Mucosal melanomas of different anatomic sites share a common global DNA methylation profile with cutaneous melanoma but show location-dependent patterns of genetic and epigenetic alterations

Cutaneous, ocular, and mucosal melanomas are histologically indistinguishable tumors that are driven by a different spectrum of genetic alterations. With current methods, identification of the site of origin of a melanoma metastasis is challenging. DNA methylation profiling has shown promise for the identification of the site of tumor origin in various settings. Here we explore the DNA methylation landscape of melanomas from different sites and analyze if different melanoma origins can be distinguished by their epigenetic profile. We performed DNA methylation analysis, next generation DNA panel sequencing, and copy number analysis of 82 non-cutaneous and 25 cutaneous melanoma samples. We further analyzed eight normal melanocyte cell culture preparations. DNA methylation analysis separated uveal melanomas from melanomas of other primary sites. Mucosal, conjunctival, and cutaneous melanomas shared a common global DNA methylation profile. Still, we observed location-dependent DNA methylation differences in cancer-related genes, such as low frequencies of RARB (7/63) and CDKN2A promoter methylation (6/63) in mucosal melanomas, or a high frequency of APC promoter methylation in conjunctival melanomas (6/9). Furthermore, all investigated melanomas of the paranasal sinus showed loss of PTEN expression (9/9), mainly caused by promoter methylation. This was less frequently seen in melanomas of other sites (24/98). Copy number analysis revealed recurrent amplifications in mucosal melanomas, including chromosomes 4q, 5p, 11q and 12q. Most melanomas of the oral cavity showed gains of chromosome 5p with TERT amplification (8/10), while 11q amplifications were enriched in melanomas of the nasal cavity (7/16). In summary, mucosal, conjunctival, and cutaneous melanomas show a surprisingly similar global DNA methylation profile and identification of the site of origin by DNA methylation testing is likely not feasible. Still, our study demonstrates tumor location-dependent differences of promoter methylation frequencies in specific cancer-related genes together with tumor site-specific enrichment for specific chromosomal changes and genetic mutations. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.     

ORCA,a values-based decision aid for selecting additional findings from genomic sequencing in adults: Efficacy results from a randomized trial

PurposeIndividuals having genomic sequencing can choose to be notified about pathogenic variants in genes unrelated to the testing indication. A decision aid can facilitate weighing one’s values before making a choice about these additional results.MethodsWe conducted a randomized trial (N = 231) comparing informed values-choice congruence among adults at risk for a hereditary cancer syndrome who viewed either the Optional Results Choice Aid (ORCA) or web-based additional findings information alone. ORCA is values-focused with a low-literacy design.ResultsIndividuals in both arms had informed values-choice congruence (75% and 73% in the decision aid and web-based groups, respectively; odds ratio [OR] = 1.10, 95% CI = 0.58-2.08). Most participants had adequate knowledge (79% and 76% in the decision aid and web-based groups, respectively; OR = 1.20, 95% CI = 0.61-2.34), with no significant difference between groups. Most had information-seeking values (97% and 98% in the decision aid and web-based groups, respectively; OR = 0.59, 95% CI = 0.10-3.61) and chose to receive additional findings.ConclusionThe ORCA decision aid did not significantly improve informed values-choice congruence over web-based information in this cohort of adults deciding about genomic results. Both web-based approaches may be effective for adults to decide about receiving medically actionable additional results.     

Cortical thinning in former professional soccer players

Soccer is the most popular sport in the world. Soccer players are at high risk for repetitive subconcussive head impact when heading the ball. Whether this leads to long-term alterations of the brain’s structure associated with cognitive decline remains unknown. The aim of this study was to evaluate cortical thickness in former professional soccer players using high-resolution structural MR imaging. Fifteen former male professional soccer players (mean age 49.3 [SD 5.1] years) underwent high-resolution structural 3 T MR imaging, as well as cognitive testing. Fifteen male, age-matched former professional non-contact sport athletes (mean age 49.6 [SD 6.4] years) served as controls. Group analyses of cortical thickness were performed using voxel-based statistics. Soccer players demonstrated greater cortical thinning with increasing age compared to controls in the right inferolateral-parietal, temporal, and occipital cortex. Cortical thinning was associated with lower cognitive performance as well as with estimated exposure to repetitive subconcussive head impact. Neurocognitive evaluation revealed decreased memory performance in the soccer players compared to controls. The association of cortical thinning and decreased cognitive performance, as well as exposure to repetitive subconcussive head impact, further supports the hypothesis that repetitive subconcussive head impact may play a role in early cognitive decline in soccer players. Future studies are needed to elucidate the time course of changes in cortical thickness as well as their association with impaired cognitive function and possible underlying neurodegenerative process.     

Low-Affinity Neurotrophin Receptor p75 Promotes the Transduction of Targeted Lentiviral Vectors to Cholinergic Neurons of Rat Basal Forebrain

Basal forebrain cholinergic neurons (BFCNs) are one of the most affected neuronal types in Alzheimer’s disease (AD), with their extensive loss documented at late stages of the pathology. While discriminatory provision of neuroprotective agents and trophic factors to these cells is thought to be of substantial therapeutic potential, the intricate topography and structure of the forebrain cholinergic system imposes a major challenge. To overcome this, we took advantage of the physiological enrichment of BFCNs with a low-affinity p75 neurotrophin receptor (p75^NTR) for their targeting by lentiviral vectors within the intact brain of adult rat. Herein, a method is described that affords selective and effective transduction of BFCNs with a green fluorescence protein (GFP) reporter, which combines streptavidin–biotin technology with anti-p75^NTR antibody-coated lentiviral vectors. Specific GFP expression in cholinergic neurons was attained in the medial septum and nuclei of the diagonal band Broca after a single intraventricular administration of such targeted vectors. Bioelectrical activity of GFP-labeled neurons was proven to be unchanged. Thus, proof of principle is obtained for the utility of the low-affinity p75^NTR for targeted transduction of vectors to BFCNs in vivo.     

Cell-free fetal DNA in amniotic fluid: unique fragmentation signatures in euploid and aneuploid fetuses

BACKGROUND: Circulating cell-free fetal deoxyribonucleic acids (cffDNA) are novel biomarkers with many clinical applications. Amniotic fluid (AF) is a rich source of cffDNA. We investigated the biophysical characteristics of cffDNA in AF, hypothesizing that they would differ from cffDNA in maternal plasma. METHODS: We obtained 10 mL of fresh AF supernatant from women carrying euploid fetuses (n = 39) and aneuploid fetuses (n = 4). To test the effects of storage and karyotype, samples from euploid fetuses (n = 19) and aneuploid fetuses with trisomies 21 (n = 16), 18 (n = 9), or 13 (n = 3); triploidy (n = 4); or monosomy X (n = 2) were frozen at -80 degrees C. AF cffDNA was characterized by real-time quantitative PCR amplification of glyceraldehyde-3-phosphate dehydrogenase, gel electrophoresis, and analysis of the DNA fragmentation signature. RESULTS: We observed a significant correlation of concentration with gestational age for fresh AF cffDNA from euploid fetuses (R(2) = 0.77, P <0.0001) but not for frozen cffDNA (P = 0.63). The median amount of cffDNA in frozen euploid samples was significantly lower than in fresh samples (P <0.0001). After adjustment for gestational age, there was a statistically significant decrease in the median amount of cffDNA in frozen aneuploidy samples compared with frozen euploid samples (P = 0.0005). Analysis of the cffDNA size distribution showed different and qualitatively unique patterns for each karyotype. CONCLUSIONS: Gestational age, karyotype, and sample storage time affect concentrations and fragment size of AF cff DNA. These effects may be attributable to fundamental differences in tissue sources, excretion modes, or kinetic pathways. Characteristic signature patterns for each common aneuploidy offer the possibility of using DNA fragmentation analysis as a means of triaging AF samples.     

Pharmacodynamic effects of telavancin against methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains in the presence of human albumin or serum and in an in vitro kinetic model

Telavancin is a novel bactericidal lipoglycopeptide with multiple mechanisms of action against gram-positive pathogens. The aim of this study was to describe the dynamics of the antimicrobial effect of telavancin against two strains of Staphylococcus aureus (methicillin susceptible and methicillin resistant) in an in vitro kinetic model with simulated human pharmacokinetics. Also, static experiments were performed to determine the rate and extent of killing by telavancin in the presence and absence of human albumin and human serum. Experiments in broth and in nutrient-depleted medium were performed to study the rate and extent of killing by telavancin of bacteria in different growth phases. In the in vitro kinetic model regrowth was noted at 24 h for both strains when exposed to initial concentrations below 5 mg/liter. There was a >3-log(10) killing at all concentrations from 0.5x MIC and above at 24 h both in broth and in the presence of 40-g/liter human albumin. In contrast to the methicillin-susceptible strain, the methicillin-resistant strain in 40-g/liter human albumin showed a regrowth at concentrations of 0.5x MIC and 1x MIC at 24 h. At all the other concentrations >3-log(10) killing was seen at 24 h. Concordant results were seen in 50% human serum. At a target area under the curve/MIC ratio of 50 (corresponding to the human dose of 10 mg/kg of body weight, administered intravenously), >3-log(10) killing was observed at 6 to 8 h. Unlike most antibiotics, telavancin was able to kill both strains in a nongrowing phase.     

Gamma Secretase Inhibitors in Cancer: A Current Perspective on Clinical Performance

Gamma secretase inhibitors (GSIs), initially developed as Alzheimer''s therapies, have been repurposed as anticancer agents given their inhibition of Notch receptor cleavage. The success of GSIs in preclinical models has been ascribed to induction of cancer stem‐like cell differentiation and apoptosis, while also impairing epithelial‐to‐mesenchymal transition and sensitizing cells to traditional chemoradiotherapies. The promise of these agents has yet to be realized in the clinic, however, as GSIs have failed to demonstrate clinical benefit in most solid tumors with the notable exceptions of CNS malignancies and desmoid tumors. Disappointing clinical performance to date reflects important questions that remain to be answered. For example, what is the net impact of these agents on antitumor immune responses, and will they require concurrent targeting of tumor‐intrinsic compensatory pathways? Addressing these limitations in our current understanding of GSI mechanisms will undoubtedly facilitate their rational incorporation into combinatorial strategies and provide a valuable tool with which to combat Notch‐dependent cancers. In the present review, we provide a current understanding of GSI mechanisms, discuss clinical performance to date, and suggest areas for future investigation that might maximize the utility of these agents.Implications for PracticeThe performance of gamma secretase inhibitors (GSIs) in clinical trials generally has not reflected their encouraging performance in preclinical studies. This review provides a current perspective on the clinical performance of GSIs across various solid tumor types alongside putative mechanisms of antitumor activity. Through exploration of outstanding gaps in knowledge as well as reasons for success in certain cancer types, the authors identify areas for future investigation that will likely enable incorporation of GSIs into rational combinatorial strategies for superior tumor control and patient outcomes.     

Pharmacodynamics of moxifloxacin against Streptococcus pyogenes in an in vitro kinetic model

The aim of the present study was to investigate the pharmacodynamics of moxifloxacin against strains of Streptococcus pyogenes with different susceptibilities to erythromycin by using an in vitro kinetic model simulating human pharmacokinetics of moxifloxacin at oral doses of 400 and 200 mg, respectively. When the different strains of S. pyogenes were exposed to the higher dose, the number of bacteria was reduced below the detection limit after 12 h and no regrowth was noted during the following 12 h. At the lower dose there was regrowth of the strains with constitutive and inducible erythromycin resistance of the MLS(B) phenotype. Replication assays of the regrowing bacteria indicated that the failure of moxifloxacin to kill the MLS(B) strains at the lower dose was likely caused by the emergence of preexisting resistant subpopulations. Thus, the present study indicates that the presently used 400-mg dose seems to have an advantage over the lower dose in that the risk for selection of resistant subpopulations is minimized.