Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: a phase 1/2 trial.

Programmed cell death protein 1 (PD-1) inhibitors have modest efficacy as a monotherapy in hepatocellular carcinoma (HCC). A personalized therapeutic cancer vaccine (PTCV) may enhance responses to PD-1 inhibitors through the induction of tumor-specific immunity. We present results from a single-arm, open-label, phase 1/2 study of a DNA plasmid PTCV (GNOS-PV02) encoding up to 40 neoantigens coadministered with plasmid-encoded interleukin-12 plus pembrolizumab in patients with advanced HCC previously treated with a multityrosine kinase inhibitor. Safety and immunogenicity were assessed as primary endpoints, and treatment efficacy and feasibility were evaluated as secondary endpoints. The most common treatment-related adverse events were injection-site reactions, observed in 15 of 36 (41.6%) patients. No dose-limiting toxicities or treatment-related grade ≥3 events were observed. The objective response rate (modified intention-to-treat) per Response Evaluation Criteria in Solid Tumors 1.1 was 30.6% (11 of 36 patients), with 8.3% (3 of 36) of patients achieving a complete response. Clinical responses were associated with the number of neoantigens encoded in the vaccine. Neoantigen-specific T cell responses were confirmed in 19 of 22 (86.4%) evaluable patients by enzyme-linked immunosorbent spot assays. Multiparametric cellular profiling revealed active, proliferative and cytolytic vaccine-specific CD4+ and CD8+ effector T cells. T cell receptor ß-chain (TCRß) bulk sequencing results demonstrated vaccination-enriched T cell clone expansion and tumor infiltration. Single-cell analysis revealed posttreatment T cell clonal expansion of cytotoxic T cell phenotypes. TCR complementarity-determining region cloning of expanded T cell clones in the tumors following vaccination confirmed reactivity against vaccine-encoded neoantigens. Our results support the PTCV's mechanism of action based on the induction of antitumor T cells and show that a PTCV plus pembrolizumab has clinical activity in advanced HCC. ClinicalTrials.gov identifier: NCT04251117 .

Analytical validation of NeXT Personal®, an ultra-sensitive personalized circulating tumor DNA assay.

We describe the analytical validation of NeXT Personal®, an ultra-sensitive, tumor-informed circulating tumor DNA (ctDNA) assay for detecting residual disease, monitoring therapy response, and detecting recurrence in patients diagnosed with solid tumor cancers. NeXT Personal uses whole genome sequencing of tumor and matched normal samples combined with advanced analytics to accurately identify up to ~1,800 somatic variants specific to the patient's tumor. A personalized panel is created, targeting these variants and then used to sequence cell-free DNA extracted from patient plasma samples for ultra-sensitive detection of ctDNA. The NeXT Personal analytical validation is based on panels designed from tumor and matched normal samples from two cell lines, and from 123 patients across nine cancer types. Analytical measurements demonstrated a detection threshold of 1.67 parts per million (PPM) with a limit of detection at 95% (LOD95) of 3.45 PPM. NeXT Personal showed linearity over a range of 0.8 to 300,000 PPM (Pearson correlation coefficient = 0.9998). Precision varied from a coefficient of variation of 12.8% to 3.6% over a range of 25 to 25,000 PPM. The assay targets 99.9% specificity, with this validation study measuring 100% specificity and in silico methods giving us a confidence interval of 99.92 to 100%. In summary, this study demonstrates NeXT Personal as an ultra-sensitive, highly quantitative and robust ctDNA assay that can be used to detect residual disease, monitor treatment response, and detect recurrence in patients.

Analytic validation of NeXT Dx™, a comprehensive genomic profiling assay.

We describe the analytic validation of NeXT Dx, a comprehensive genomic profiling assay to aid therapy and clinical trial selection for patients diagnosed with solid tumor cancers. Proprietary methods were utilized to perform whole exome and whole transcriptome sequencing for detection of single nucleotide variants (SNVs), insertions/deletions (indels), copy number alterations (CNAs), and gene fusions, and determination of tumor mutation burden and microsatellite instability. Variant calling is enhanced by sequencing a patient-specific normal sample from, for example, a blood specimen. This provides highly accurate somatic variant calls as well as the incidental reporting of pathogenic and likely pathogenic germline alterations. Fusion detection via RNA sequencing provides more extensive and accurate fusion calling compared to DNA-based tests. NeXT Dx features the proprietary Accuracy and Content Enhanced technology, developed to optimize sequencing and provide more uniform coverage across the exome. The exome was validated at a median sequencing depth of >500x. While variants from 401 cancer-associated genes are currently reported from the assay, the exome/transcriptome assay is broadly validated to enable reporting of additional variants as they become clinically relevant. NeXT Dx demonstrated analytic sensitivities as follows: SNVs (99.4%), indels (98.2%), CNAs (98.0%), and fusions (95.8%). The overall analytic specificity was >99.0%.

Surface for catalysis by poliovirus RNA-dependent RNA polymerase.

The poliovirus RNA-dependent RNA polymerase, 3Dpol, replicates the viral genomic RNA on the surface of virus-induced intracellular membranes. Macromolecular assemblies of 3Dpol form linear arrays of subunits that propagate along a strong protein-protein interaction called interface-I, as was observed in the crystal structure of wild-type poliovirus polymerase. These "filaments" recur with slight modifications in planar sheets and, with additional modifications that accommodate curvature, in helical tubes of the polymerase, by packing filaments together via a second set of interactions. Periodic variations of subunit orientations within 3Dpol tubes give rise to "ghost reflections" in diffraction patterns computed from electron cryomicrographs of helical arrays. The ghost reflections reveal that polymerase tubes are formed by bundles of four to five interface-I filaments, which are then connected to the next bundle of filaments with a perturbation of interface interactions between bundles. While enzymatically inactive polymerase is also capable of oligomerization, much thinner tubes that lack interface-I interactions between adjacent subunits are formed, suggesting that long-range allostery produces conformational changes that extend from the active site to the protein-protein interface. Macromolecular assemblies of poliovirus polymerase show repeated use of flexible interface interactions for polymerase lattice formation, suggesting that adaptability of polymerase-polymerase interactions facilitates RNA replication. In addition, the presence of a positively charged groove identified in polymerase arrays may help position and stabilize the RNA template during replication.

Cohort profile: the Western Australian Sleep Health Study.

BACKGROUND: Epidemiologic and genetic studies of obstructive sleep apnoea (OSA) are limited by a lack of large-scale, well-characterized OSA cohorts. These studies require large sample size to provide adequate power to detect differences between groups. This study describes the development of such a cohort (The Western Australian Sleep Health Study) in OSA patients of Caucasian-European origin attending the only public sleep clinic in Western Australia (WA). AIMS: The main aim of the study is to phenotype 4,000 OSA patients in order to define the genetics of OSA and its co-morbidities. METHODS: Almost all underwent laboratory-based attended polysomnography (PSG). RESULTS: Currently complete data (questionnaire, biochemistry, DNA, and PSG) has been obtained on over 3,000 individuals and will reach the target of 4,000 individuals by the end of 2010. In a separate but related study, we have developed a sleep study database containing data from all patients who have undergone PSG at the sleep laboratory since its inception in 1988 until the present day (over 30,000 PSG studies representing data from approximately 20,000 individuals). In addition, data from both cohorts have been linked prospectively to statutory health data collected by the WA Department of Health. CONCLUSION: This study will be the largest sleep clinic cohort database internationally with access to genetic and epidemiological data. It is unique among sleep clinic cohorts because of its size, the breadth of data collected and the ability to link prospectively to statutory health data. It will be a major tool to comprehensively assess genetic and epidemiologic factors determining OSA and its co-morbidities.

Real-time DNA sequencing from single polymerase molecules.

We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.

Mixing methods in assessing coaches' decision making.

Mixing methods has recently achieved respectability as an appropriate approach to research design, offering a variety of advantages (Tashakkori & Teddlie, 2003). The purpose of this paper is to outline and evaluate a mixed methods approach within the domain of coaches' decision making. Illustrated with data from a policy-capturing study on coaches' decisions about an injured gymnast's participation in competition, the approach involves the concurrent collection of quantitative and qualitative data and a three-phase process of data analysis. It is argued that (a) the method described can provide additional insights into the factors involved in coaches' decision making, beyond those provided via quantitative or qualitative methods alone, and (b) mixing methods holds promise for coaching research more generally.

UCSF tomography: an integrated software suite for real-time electron microscopic tomographic data collection, alignment, and reconstruction.

A real-time alignment and reconstruction scheme for electron microscopic tomography (EMT) has been developed and integrated within our UCSF tomography data collection software. This newly integrated software suite provides full automation from data collection to real-time reconstruction by which the three-dimensional (3D) reconstructed volume is immediately made available at the end of each data collection. Real-time reconstruction is achieved by calculating a weighted back-projection on a small Linux cluster (five dual-processor compute nodes) concurrently with the UCSF tomography data collection running on the microscope's computer, and using the fiducial-marker free alignment data generated during the data collection process. The real-time reconstructed 3D volume provides users with immediate feedback to fully asses all aspects of the experiment ranging from sample choice, ice thickness, experimental parameters to the quality of specimen preparation. This information can be used to guide subsequent data collections. Access to the reconstruction is especially useful in low-dose cryo EMT where such information is very difficult to obtain due to extraordinary low signal to noise ratio in each 2D image. In our environment, we generally collect 2048 x 2048 pixel images which are subsequently computationally binned four-fold for the on-line reconstruction. Based upon experiments performed with thick and cryo specimens at various CCD magnifications (50000x-80000x), alignment accuracy is sufficient to support this reduced resolution but should be refined before calculating a full resolution reconstruction. The reduced resolution has proven to be quite adequate to assess sample quality, or to screen for the best data set for full-resolution reconstruction, significantly improving both productivity and efficiency of system resources. The total time from start of data collection to a final reconstructed volume (512 x 512 x 256 pixels) is about 50 min for a +/-70 degrees 2k x 2k pixel tilt series acquired at every 1 degrees.

Intramolecular and intermolecular uridylylation by poliovirus RNA-dependent RNA polymerase.

The 22-amino-acid protein VPg can be uridylylated in solution by purified poliovirus 3D polymerase in a template-dependent reaction thought to mimic primer formation during RNA amplification in infected cells. In the cell, the template used for the reaction is a hairpin RNA termed 2C-cre and, possibly, the poly(A) at the 3' end of the viral genome. Here, we identify several additional substrates for uridylylation by poliovirus 3D polymerase. In the presence of a 15-nucleotide (nt) RNA template, the poliovirus polymerase uridylylates other polymerase molecules in an intermolecular reaction that occurs in a single step, as judged by the chirality of the resulting phosphodiester linkage. Phosphate chirality experiments also showed that VPg uridylylation can occur by a single step; therefore, there is no obligatory uridylylated intermediate in the formation of uridylylated VPg. Other poliovirus proteins that could be uridylylated by 3D polymerase in solution were viral 3CD and 3AB proteins. Strong effects of both RNA and protein ligands on the efficiency and the specificity of the uridylylation reaction were observed: uridylylation of 3D polymerase and 3CD protein was stimulated by the addition of viral protein 3AB, and, when the template was poly(A) instead of the 15-nt RNA, the uridylylation of 3D polymerase itself became intramolecular instead of intermolecular. Finally, an antiuridine antibody identified uridylylated viral 3D polymerase and 3CD protein, as well as a 65- to 70-kDa host protein, in lysates of virus-infected human cells.

Allosteric effects of ligands and mutations on poliovirus RNA-dependent RNA polymerase.

Protein priming of viral RNA synthesis plays an essential role in the replication of picornavirus RNA. Both poliovirus and coxsackievirus encode a small polypeptide, VPg, which serves as a primer for addition of the first nucleotide during synthesis of both positive and negative strands. This study examined the effects on the VPg uridylylation reaction of the RNA template sequence, the origin of VPg (coxsackievirus or poliovirus), the origin of 3D polymerase (coxsackievirus or poliovirus), the presence and origin of interacting protein 3CD, and the introduction of mutations at specific regions in the poliovirus 3D polymerase. Substantial effects associated with VPg origin were traced to differences in VPg-polymerase interactions. The effects of 3CD proteins and mutations at polymerase-polymerase intermolecular Interface I were most consistent with allosteric effects on the catalytic 3D polymerase molecule. In conclusion, the efficiency and specificity of VPg uridylylation by picornavirus polymerases is greatly influenced by allosteric effects of ligand binding that are likely to be relevant during the viral replicative cycle.

Visualization and functional analysis of RNA-dependent RNA polymerase lattices.

Positive-strand RNA viruses such as poliovirus replicate their genomes on intracellular membranes of their eukaryotic hosts. Electron microscopy has revealed that purified poliovirus RNA-dependent RNA polymerase forms planar and tubular oligomeric arrays. The structural integrity of these arrays correlates with cooperative RNA binding and RNA elongation and is sensitive to mutations that disrupt intermolecular contacts predicted by the polymerase structure. Membranous vesicles isolated from poliovirus-infected cells contain structures consistent with the presence of two-dimensional polymerase arrays on their surfaces during infection. Therefore, host cytoplasmic membranes may function as physical foundations for two-dimensional polymerase arrays, conferring the advantages of surface catalysis to viral RNA replication.

Similar structural basis for membrane localization and protein priming by an RNA-dependent RNA polymerase.

Protein primers are used to initiate genomic synthesis of several RNA and DNA viruses, although the structural details of the primer-polymerase interactions are not yet known. Poliovirus polymerase binds with high affinity to the membrane-bound viral protein 3AB but uridylylates only the smaller peptide 3B in vitro. Mutational analysis of the polymerase identified four surface residues on the three-dimensional structure of poliovirus polymerase whose wild-type identity is required for 3AB binding. These mutants also decreased 3B uridylylation, arguing that the binding sites for the membrane tether and the protein primer overlap. Mutation of flanking residues between the 3AB binding site and the polymerase active site specifically decreased 3B uridylylation, likely affecting steps subsequent to binding. The physical overlap of sites for protein priming and membrane association should facilitate replication initiation in the membrane-associated complex.