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Intro: High-Grade Gliomas (HGGs) are the most common and aggressive primary brain tumors in adults and are almost always fatal. Liquid biopsy provides a noninvasive window into the cancer genome and the underlying biology of the tumor. Circulating-tumor DNA (ctDNA) is a versatile analyte for tumor diagnosis, monitoring treatment response, detecting resistance, and tracking tumor evolution. The central hypothesis of our work is that detection of cerebrospinal fluid (CSF) ctDNA correlates with clinically significant events and can be used as a surrogate for tissue biopsy to guide treatment decisions in the clinic. Methods: Our study includes CSF ctDNA samples from 140 recurrent glioma patients at Memorial Sloan Kettering Cancer Center who underwent collection of CSF as part of their clinical evaluation for neurological signs and symptoms. For each patient, glioma subtype and grade were confirmed by a neuropathologist. Overall survival was calculated as the time interval between the date of diagnosis and the date of death. All samples were sequenced using the MSK-IMPACT targeted sequencing assay (468 genes). Allele-specific copy number calls were assessed using the FACETS algorithm. Mutations were classified as drivers based on OncoKB. To evaluate the relationship between CSF ctDNA detection and clinico-pathologic correlates, CSF ctDNA status was determined positive by the presence of at least one somatic mutation and CSF ctDNA status was correlated with clinico-pathologic features. Results: Within this cohort, we found 68 CSF ctDNA positive and 72 CSF negative samples. The most frequently mutated genes were: TERT (58.2%), TP53 (47.8%), IDH1 (20.9%) and EGFR (26.9%). We observed a concordance between contemporaneously sampled tumor and CSF. In a multivariate analysis accounting for established prognostic factors including: % extent of resection at diagnosis; tumor burden at the time of lumbar puncture; and IDH status, we found that CSF ctDNA positivity was negatively correlated with overall survival (HR: 2.52, p Conclusion: ctDNA from CSF depicts a powerful analyte with the potential to alter the standard of care. We have established a robust liquid biopsy program across the neuro-oncology department at MSK, and MSK-IMPACT is now certified by NYS DOH for use on CSF ctDNA enabling routine integration into clinical care. In summary, we are now able to monitor the changing genome along the disease course and have the potential to detect disease occurrence at an earlier time point, however further validation of CSF ctDNA for disease monitoring is needed. Additionally, our data suggests that CSF ctDNA may be used as a prognostic biomarker for survival, but confirmation requires further validation in a prospective study. Citation Format: Subhiksha Nandakumar, Christoph Kreitzer, Charli Ann Hertz, Johnathan Rafailov, Timothy Song, Nicholas D. Socci, A. Rose Brannon, Maria E. Arcila, David B. Solit, Michael F. Berger, Nikolaus Schultz, Ingo K. Mellinghoff, Alexandra M. Miller. Circulating tumor DNA from cerebrospinal fluid (CSF) allows for characterization and monitoring of glioma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1053.

Our results suggest that there is a significant association between the composition of the microbiota in the nasopharynx and the influenza virus type causing the infection. We observe that vaccination status, especially in more senior individuals, also has an association with the microbial community profile. This indicates that vaccination against influenza, even when ineffective to prevent disease, could play a role in controlling secondary bacterial complications., Factors that contribute to enhanced susceptibility to severe bacterial disease after influenza virus infection are not well defined but likely include the microbiome of the respiratory tract. Vaccination against influenza, while having variable effectiveness, could also play a role in microbial community stability. We collected nasopharyngeal samples from 215 individuals infected with influenza A/H3N2 or influenza B virus and profiled the microbiota by target sequencing of the 16S rRNA gene. We identified signature taxonomic groups by performing linear discriminant analysis and effective size comparisons (LEfSe) and defined bacterial community types using Dirichlet multinomial mixture (DMM) models. Influenza infection was shown to be significantly associated with microbial composition of the nasopharynx according to the virus type and the vaccination status of the patient. We identified four microbial community types across the combined cohort of influenza patients and healthy individuals with one community type most representative of the influenza virus-infected group. We also identified microbial taxa for which relative abundance was significantly higher in the unvaccinated elderly group; these taxa include species known to be associated with pneumonia.

In October 2018, the order Mononegavirales was amended by the establishment of three new families and three new genera, abolishment of two genera, and creation of 28 novel species. This article presents the updated taxonomy of the order Mon-onegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

International audience; In 2018, the order Mononegavirales was expanded by inclusion of 1 new genus and 12 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.

Influenza A virus is characterized by high genetic diversity. However, most of what is known about influenza evolution has come from consensus sequences sampled at the epidemiological scale that only represent the dominant virus lineage within each infected host. Less is known about the extent of within-host virus diversity and what proportion of this diversity is transmitted between individuals. To characterize virus variants that achieve sustainable transmission in new hosts, we examined within-host virus genetic diversity in household donor-recipient pairs from the first wave of the 2009 H1N1 pandemic when seasonal H3N2 was co-circulating. Although the same variants were found in multiple members of the community, the relative frequencies of variants fluctuated, with patterns of genetic variation more similar within than between households. We estimated the effective population size of influenza A virus across donor-recipient pairs to be approximately 100-200 contributing members, which enabled the transmission of multiple lineages, including antigenic variants.

On November 2017, the US Food and Drug Administration (FDA) authorized Memorial Sloan Kettering Cancer Center's (MSKCC) IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets) tumor profiling assay and bioinformatics pipeline for clinical decisions in treating all solid tumors. The IMPACT test uses next-generation sequencing (NGS) to identify the presence of mutations in 468 unique genes, fusions at known sites, copy-number alterations, and microsatellite instability (MSI). A version freeze of the bioinformatics pipeline was made public in May 2017 in Zehir et al. "Mutational Landscape of Metastatic Cancer Revealed from Prospective Clinical Sequencing of 10,000 Patients", Nat Med. 23(6): 703-713. Active development of the pipeline has continued in its CLIA-compliant computing environment, and the resulting improvements have been incorporated into an open-source portable research-grade bioinformatics pipeline. Here, we present results from Roslin, our research bioinformatics pipeline built to be functionally equivalent to the IMPACT clinical reporting workflow that includes manual filtering/annotation and signout by molecular oncologists. Roslin can be installed and executed on laptops, workstations, local compute clusters, or cloud compute servers. As a research-grade pipeline it is flexible enough to handle new assays including exomes/genomes, and extensible enough to add or replace components like reference genomes, sequence aligners, variant callers, false-positive filters, and functional/clinical annotation. Tracking all components and parameters used in each pipeline run allows researchers to easily generate method sections for manuscripts that are sufficiently detailed for peer-review and reproducibility of published results. Roslin has been deployed and tested on multiple high-performance clusters and cloud computing resources. It offers end users GUI driven workflow logging, run reporting, and real-time tracking (https://github.com/mskcc/roslin). Evaluated on NGS data from 128 tumors with signed out IMPACT clinical reports, Roslin achieves a recall rate of 98% for both somatic substitutions and indels, and 60% for fusions. Precision was 96% for substitutions, and 86% for indels. We present our work in maximizing precision and recall rates while retaining the portability, modularity, and reproducibility of Roslin. Citation Format: Zuojian Tang, Nikhil Kumar, Allan Bolipata, Timothy Song, Cyriac Kandoth, Feng Liu, David Mcmanamon, Oliver Hampton, Nicholas Socci, David Solit. A portable bioinformatics pipeline for the FDA authorized IMPACT DNAseq assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2482.

Resistance following antiviral therapy is commonly observed in human influenza viruses. Although this evolutionary process is initiated within individual hosts, little is known about the pattern, dynamics, and drivers of antiviral resistance at this scale, including the role played by reassortment. In addition, the short duration of human influenza virus infections limits the available time window in which to examine intrahost evolution. Using single-molecule sequencing, we mapped, in detail, the mutational spectrum of an H3N2 influenza A virus population sampled from an immunocompromised patient who shed virus over a 21-month period. In this unique natural experiment, we were able to document the complex dynamics underlying the evolution of antiviral resistance. Individual resistance mutations appeared weeks before they became dominant, evolved independently on cocirculating lineages, led to a genome-wide reduction in genetic diversity through a selective sweep, and were placed into new combinations by reassortment. Notably, despite frequent reassortment, phylogenetic analysis also provided evidence for specific patterns of segment linkage, with a strong association between the hemagglutinin (HA)- and matrix (M)-encoding segments that matches that previously observed at the epidemiological scale. In sum, we were able to reveal, for the first time, the complex interaction between multiple evolutionary processes as they occur within an individual host., IMPORTANCE Understanding the evolutionary forces that shape the genetic diversity of influenza virus is crucial for predicting the emergence of drug-resistant strains but remains challenging because multiple processes occur concurrently. We characterized the evolution of antiviral resistance in a single persistent influenza virus infection, representing the first case in which reassortment and the complex patterns of drug resistance emergence and evolution have been determined within an individual host. Deep-sequence data from multiple time points revealed that the evolution of antiviral resistance reflects a combination of frequent mutation, natural selection, and a complex pattern of segment linkage and reassortment. In sum, these data show how immunocompromised hosts may help reveal the drivers of strain emergence.

Mosquito-borne chikungunya virus (CHIKV) is a positive-sense, single-stranded RNA virus from the genus Alphavirus, family Togaviridae, which causes fever, rash and severe persistent polyarthralgia in humans. Since there are currently no FDA licensed vaccines or antiviral therapies for CHIKV, the development of vaccine candidates is of critical importance. Historically, live-attenuated vaccines (LAVs) for protection against arthropod-borne viruses have been created by blind cell culture passage leading to attenuation of disease, while maintaining immunogenicity. Attenuation may occur via multiple mechanisms. However, all examined arbovirus LAVs have in common the acquisition of positively charged amino acid substitutions in cell-surface attachment proteins that render virus infection partially dependent upon heparan sulfate (HS), a ubiquitously expressed sulfated polysaccharide, and appear to attenuate by retarding dissemination of virus particles in vivo. We previously reported that, like other wild-type Old World alphaviruses, CHIKV strain, La Réunion, (CHIKV-LR), does not depend upon HS for infectivity. To deliberately identify CHIKV attachment protein mutations that could be combined with other attenuating processes in a LAV candidate, we passaged CHIKV-LR on evolutionarily divergent cell-types. A panel of single amino acid substitutions was identified in the E2 glycoprotein of passaged virus populations that were predicted to increase electrostatic potential. Each of these substitutions was made in the CHIKV-LR cDNA clone and comparisons of the mutant viruses revealed surface exposure of the mutated residue on the spike and sensitivity to competition with the HS analog, heparin, to be primary correlates of attenuation in vivo. Furthermore, we have identified a mutation at E2 position 79 as a promising candidate for inclusion in a CHIKV LAV., Author Summary With the adaptation of chikungunya virus (CHIKV) to transmission by the Aedes albopictus mosquito, a pandemic has occurred resulting in four to six million human infections, and the virus continues to become endemic in new regions, most recently in the Caribbean. CHIKV can cause debilitating polyarthralgia, lasting for weeks to years, and there are currently no licensed vaccines or antiviral therapies available. While an investigational live-attenuated vaccine (LAV) exists, problems with reactogenicity have precluded its licensure. The purpose of the current study was to: i) devise an in vitro passage procedure that reliably generates a panel of CHIKV envelope glycoprotein mutations for screening as vaccine candidates; ii) determine the position of the mutations in the three-dimensional structure of the alphavirus spike complex and their effect on electrostatic potential; iii) determine the attenuation characteristics of each mutation in a murine model of CHIKV musculoskeletal disease; and iv) to identify in vitro assays examining the dependency of infection upon HS that correlate with attenuation and localization in the glycoprotein spike. This approach provides a paradigm for the rational design of future LAVs for CHIKV and other mosquito-borne viruses, by deliberately selecting and combining attenuating processes.

The increasing number of annotated genome sequences in public databases has made it possible to study the length distributions and domain composition of proteins at unprecedented resolution. To identify factors that influence protein length in metazoans, we performed an analysis of all domain­annotated proteins from a total of 49 animal species from Ensembl (v.56) or EnsemblMetazoa (v.3). Our results indicate that protein length constraints are not fixed as a linear function of domain count and can vary based on domain content. The presence of repeating domains was associated with relaxation of the constraints that govern protein length. Conversely, for proteins with unique domains, length constraints were generally maintained with increased domain counts. It is clear that mean (and median) protein length and domain composition vary significantly between metazoans and other kingdoms; however, the connections between function, domain content, and length are unclear. We incorporated Gene Ontology (GO) annotation to identify biological processes, cellular components, or molecular functions that favor the incorporation of multi­domain proteins. Using this approach, we identified multiple GO terms that favor the incorporation of multi-domain proteins; interestingly, several of the GO terms with elevated domain counts were not restricted to a single gene family. The findings presented here represent an important step in resolving the complex relationship between protein length, function, and domain content. The comparison of the data presented in this work to data from other kingdoms is likely to reveal additional differences in the regulation of protein length.

Gregory Bateson (1972, 1979) established an epistemology that integrates mind and nature as a necessary unity, a unity in which learning and evolution share fundamental principles and in which criteria for mental process are explicitly specified. E42 is a suite of freely available Java applets that constitute an online research lab for creating and interacting with simulations of the Boolean systems developed by Kauffman (1993) in his study of evolution where he proposed that self-organization and natural selection are co-principles "weaving the tapestry of life." This paper maps Boolean systems, developed in the study of evolution, onto Bateson's epistemology in general and onto his criteria of mental process in particular.

CD47 is a signaling receptor for thrombospondin-1 and the counter-receptor for signal-regulatory protein-α (SIRPα). By inducing inhibitory SIRPα signaling, elevated CD47 expression by some cancers prevents macrophage phagocytosis. The anti-human CD47 antibody B6H12 inhibits tumor growth in several xenograft models, presumably by preventing SIRPα engagement. However, CD47 signaling in nontransformed and some malignant cells regulates self-renewal, suggesting that CD47 antibodies may therapeutically target cancer stem cells (CSCs). Treatment of MDA-MB-231 breast CSCs with B6H12 decreased proliferation and asymmetric cell division. Similar effects were observed in T47D CSCs but not in MCF7 breast carcinoma or MCF10A breast epithelial cells. Gene expression analysis in breast CSCs treated with B6H12 showed decreased expression of epidermal growth factor receptor (EGFR) and the stem cell transcription factor KLF4. EGFR and KLF4 mRNAs are known targets of microRNA-7, and B6H12 treatment correspondingly enhanced microRNA-7 expression in breast CSCs. B6H12 treatment also acutely inhibited EGF-induced EGFR tyrosine phosphorylation. Expression of B6H12-responsive genes correlated with CD47 mRNA expression in human breast cancers, suggesting that the CD47 signaling pathways identified in breast CSCs are functional in vivo. These data reveal a novel SIRPα-independent mechanism by which therapeutic CD47 antibodies could control tumor growth by autonomously forcing differentiation of CSC.