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 TERT 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 TERT promoter. We analyzed 21 stage III and IV melanoma patient samples who were treatment-naïve or on therapy. The overall detection rate of the custom panel, based on BRAF/NRAS/TERT promoter mutations, was 14/21 (67%) patient samples which included a TERT C250T mutation in one BRAF and NRAS mutation negative sample. A BRAF or NRAS mutation was detected in the ctDNA of 13/21 (62%) patients while TERT promoter mutations were detected in 10/21 (48%) patients. Co-occurrence of TERT promoter mutations with BRAF or NRAS mutations was found in 9/10 (90%) patients. The custom ctDNA panel showed a concordance of 16/21 (76%) with tissue based-detection and included 12 BRAF/NRAS mutation positive and 4 BRAF/NRAS mutation negative patients. The ctDNA mutation detection rate for stage IV was 12/16 (75%) and for stage III was 1/5 (20%). Based on BRAF, NRAS and TERT promoter mutations, the custom melanoma panel displayed a limit of detection of ~0.2% mutant allele frequency and showed significant correlation with droplet digital PCR. For one patient, a novel MAP2K1 H119Y mutation was detected in an NRAS/BRAF/TERT promoter mutation negative background. To increase the detection rate to >90% for stage IV melanoma patients, we plan to expand our custom panel to 50 genes. This study represents one of the first to successfully detect TERT promoter mutations in ctDNA from cutaneous melanoma patients using a targeted NGS panel.

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. To date, few cases of nodal blue nevi have been reported in the literature, and those reports have had limited clinical follow-up and supporting molecular data. This study sought to determine the clinical, pathologic, and molecular features of blue nevi involving lymph nodes, clarify their clinical significance, provide evidence for understanding their pathogenesis, and highlight potential pitfalls in the interpretation of lymph nodes with an ultimate aim of improving patient care. Thirteen cases of blue nevi involving lymph nodes were identified in the archives of Royal Prince Alfred Hospital, Sydney, Australia (1984-2018). A detailed assessment of the clinical and pathologic features of each case was performed, including an evaluation of all available immunohistochemical stains. Extended clinical follow-up was available for 9 patients. Droplet digital polymerase chain reaction for GNAQ Q209L, Q209P and GNA11 Q209L mutations was performed on 7 cases of blue nevi within lymph nodes together with matching cutaneous (presumed primary) blue nevi in 2 cases. All cases showed typical histologic features of blue nevi. BAP1 was retained in all cases (n=7). There were no recurrence or metastasis of blue nevus in any case on long-term clinical follow-up (n=9, median follow-up, 12 y). The majority of cases (n=5 of 7 evaluated) had GNAQ and GNA11 driver mutations. The 2 patients with a matched primary cutaneous blue nevus and regionally associated nodal blue nevus had the same GNAQ Q209L mutation in both sites in each patient. We conclude that blue nevi can involve lymph nodes and are associated with benign clinical behavior, and probably represent so-called "benign" metastasis. Awareness of these lesions is important when evaluating lymph nodes to avoid misdiagnosis as metastatic melanoma.

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. These mutations activate downstream and targetable signaling pathways, including the protein kinase C (PKC) cascade. PKC inhibitors have been used in clinical trials for metastatic UM but have shown limited efficacy. In this study, we examined the signaling and functional effects of two PKC inhibitors (AEB071 and IDE196) in a panel of UM cell models. In response to PKC inhibition, all UM cell lines showed potent suppression of PKC activity, but this was not sufficient to predict PKC inhibitor sensitivity and only two UM cell lines showed substantial PKC inhibitor-induced cell death. The differences in UM cell responses to PKC inhibition were not attributable to the degree or timing of PKC suppression or inhibition of the downstream mitogen-activated protein kinase (MAPK) or phosphatidylinositol-3-kinase (PI3K) pathways. Instead, UM cell show complex, PKC-independent signaling pathways that contribute to their survival and resistance to targeted therapies.

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. Subsequent study of a recombinant HSV-1 gE WIRS-mutant found no significant changes to viral production and release based on growth kinetics studies, or changes to plaque and comet size in various cell types, suggesting no function for the motif in cell-to-cell spread. GFP-Trap pulldown and proximity ligation assays were unable to confirm a WIRS-dependent interaction between gE and the WRC in human cell lines though the WIRS-independent interaction observed in situ warrants further study. Confocal microscopy of infected cells of neuronal origin identified no impairment of gE WIRS-mutant HSV-1 anterograde transport along axons. We propose that the identified gE WIRS motif does not function directly in recruitment of the WRC in human cells, in cell-to-cell spread of virus or in anterograde transport along axons. Further studies are needed to understand how HSV-1 manipulates and traverses the actin cytoskeleton and how gE may contribute to these processes in a WIRS-independent manner.

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. Longitudinal plasma samples were analyzed for ctDNA in 17 metastatic UM patients treated with PKCi-based therapy in a phase 1 clinical trial setting. Plasma ctDNA was assessed using digital droplet PCR (ddPCR) and a custom melanoma gene panel for targeted next generation sequencing (NGS). Baseline ctDNA strongly correlated with baseline lactate dehydrogenase (LDH) (p < 0.001) and baseline disease burden (p = 0.002). Early during treatment (EDT) ctDNA accurately predicted patients with clinical benefit to PKCi using receiver operator characteristic (ROC) curves (AUC 0.84, [95% confidence interval 0.65-1.0, p = 0.026]). Longitudinal ctDNA assessment was informative for establishing clinical benefit and detecting disease progression with 7/8 (88%) of patients showing a rise in ctDNA and targeted NGS of ctDNA revealed putative resistance mechanisms prior to radiological progression. The inclusion of longitudinal ctDNA monitoring in metastatic UM can advance adaptive clinical trial design.

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. Hence, a robust, rapid and facile strategy is needed for detecting multiple ctDNA mutations to improve the management of CRC patients. To address this significant problem, herein, we propose a new application of multiplex PCR/SERS (surface-enhanced Raman scattering) assay for the detection of ctDNA in CRC, in a fast and non-invasive manner to diagnose and stratify patients for effective treatment. Methods: To discriminate ctDNA mutations from wild-type cfDNA, allele-specific primers were designed for the amplification of three clinically important DNA point mutations in CRC including KRAS G12V, KRAS G13D and BRAF V600E. Surface-enhanced Raman scattering (SERS) nanotags were labelled with a short and specific sequence of oligonucleotide, which can hybridize with the corresponding PCR amplicons. The PCR/SERS assay was implemented by firstly amplifying the multiple mutations, followed by binding with multicolor SERS nanotags specific to each mutation, and subsequent enrichment with magnetic beads. The mutation status was evaluated using a portable Raman spectrometer where the fingerprint spectral peaks of the corresponding SERS nanotags indicate the presence of the mutant targets. The method was then applied to detect ctDNA from CRC patients under a blinded test, the results were further validated by ddPCR. Results: The PCR/SERS strategy showed high specificity and sensitivity for genotyping CRC cell lines and plasma ctDNA, where as few as 0.1% mutant alleles could be detected from a background of abundant wild-type cfDNA. The blinded test using 9 samples from advanced CRC patients by PCR/SERS assay was validated with ddPCR and showed good consistency with pathology testing results. Conclusions: With ddPCR-like sensitivity yet at the convenience of standard PCR, the proposed assay shows great potential in sensitive detection of multiple ctDNA mutations for clinical decision-making.

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. At the recommended cfDNA input quantity of 20 ng (available in 28/74 patients), at least one cancer-associated mutation was detected in the ctDNA of 84% of stage IV patients and 47% of stage III patients with a limit of detection for mutant allele frequency (MAF) of 0.2%. This custom melanoma panel showed significant correlation with droplet digital PCR (ddPCR) and provided a more comprehensive melanoma mutation profile. Our custom panel could be further optimized by replacing amplicons spanning the TERT promoter, which did not perform well due to the high GC content. To increase the detection rate to 90% of stage IV melanoma and decrease the sensitivity to 0.1% MAF, we recommend increasing the volume of plasma to 8 mL to achieve minimal recommended cfDNA input and the refinement of poorly performing amplicons. Our panel can also be expanded to include new targetable and treatment resistance mutations to improve the tracking of treatment response and resistance in melanoma patients treated with systemic drug therapies.

Longitudinal Monitoring of ctDNA in Patients with Melanoma and Brain Metastases Treated with Immune Checkpoint Inhibitors.

PURPOSE: Brain involvement occurs in the majority of patients with metastatic melanoma. The potential of circulating tumor DNA (ctDNA) for surveillance and monitoring systemic therapy response in patients with melanoma brain metastases merits investigation. EXPERIMENTAL DESIGN: This study examined circulating BRAF, NRAS, and c-KIT mutations in patients with melanoma with active brain metastases receiving PD-1 inhibitor-based therapy. Intracranial and extracranial disease volumes were measured using the sum of product of diameters, and response assessment performed using RECIST. Longitudinal plasma samples were analyzed for ctDNA over the first 12 weeks of treatment (threshold 2.5 copies/mL plasma). RESULTS: Of a total of 72 patients, 13 patients had intracranial metastases only and 59 patients had concurrent intracranial and extracranial metastases. ctDNA detectability was 0% and 64%, respectively, and detectability was associated with extracranial disease volume (P < 0.01). Undetectable ctDNA on-therapy was associated with extracranial response (P < 0.01) but not intracranial response. The median overall survival in patients with undetectable (n = 34) versus detectable (n = 38) ctDNA at baseline was 39.2 versus 10.6 months [HR, 0.51; 95% confidence interval (CI), 0.28-0.94; P = 0.03] and on-therapy was 39.2 versus 9.2 months (HR, 0.32; 95% CI, 0.16-0.63; P < 0.01). CONCLUSIONS: ctDNA remains a strong prognostic biomarker in patients with melanoma with brain metastases, especially in patients with concurrent extracranial disease. However, ctDNA was not able to detect or monitor intracranial disease activity, and we recommend against using ctDNA as a sole test during surveillance and therapeutic monitoring in patients with 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. However, because of the high heterogeneity and low level of small EVs in body fluids, there is a high demand for sensitive detection and characterization of such vesicles at a molecular level. In this study, we have developed a sensitive and effective approach to simultaneously profile multiple protein biomarkers expressed on cancer-derived small EVs using surface-enhanced Raman spectroscopy (SERS) nanotags in a single test, without complex isolation steps. Rapid and multiplexed phenotypic profiling of small EVs is achieved by mixing specific detection antibody-coated SERS nanotags, filtered conditioned EV-suspended medium (conditioned EVs), and capture antibody (CD63)-conjugated magnetic beads to form a sandwich immunoassay. As a proof-of-concept demonstration, we applied this approach to characterize pancreatic cancer-derived EVs by simultaneously detecting three specific EV surface receptors including Glypican-1, epithelial cell adhesion molecules (EpCAMs), and CD44 variant isoform 6 (CD44V6). The sensitivity of this method was measured down to 2.3 × 106 particles/mL, which is more sensitive and shows higher multiplexing capability than most other reported EV profiling techniques, such as western blot, enzyme-linked immunosorbent assay, and flow cytometry. Furthermore, phenotypic profiling of small EVs from colorectal cancer and bladder cancer cell lines (SW480 and C3) was conducted and compared to those derived from pancreatic cancer (Panc-1), highlighting the significant difference in EV phenotypes for various cancer cell types suspended in both phosphate-buffered saline and plasma. Thus, we believe that this technology enables a comprehensive evaluation of small secreted EV heterogeneity with high sensitivity, offering strong potential for accurate noninvasive cancer diagnosis and monitoring of drug treatment. In addition, this assay provides point-of-care use because of the easy sample preparation and portable nature of the Raman spectrometer.

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.

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. WES parameters such as sequencing coverage and total sequencing reads were comparable between gDNA and ctDNA. The mutant allele frequency of common single nucleotide variants was lower in ctDNA, reflecting the lower read depth and minor fraction of ctDNA within the total circulating free DNA pool. There was also variable concordance between gDNA and ctDNA based on the total number and identity of detected variants and this was independent of the tumor biopsy site. Nevertheless, established melanoma driver mutations and several other melanoma-associated mutations were concordant between matched gDNA and ctDNA. This study highlights that WES of ctDNA could capture clinically relevant mutations present in melanoma metastases and that enhanced sequencing sensitivity will be required to identify low frequency mutations.

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. The use of polymerase chain reaction (PCR)-based methods, such as droplet digital PCR, allows for a very sensitive analysis of circulating tumour DNA, but typically only a limited number of gene mutations can be detected in parallel. In contrast, next-generation sequencing allows for parallel analysis of multiple mutations in many genes. The development of targeted next-generation sequencing cancer gene panels optimised for the detection of circulating free DNA now provides both the flexibility of multiple mutation analysis coupled with a sensitivity that approaches or even matches droplet digital PCR. In this review, we discuss the advantages and disadvantages of these current molecular technologies in conjunction with how this field is evolving in the context of melanoma diagnosis, prognosis, and monitoring of response to therapy.

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. The following commercial cfDNA purification kits were analysed in this study: QIAamp circulating nucleic acid kit (Qiagen, Germany); Plasma/serum cell-free circulating DNA Purification midi kit (Norgen Biotek, Canada); QIAamp minElute ccfDNA mini kit (Qiagen); Maxwell RSC ccfDNA plasma kit (Promega, USA); MagMAX cell-free DNA isolation kit (Applied Biosystems, USA); and NextPrep-Mag cfDNA isolation kit (Bioo Scientific, USA). Extracted DNA from the plasma of healthy individuals, either nonspiked or spiked with DNA fragments or cfDNA, was evaluated for recovery using either a BioRad Experion or ddPCR analysis. This study represents the first to use a comprehensive size distribution of spiked-in DNA fragments to evaluate commercial cfDNA kits. The commonly used spin column-based Qiagen QIAamp circulating nucleic acid kit was found to be the most consistent performing kit across the two evaluation assays employed. The Qiagen QIAamp minElute ccfDNA mini kit represented the best performing magnetic bead-based kit and provides an alternative based on lower cost/sample with a simpler workflow than spin column-based kits.

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 trans-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. We have established a novel imaging technology to track virus transport that uses photoconvertible fluorescent recombinant viruses to track subsets of virus particles from their site of origin and determine their destination. Here we image virus exit from the TGN and their rate of egress to the cell periphery. We demonstrate a role for kinesin-1 engagement in regulating virus exit from the TGN by removing A36 and F12 function, critical viral mediators of kinesin-1 recruitment to virus particles. Phototracking viral particles and components during infection is a powerful new imaging approach to elucidate mechanisms of virus replication.

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. Additional cytogenetic and genetic changes, however, including chromosome 3 monosomy, mutations in the BAP1 tumor suppressor gene, alterations in the splicing factors SRSF2/SF3B1, and mutations in the translation initiation factor EIF1AX, modulate signaling output in uveal tumors and modify the risk of metastases. Here, we review the complex interactions between genetic, molecular signaling, and prognostic profiles in uveal melanoma; the clinical implications of these interactions; and the latest potential targets for rational therapy.

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. This review will focus on the currently known mechanisms utilized by HSV-1 to harness the neuronal cytoskeleton, molecular motors, and the secretory and exocytic pathways for efficient virus entry, axonal transport, replication, assembly, and exit from the distinct functional compartments (cell body and axon) of the highly polarized sensory neurons.

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. Human-tropic alphaherpesviruses are prevalent pathogens carried by more than half of the world's population; comprising herpes simplex virus (types 1 and 2) and varicella-zoster virus, these viruses are characterised by their ability to establish latency in sensory neurons. This review will discuss the known mechanisms involved in subversion of and transport via the cytoskeleton during alphaherpesvirus infections, focusing on protein-protein interactions and pathways that have recently been identified. Studies on related alphaherpesviruses whose primary host is not human, along with comparisons to more distantly related beta and gammaherpesviruses, are also presented in this review. The need to decipher as-yet-unknown mechanisms exploited by viruses to hijack cytoskeletal components-to reveal the hidden cytoskeletons in the closet-will also be addressed.

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. In this era of effective molecular targeted treatments and immunotherapies, there is now an urgent need to implement use of these circulating biomarkers in the clinic to facilitate personalized therapy. In this review, we present recent findings in circulating melanoma biomarkers, examine the challenges and promise of evolving technologies used for liquid biomarker discovery, and discuss future directions and perspectives in melanoma biomarker research.

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. We use high resolution imaging approaches including fluorescence recovery after photobleaching and other approaches to document interaction between Vpr and the dynein light chain protein, DYNLT1, and impaired interaction of the F72L mutant with DYNLT1. The results implicate MTs/DYNLT1 as drivers of Vpr nuclear import and HIV infection, with important therapeutic implications.