Circulating tumour DNA dynamics predict recurrence in stage III melanoma patients receiving neoadjuvant immunotherapy.

Background: Neoadjuvant therapy improves recurrence-free survival (RFS) in resectable stage III cutaneous melanoma. However, accurately predicting individual recurrence risk remains a significant challenge. We investigated circulating tumour DNA (ctDNA) as a biomarker for recurrence in measurable stage IIIB/C melanoma patients undergoing neoadjuvant immunotherapy.

Anchored Multiplex PCR Custom Melanoma Next Generation Sequencing Panel for Analysis of Circulating Tumor DNA.

Detection of melanoma mutations using circulating tumor DNA (ctDNA) is a potential alternative to using genomic DNA from invasive tissue biopsies. To date, mutations in the GC-rich promoter region, which is commonly mutated in melanoma, have been technically difficult to detect in ctDNA using next-generation sequencing (NGS) panels. In this study, we developed a custom melanoma NGS panel for detection of ctDNA, which encompasses the top 15 gene mutations in melanoma including the promoter.

Comprehensive Clinical, Histopathologic, and Molecular Analysis and Long-term Follow-up of Patients With Nodal Blue Nevi.

Blue nevi are benign, melanocytic neoplasms that show a range of clinical and morphologic patterns and include common/dendritic, cellular, and atypical cellular subtypes. Like other nevi, they most commonly occur in skin but can occasionally involve lymph nodes where they may be misinterpreted as representing metastatic melanoma. Moreover, whether benign blue nevi can metastasize to lymph nodes and their natural history and prognostic significance has been the subject of great controversy.

Protein kinase inhibitor responses in uveal melanoma reflects a diminished dependency on PKC-MAPK signaling.

Uveal melanoma (UM) is a rare cancer arising from melanocytes in the uveal tract of the eye. Despite effective primary treatment, there is no approved therapy for metastatic UM and prognosis and survival remain poor. Over 90% of UM are driven by mutations affecting the Gα subunits encoded by the GNAQ and GNA11 genes.

A putative WAVE regulatory complex (WRC) interacting receptor sequence (WIRS) in the cytoplasmic tail of HSV-1 gE does not function in WRC recruitment or neuronal transport.

HSV-1 envelope glycoprotein E (gE) is important for viral egress and cell-to-cell spread but the host protein(s) involved in these functions have yet to be determined. We aimed to investigate a role for the Arp2/3 complex and actin regulation in viral egress based on the identification of a WAVE Regulatory Complex (WRC) Interacting Receptor Sequence (WIRS) in the cytoplasmic tail (CT) of gE. A WIRS-dependent interaction between the gE(CT) and subunits of the WRC was demonstrated by GST-pulldown assay and a role for the Arp2/3 complex in cell-to-cell spread was also observed by plaque assay.

Circulating Tumor DNA Reflects Uveal Melanoma Responses to Protein Kinase C Inhibition.

The prognosis for patients with UM is poor, and recent clinical trials have failed to prolong overall survival (OS) of these patients. Over 95% of UM harbor activating driver mutations, and this allows for the investigation of ctDNA. In this study, we investigated the value of ctDNA for adaptive clinical trial design in metastatic UM.

Multiplex detection of ctDNA mutations in plasma of colorectal cancer patients by PCR/SERS assay.

Molecular diagnostic testing of KRAS and BRAF mutations has become critical in the management of colorectal cancer (CRC) patients. Some progress has been made in liquid biopsy detection of mutations in circulating tumor DNA (ctDNA), which is a fraction of circulating cell-free DNA (cfDNA), but slow analysis for DNA sequencing methods has limited rapid diagnostics. Other methods such as quantitative PCR and more recently, droplet digital PCR (ddPCR), have limitations in multiplexed capacity and the need for expensive specialized equipment.

Design and Testing of a Custom Melanoma Next Generation Sequencing Panel for Analysis of Circulating Tumor DNA.

Detection of melanoma-associated mutations using circulating tumor DNA (ctDNA) from plasma is a potential alternative to using genomic DNA from invasive tissue biopsies. In this study, we developed a custom melanoma next-generation sequencing (NGS) panel which includes 123 amplicons in 30 genes covering driver and targetable mutations and alterations associated with treatment resistance. Analysis of a cohort of 74 stage III and IV treatment-naïve melanoma patients revealed that sensitivity of ctDNA detection was influenced by the amount of circulating-free DNA (cfDNA) input and stage of melanoma.

Enabling Sensitive Phenotypic Profiling of Cancer-Derived Small Extracellular Vesicles Using Surface-Enhanced Raman Spectroscopy Nanotags.

Circulating cancer-derived small extracellular vesicles (EVs) are nanoscale membranous vesicles shed from cancer cells that are released into surrounding body fluids. Small EVs contain biomolecules associated with cancer such as DNA and proteins for cell-to-cell communication. Therefore, small EVs have been regarded as important cancer biomarkers for liquid biopsy-based cancer diagnosis and drug treatment monitoring.

Analysis of the Whole-Exome Sequencing of Tumor and Circulating Tumor DNA in Metastatic Melanoma.

The use of circulating tumor DNA (ctDNA) to monitor cancer progression and response to therapy has significant potential but there is only limited data on whether this technique can detect the presence of low frequency subclones that may ultimately confer therapy resistance. In this study, we sought to evaluate whether whole-exome sequencing (WES) of ctDNA could accurately profile the mutation landscape of metastatic melanoma. We used WES to identify variants in matched, tumor-derived genomic DNA (gDNA) and plasma-derived ctDNA isolated from a cohort of 10 metastatic cutaneous melanoma patients.

Tour de Herpes: Cycling Through the Life and Biology of HSV-1.

Herpes simplex virus type 1 (HSV-1) is a prevalent and important human pathogen that has been studied in a wide variety of contexts. This book provides protocols currently in use in leading laboratories in many fields of HSV-1 research. This introductory chapter gives a brief overview of HSV-1 biology and life cycle, covering basic aspects of virus structure, the prevalence of and diseases caused by the virus, replication in cultured cells, viral latency, antiviral defenses, and the mechanisms that the virus uses to counteract these defenses.

Evaluation of commercial kits for purification of circulating free DNA.

Analysis of liquid biopsies and the identification of non-invasive biomarkers for the diagnosis and prognosis of solid tumors has grown exponentially over the last few years. This has led to an increasing number of commercial kits optimised for the purification of circulating free (cf) DNA and RNA/miRNA from biofluids such as plasma, serum and urine. To optimise and standardise current practices we sought to evaluate the performance of spin column-based and magnetic bead-based commercial kits.

Monitoring Melanoma Using Circulating Free DNA.

Genetic material derived from tumours is constantly shed into the circulation of cancer patients both in the form of circulating free nucleic acids and within circulating cells or extracellular vesicles. Monitoring cancer-specific genomic alterations, particularly mutant allele frequencies, in circulating nucleic acids allows for a non-invasive liquid biopsy for detecting residual disease and response to therapy. The advent of molecular targeted treatments and immunotherapies with increasing effectiveness requires corresponding effective molecular biology methods for the detection of biomarkers such as circulating nucleic acid to monitor and ultimately personalise therapy.

Phototracking Vaccinia Virus Transport Reveals Dynamics of Cytoplasmic Dispersal and a Requirement for A36R and F12L for Exit from the Site of Wrapping.

The microtubule cytoskeleton is a primary organizer of viral infections for delivering virus particles to their sites of replication, establishing and maintaining subcellular compartments where distinct steps of viral morphogenesis take place, and ultimately dispersing viral progeny. One of the best characterized examples of virus motility is the anterograde transport of the wrapped virus form of vaccinia virus (VACV) from the -Golgi network (TGN) to the cell periphery by kinesin-1. Yet many aspects of this transport event are elusive due to the speed of motility and the challenges of imaging this stage at high resolution over extended time periods.

Oncogenic signaling in uveal melanoma.

Uveal melanoma is the most common primary cancer of the eye, and despite rapidly emerging insights into the molecular profile of this disease, prognosis of patients with metastatic uveal melanoma remains poor with mortality rates unchanged in over 35 years. Early genetic events activate G protein-coupled receptor signaling in nearly all uveal tumors via mutually exclusive mutations in the GNAQ, GNA11, CYSLTR2, or PLCB4 genes. A multitude of signaling cascades downstream of G protein activation, including protein kinase C and mitogen-activated protein kinase activity, are actionable, and many ongoing clinical trials are targeting these pathways.

Infection and Transport of Herpes Simplex Virus Type 1 in Neurons: Role of the Cytoskeleton.

Herpes simplex virus type 1 (HSV-1) is a neuroinvasive human pathogen that has the ability to infect and replicate within epithelial cells and neurons and establish a life-long latent infection in sensory neurons. HSV-1 depends on the host cellular cytoskeleton for entry, replication, and exit. Therefore, HSV-1 has adapted mechanisms to promote its survival by exploiting the microtubule and actin cytoskeletons to direct its active transport, infection, and spread between neurons and epithelial cells during primary and recurrent infections.

Cytoskeletons in the Closet-Subversion in Alphaherpesvirus Infections.

Actin filaments, microtubules and intermediate filaments form the cytoskeleton of vertebrate cells. Involved in maintaining cell integrity and structure, facilitating cargo and vesicle transport, remodelling surface structures and motility, the cytoskeleton is necessary for the successful life of a cell. Because of the broad range of functions these filaments are involved in, they are common targets for viral pathogens, including the alphaherpesviruses.

Liquid biomarkers in melanoma: detection and discovery.

A vast array of tumor-derived genetic, proteomic and cellular components are constantly released into the circulation of cancer patients. These molecules including circulating tumor DNA and RNA, proteins, tumor and immune cells are emerging as convenient and accurate liquid biomarkers of cancer. Circulating cancer biomarkers provide invaluable information on cancer detection and diagnosis, prognosticate patient outcomes, and predict treatment response.

Fast track, dynein-dependent nuclear targeting of human immunodeficiency virus Vpr protein; impaired trafficking in a clinical isolate.

Nuclear import of the accessory protein Vpr is central to infection by human immunodeficiency virus (HIV). We previously identified the Vpr F72L mutation in a HIV-infected, long-term non-progressor, showing that it resulted in reduced Vpr nuclear accumulation and altered cytoplasmic localisation. Here we demonstrate for the first time that the effects of nuclear accumulation of the F72L mutation are due to impairment of microtubule-dependent-enhancement of Vpr nuclear import.

Dual Role of Herpes Simplex Virus 1 pUS9 in Virus Anterograde Axonal Transport and Final Assembly in Growth Cones in Distal Axons.

Unlabelled: The herpes simplex virus type 1 (HSV-1) envelope protein pUS9 plays an important role in virus anterograde axonal transport and spread from neuronal axons. In this study, we used both confocal microscopy and transmission electron microscopy (TEM) to examine the role of pUS9 in the anterograde transport and assembly of HSV-1 in the distal axon of human and rat dorsal root ganglion (DRG) neurons using US9 deletion (US9(-)), repair (US9R), and wild-type (strain F, 17, and KOS) viruses. Using confocal microscopy and single and trichamber culture systems, we observed a reduction but not complete block in the anterograde axonal transport of capsids to distal axons as well as a marked (∼90%) reduction in virus spread from axons to Vero cells with the US9 deletion viruses.

The Basic Domain of Herpes Simplex Virus 1 pUS9 Recruits Kinesin-1 To Facilitate Egress from Neurons.

Unlabelled: The alphaherpesviral envelope protein pUS9 has been shown to play a role in the anterograde axonal transport of herpes simplex virus 1 (HSV-1), yet the molecular mechanism is unknown. To address this, we used an in vitro pulldown assay to define a series of five arginine residues within the conserved pUS9 basic domain that were essential for binding the molecular motor kinesin-1. The mutation of these pUS9 arginine residues to asparagine blocked the binding of both recombinant and native kinesin-1.

Abalone Hemocyanin Blocks the Entry of Herpes Simplex Virus 1 into Cells: a Potential New Antiviral Strategy.

A marine-derived compound, abalone hemocyanin, from Haliotis rubra was shown to have a unique mechanism of antiviral activity against herpes simplex virus 1 (HSV-1) infections. In vitro assays demonstrated the dose-dependent and inhibitory effect of purified hemocyanin against HSV-1 infection in Vero cells with a 50% effective dose (ED50) of 40 to 50 nM and no significant toxicity. In addition, hemocyanin specifically inhibited viral attachment and entry by binding selectively to the viral surface glycoproteins gD, gB, and gC, probably by mimicking their receptors.

Conserved tegument protein complexes: Essential components in the assembly of herpesviruses.

One of the structural components of herpesviruses is a protein layer called the tegument. Several of the tegument proteins are highly conserved across the herpesvirus family and serve as a logical focus for defining critical interactions required for viral assembly. A number of studies have helped to elucidate a role for conserved tegument proteins in the process of secondary envelopment during the course of herpesviral assembly.