artery's suitability as the run-off vessel. RESULTS: interobserver agreement for DSA (weighted Kappa 0.63, CI 0.53-0.73 and CE-MRA (weighted kappa 0.60, CI 0.5-0.7) was moderate to substantial. CE-MRA depicted significantly more vascular segments than DSA (p congruent with 0.0001).In the prediction of the distal outflow vessel duplex and CE-MRA proved to be superior to DSA. CONCLUSION: because of the moderate inter-observer agreement it may be questionable to regard selective DSA as gold standard imaging procedure in preoperative pedal artery imaging. CE-MRA and duplex are very helpful in assessing the pedal artery morphology and should be used if selective DSA does not sufficiently depict the pedal vasculature.