Transcriptional regulation of S100B and identification of novel melanoma biomarkers

Summary Introduction Cutaneous melanoma is an aggressive disease, which is recognised as the most common fatal skin cancer worldwide. Approximately 450 cases are diagnosed in Ireland per year. Between 1994 and 2004, melanoma demonstrated a higher rate of increase in mortality than any other cancer in this country. Although the prognosis for early melanoma is favorable, less than 20 percent of patients with metastatic melanoma survive for five years. S100B is a calcium sensor protein that modulates biological activity via calcium binding, which is routinely used in histological diagnosis of malignant melanoma and which is also a well-recognized serum marker of the disease. HOX proteins are members of the homeodomain family of transcription factors, which are involved in a host of cellular functions including organogenesis, cellular differentiation, cell cycle and apoptosis. As transcription factors, HOX proteins require co-activator proteins to achieve their full function. SRC-1 is one such coactivator and our group has extensively explored its function. In particular, functional interactions between SRC-1 and HOXCI 1 in breast cancer cell lines and tissue have been described. HOXCI 1 is known to enhance expression of the secreted serum marker S100B. Given the strong association between S100B and malignant melanoma, we believe that this pathway may also have a role in melanoma tumour genesis. S100B is a well-described biomarker in melanoma and serum levels have been shown to correlate with disease stage and response to treatment. In spite of this and the availability other prognostic indicators, many patients go on to develop an unpredictable disease course. For this reason, identification of novel biomarkers is an active area of melanoma research. In the second part of this work, an autoantibody microarray screen was undertaken to identify differentially expressed biomarkers in sera from patients with melanoma. Hypothesis Production of S100B in malignant melanoma is regulated by the transcription factor HOXCI 1, in cooperation with coactivator SRC-1. Protein microarray technology may provide a useful means of identification of autoantibody biomarkers in serum from patients with melanoma. Aims To define the molecular role of HOXC11 and SRC-1 in the transcriptional control of S100B in malignant melanoma. To characterise the effect of manipulation of HOXC11 and SRC-1 on S100B expression. To identify new biomarkers in sera from patients with malignant melanoma. Results Expression of SIOOB, HOXC11 and SRC-1 protein in primary (SKMe128) and metastatic (MeWo) melanoma cell lines was confirmed by Western blotting and quantitative Real Time PCR (qRT-PCR) analysis. Colocalisation of HOXCI 1 and SRC-1 in melanoma cells was confirmed by immunofluorescence. Co-immunoprecipitation was carried out and demonstrated interaction of HOXC11 and SRC-1 in cell lysates. Paraffinembedded melanoma and nevi samples were examined by immunofluorescence and a significantly higher nuclear expression of HOXCI 1 and SRC-1 was observed in the melanoma cohort. Colocalisation of the two proteins was also demonstrated in a series of melanoma primary culture specimens. Chromatin-immunoprecipitation was employed to confirm recruitment of HOXCI 1 to the promoter region of the S100B gene. To determine the ability of HOXCI 1 to regulate expression of S1 OOB, HOXCI 1 was transfected into the SKMe128 cell line and it was found to significantly increase the expression of the target gene S100B. When concomitant HOXCI 1 and SRC- 1 knockdowns were performed, a significant reduction in the presence of S100B was noted. Treatment of cell lines with the phospho-Src inhibitor, dasatinib, resulted in decreased coassociation between HOXCI 1 and SRC-1 in both primary and metastatic cell lines as well as decreased expression of S100B in SKMe128 cells. Protein microarray analysis of sera from patients with melanoma and control patients was carried out. A series of differentially expressed autoantibodies was identified and the non-receptor tyrosine kinase, BMX was chosen for further study. Elevated expression of anti-BMX autoantibody in sera from a larger cohort of melanoma patients was confirmed. Furthermore, expression of BMX protein in melanoma cell lines and frozen tissue samples was confirmed by Western blotting. Conclusion In the absence of effective treatment for advanced melanoma, elucidation of novel signalling pathways and therapeutic targets remains at the forefront of molecular research. In this work, translational techniques have provided an insight in to the transcriptional regulation of S100B in melanoma. Furthermore, protein microarray analysis has been utilised to identify potentially useful autoantibody biomarkers. These findings constitute a small fragment of all the potential genetic aberrations that may be implicated in melanoma turnourgenesis. Advanced melanoma is likely to present a significant therapeutic challenge to clinicians and academics for many years to come. What is certain is that translational research methods, as have been employed here, are essential in pushing forward the boundaries of our molecular understanding of this fascinating disease

NovoSorb Biodegradable Temporizing Matrix for Reconstruction of Multiplanar Degloving Injury of the Upper Limb

Originally described as “wringer injuries” by MacCollum in 1938,1 traumatic multiplanar degloving injuries that occur as the result of the hand, forearm or arm being drawn between the rollers of a machine are functionally devastating and present a significant reconstructive challenge. Revascularization and comprehensive excision of devitalized bone and soft tissue, followed by appropriate skeletal fixation and vascularized soft tissue cover are the mainstays of management. To date, published case series have described local flaps and free tissue transfer for coverage of wounds that involve exposed vital structures such as nerves, vessels, and tendons.2 NovoSorb biodegradable temporizing matrix (BTM; PolyNovo Biomaterials Pty Ltd, Melbourne, Australia) is a bilayer bioabsorbable synthetic polymer dermal substitute, which has the ability to integrate into large wound beds and is resistant to infection.3 BTM comprises a bioabsorbable, polyurethane matrix that allows for cellular infiltration and a temporary nonbiodegradable, nonporous polyurethane layer, which limits moisture loss and provides a barrier to bacteria. Here we describe the successful use of BTM in the staged reconstruction of a high-energy industrial roller injury in an adolescent patient

Feasibility of cleft lip and palate repair in personal protective equipment (PPE).

Elective surgery during the evolving COVID-19 pandemic presents unprecedented logistical challenges to surgical teams. Cleft surgery may be considered an aerosol generating procedure (AGP), which may lead to small-droplet transmission of virions. Strict adherence to personal protective equipment (PPE) policy is used with the hope of preventing transmission of the virus between patients and operating theatre staff. </p

Non-interventional factors influencing velopharyngeal function for speech in initial cleft palate repair: a systematic review protocol

Background: This systematic review aims to inform the development of a screening tool which pre-operatively predicts which children are likely to develop velopharyngeal insufficiency, one of the causes of poor speech outcomes, following cleft palate repair. This would be highly beneficial as it would inform pre-operative counselling of parents, allow targeted speech and language therapy, and enable meaningful comparison of outcomes between surgeons, techniques, and institutions. Currently, it is unclear which factors influence speech outcomes. A systematic review investigating the non-interventional factors which potentially influence speech outcomes following cleft palate repair is warranted. This may be illuminating in itself or provide foundations for future studies. Methods: A systematic review will be carried out according to Cochrane methodology and reported according to PRISMA guidelines (PLoS Med 6: e1000097, 2009). Systematic review software will be used to facilitate three-stage screening by two independent reviewers experienced in cleft lip and palate. Thereafter, data extraction and GRADE assessment will be performed in duplicate by five independent reviewers experienced in cleft lip and palate. Studies reporting the proportion of patients who were recommended or underwent secondary speech surgery for velopharyngeal insufficiency following primary surgery for cleft palate will be included. The study findings will be tabulated and summarised. The primary outcome measure will be further speech surgery (either recommended or performed). The secondary outcome measure will be perceptual speech assessment for the presence of velopharyngeal insufficiency. A meta-analysis is planned. However, if this is not possible, due to the anticipated marked heterogeneity of study characteristics, pre-operative assessment, and the recorded outcome measures, a narrative synthesis will be undertaken. Discussion: This systematic review may provide sufficient data to inform the development of a screening tool to predict the risk of velopharyngeal insufficiency prior to cleft palate repair. However, it is anticipated that these findings will provide the foundation for future studies in this area. Systematic review registration: Registered on 19 December 2016 with PROSPERO CRD42017051624.</p

Protocol for the development of a core outcome set for reporting outcomes of management of velopharyngeal dysfunction

Introduction: Velopharyngeal dysfunction (VPD) is present in up to 40% of patients following cleft palate repair. Children with VPD display hypernasal speech, nasal air emission and are at a high risk for developing articulation disorders. The overall result is decreased intelligibility and acceptability of speech, as well as significant functional and social impairments. Although there are several surgical approaches for the management of children with VPD, standard treatment protocols have not been well defined. There is a need for a core outcome set (COS) to reduce outcome reporting bias and heterogeneity across studies of VPD. The COS-VPD Initiative is an international effort to establish a COS for the reporting of studies of the management of VPD. Methods and analysis: The study has been developed according to the Core Outcome Set-STAandards for Development standards for the design of a COS study and will be carried out according to the guidance of the Core Outcome Measures in Effectiveness Trials (COMET) initiative. A long list of clinical and patient-reported outcomes will be identified from a systematic review of the literature. A two-stage Delphi consensus process will be used to refine this list into a COS. An international panel of key stakeholders including patients, parents and multidisciplinary clinical and academic experts will be invited to participate in this process. Consensus criteria will be specified a priori and the steering group will ratify the final COS. Ethics and dissemination: The study has ethical approval through Children's Health Ireland at Crumlin Research and Ethics Committee, Ref: GEN/683/18. The study is registered with the COMET Initiative (http://www.cometinitiative.org/studies/details/1146?result=true). The COS will be disseminated by publication in the peer-reviewed literature, presentation at international research meetings and distribution to patient-representative organisations. This will facilitate the application of the COS in future studies of the management of VPD.</p