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Burkitt lymphoma (BL) accounts for most pediatric non-Hodgkin lymphomas, being less common but significantly more lethal when diagnosed in adults. Much of the knowledge of the genetics of BL thus far has originated from the study of pediatric BL (pBL), leaving its relationship to adult BL (aBL) and other adult lymphomas not fully explored. We sought to more thoroughly identify the somatic changes that underlie lymphomagenesis in aBL and any molecular features that associate with clinical disparities within and between pBL and aBL. Through comprehensive whole-genome sequencing of 230 BL and 295 diffuse large B-cell lymphoma (DLBCL) tumors, we identified additional significantly mutated genes, including more genetic features that associate with tumor Epstein-Barr virus status, and unraveled new distinct subgroupings within BL and DLBCL with 3 predominantly comprising BLs: DGG-BL (DDX3X, GNA13, and GNAI2), IC-BL (ID3 and CCND3), and Q53-BL (quiet TP53). Each BL subgroup is characterized by combinations of common driver and noncoding mutations caused by aberrant somatic hypermutation. The largest subgroups of BL cases, IC-BL and DGG-BL, are further characterized by distinct biological and gene expression differences. IC-BL and DGG-BL and their prototypical genetic features (ID3 and TP53) had significant associations with patient outcomes that were different among aBL and pBL cohorts. These findings highlight shared pathogenesis between aBL and pBL, and establish genetic subtypes within BL that serve to delineate tumors with distinct molecular features, providing a new framework for epidemiologic, diagnostic, and therapeutic strategies.

Burkitt lymphoma (BL) accounts for the majority of pediatric non-Hodgkin lymphomas (NHL) and is relatively rare but significantly more lethal when diagnosed in adults. The global incidence is highest in Sub-Saharan Africa, where Epstein-Barr virus (EBV) positivity is observed in 95% of all tumors. Both pediatric (pBL) and adult (aBL) cases are known to share some driver mutations, for example MYC translocations, which are seen in > 90% of cases. Sequencing efforts have identified many common somatic alterations that cooperate with MYC in lymphomagenesis with approximately 30 significantly mutated genes (SMG) reported thus far. Recent analyses revealed non-coding mutation patterns in pBL that were attributed to aberrant somatic hypermutation (aSHM). We sought to identify genomic and molecular features that may explain clinical disparities within and between aBL and pBL in an effort to delineate BL subtypes that may allow for the stratification of patients with shared pathobiology. Through comprehensive sequencing of BL genomes, we found additional SMGs, including more genetic features that associate with tumor EBV status, and established three new genetic subgroups that span pBL and aBL. Direct comparisons between pBL and aBL revealed only marginal differences and the mutational profiles were consistently better explained by EBV status. Using an unsupervised clustering approach to identify subgroupings within BL and diffuse large B-cell lymphoma (DLBCL), we have defined three genetic subgroups that predominantly comprise BL tumors. Akin to the recently defined DLBCL subgroups, each BL subgroup is characterized by combinations of common driver mutations and non-coding mutations caused by aSHM. Two of these subgroups and their prototypical genetic features (ID3 and TP53) had significant associations with patient outcomes that were different among the aBL and pBL cohorts. These findings highlight not only a shared pathogenesis between aBL and pBL, but also establish genetic subtypes within BL that serve to delineate tumors with distinct molecular features, providing a new framework for epidemiological studies, and diagnostic and therapeutic strategies.

Cervical cancer is the most common cancer affecting sub-Saharan African women and is prevalent among HIV-positive (HIV+) individuals. No comprehensive profiling of cancer genomes, transcriptomes or epigenomes has been performed in this population thus far. We characterized 118 tumors from Ugandan patients, of whom 72 were HIV+, and performed extended mutation analysis on an additional 89 tumors. We detected human papillomavirus (HPV)-clade-specific differences in tumor DNA methylation, promoter- and enhancer-associated histone marks, gene expression and pathway dysregulation. Changes in histone modification at HPV integration events were correlated with upregulation of nearby genes and endogenous retroviruses.

Introduction: Burkitt lymphoma (BL) accounts for approximately 50% of all pediatric non-Hodgkin lymphomas compared to 1-2% in adults. Adult BL (aBL) remains a poorly understood entity and its relationship to pediatric BL (pBL) and to DLBCL has not been fully elucidated. The variable treatment outcomes between these entities necessitate a more thorough understanding of the genetic and molecular features underlying their biology to enable better prognostication and more effective treatments. We sought to comprehensively determine genetic features shared with DLBCL and those that are unique to BL, to further delineate genetic subgroupings within each entity. Methods: Samples for this study were collected through the Burkitt Lymphoma Genome Sequencing Project (BLGSP). We sequenced the tumor genomes of 139 pBL and 92 aBL, consisting of both EBV-positive (EBV+) and EBV-negative (EBV-) BLs, and compared these to the genomes of 252 DLBCL patients. All cases were analyzed for simple somatic mutations (SSM), recurrent copy number variations (CNV), structural variations (SV), aberrant somatic hypermutation (aSHM), and SSM hotspots. Mutations were used as features for the identification of genetic subgroups using non-negative matrix factorization (NMF) clustering. Results: Clustering of BL and DLBCL revealed six distinct genetic subgroups (Figure 1) with three primarily representing DLBCLs (DLBCL-1, DLBCL-2, and DLBCL-3) and three predominantly comprising BLs (M53-BL, IC-BL, and DGG-BL). The DLBCL-predominant subgroups partially overlapped with those previously described and resembled features of EZB and ST2-like subgroups. The frequency of aBLs within these subgroups was higher than that of pBL patients (p=0.0005). The new cluster M53-BL consists of both pBL (9/27) and aBL (13/27) samples and is characterized by the highest prevalence of mutations in TP53 accompanied by the paucity of other driver mutations but without the aneuploidy associated with the A53 subgroup described in DLBCL. Enrichment of EBV- samples in this cluster further corroborate our previous findings of TP53 mutations being associated with EBV- BL. IC-BL is characterized by mutations in ID3, CCND3, and SMARCA4. In contrast, DGG-BL, where 65% of the cluster consisted of EBV+ BL samples, had mutations in DDX3X, GNA13, and GNAI2. Using a linear model, we compared the rates of aSHM in BL genomes from all clusters and identified the DLBCL-3 cluster to harbor the highest aSHM rates at common sites while the M53-BL cluster harbored the lowest rates. To further establish the biological basis of unique clusters within BL, we conducted differential gene expression analyses between the two major BL genetic subgroups, DGG-BL and IC-BL. We identified a total of 86 differentially expressed genes between the two clusters (p.adj < 0.01 and |log2foldChange| > 1). Among the genes with the strongest differential expression were IRF4, SERPINA9, and TNFRSF13B. Each of these are notable as their expression is a component of the DLBCL cell-of-origin and double-hit signature classifiers. Further, we found IRF4 expression to be one of the strongest predictors of cluster membership, with high IRF4 expression associated with IC-BL membership. Using TP53 and ID3 mutations as a proxy for M53-BL and IC-BL clusters in aBL, we found mutations in TP53 to be associated with significantly inferior progression free survival (PFS) at 2 year follow up, while mutations in ID3 were associated with overall better PFS at 2 year follow up. Conclusion: This work identifies novel genetic subgroups within BL with characteristic genetic and gene expression differences and some bearing relationship to DLBCL subgroups. The three subgroups with predominantly BL samples (DGG-BL, IC-BL, and M53-BL) each comprised a mixture of aBL and pBL samples, confirming similar molecular features in these entities. The IC-BL cluster is associated with mutations in ID3 and CCND3, high IRF4 expression, and ID3 mutated cases exhibited significantly better outcomes. M53-BL is associated with TP53 mutations and inferior PFS in aBL, representing a subset of patients to be considered for novel treatment approaches. These findings highlight shared pathogenesis between aBL and pBL and establish genetic subtypes within BL that delineate cases with distinct molecular and clinical features. This provides a new framework for new diagnostic and therapeutic strategies. Figure 1 Figure 1. Disclosures Abramson: Seagen Inc.: Research Funding; Allogene Therapeutics: Consultancy; Astra-Zeneca: Consultancy; Incyte Corporation: Consultancy; BeiGene: Consultancy; Kymera: Consultancy; Kite Pharma: Consultancy; Novartis: Consultancy; Bluebird Bio: Consultancy; C4 Therapeutics: Consultancy; Morphosys: Consultancy; Genmab: Consultancy; EMD Serono: Consultancy; Bristol-Myers Squibb Company: Consultancy, Research Funding; AbbVie: Consultancy; Karyopharm: Consultancy; Genentech: Consultancy. Bartlett: Pharmacyclics: Research Funding; Millennium: Research Funding; Merck: Research Funding; Kite, a Gilead Company: Research Funding; Janssen: Research Funding; Genentech: Research Funding; Forty Seven: Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Research Funding; Autolus: Research Funding; Seagen: Consultancy, Research Funding; Roche/Genentech: Consultancy; ADC Therapeutics: Consultancy, Research Funding. Casper: EUSA Pharma: Consultancy. Gerrie: Roche: Research Funding; AbbVie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Astrazeneca: Honoraria, Research Funding; Sandoz: Honoraria. Grande: Sage Bionetworks: Current Employment. Mullighan: Illumina: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company. Noy: Epizyme: Consultancy; Rafael Parhma: Research Funding; Morphosys: Consultancy; Targeted Oncology: Consultancy; Medscape: Consultancy; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Honoraria. Scott: NanoString Technologies: Patents & Royalties: Patent describing measuring the proliferation signature in MCL using gene expression profiling.; AstraZeneca: Consultancy; Abbvie: Consultancy; Celgene: Consultancy; Incyte: Consultancy; Janssen: Consultancy, Research Funding; Rich/Genentech: Research Funding; BC Cancer: Patents & Royalties: Patent describing assigning DLBCL COO by gene expression profiling--licensed to NanoString Technologies. Patent describing measuring the proliferation signature in MCL using gene expression profiling. . Morin: Foundation for Burkitt Lymphoma Research: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Epizyme: Patents & Royalties.

Although generally curable with intensive chemotherapy in resource-rich settings, Burkitt lymphoma (BL) remains a deadly disease in older patients and in sub-Saharan Africa. Epstein-Barr virus (EBV) positivity is a feature in more than 90% of cases in malaria-endemic regions, and up to 30% elsewhere. However, the molecular features of BL have not been comprehensively evaluated when taking into account tumor EBV status or geographic origin. Through an integrative analysis of whole-genome and transcriptome data, we show a striking genome-wide increase in aberrant somatic hypermutation in EBV-positive tumors, supporting a link between EBV and activation-induced cytidine deaminase (AICDA) activity. In addition to identifying novel candidate BL genes such as SIN3A, USP7, and CHD8, we demonstrate that EBV-positive tumors had significantly fewer driver mutations, especially among genes with roles in apoptosis. We also found immunoglobulin variable region genes that were disproportionally used to encode clonal B-cell receptors (BCRs) in the tumors. These include IGHV4-34, known to produce autoreactive antibodies, and IGKV3-20, a feature described in other B-cell malignancies but not yet in BL. Our results suggest that tumor EBV status defines a specific BL phenotype irrespective of geographic origin, with particular molecular properties and distinct pathogenic mechanisms. The novel mutation patterns identified here imply rational use of DNA-damaging chemotherapy in some patients with BL and targeted agents such as the CDK4/6 inhibitor palbociclib in others, whereas the importance of BCR signaling in BL strengthens the potential benefit of inhibitors for PI3K, Syk, and Src family kinases among these patients.

Hyperbranching poly(allylamine) (PAAm) and poly(ethylene glycol) (PEG) on silicon and its effect on protein adhesion was investigated. Hyperbranching involves sequential grafting of polymers on a surface with one of the components having multiple reactive sites. In this research, PAAm provided multiple amines for grafting PEG diacrylate. Current methodologies for generating PEG surfaces include PEG-silane monolayers or polymerized PEG networks. Hyperbranching combines the nanoscale thickness of monolayers with the surface coverage afforded by polymerization. A multistep approach was used to generate the silicon-supported hyperbranched polymers. The silicon wafer surface was initially modified with a vinyl silane followed by oxidation of the terminal vinyl group to present an acid function. Carbodiimide activation of the surface carboxyl group allowed for coupling to PAAm amines to form the first polymer layer. The polymers were hyperbranched by grafting alternating PEG and PAAm layers to the surface using Michael addition chemistry. The alternating polymers were grafted up to six total layers. The substrates remained hydrophilic after each modification. Static contact angles for PAAm (32-44 degrees) and PEG (33-37 degrees) were characteristic of the corresponding individual polymer (30-50 degrees for allylamine, 34-42 degrees for PEG). Roughness values varied from approximately 1 to 8 nm, but had no apparent affect on protein adhesion. Modifications terminating with a PEG layer reduced bovine serum albumin adhesion to the surface by approximately 80% as determined by ELISA and radiolabel binding studies. The hyperbranched PAAm and PEG surfaces described in this paper are nanometer-scale, multilayer films capable of reducing protein adhesion.

With recent advances in surface chemistry, microfluidics, and data analysis, there are ever increasing reports of array-based methods for detecting and quantifying multiple targets. However, only a few systems have been described that require minimal preparation of complex samples and possess a means of quantitatively assessing matrix effects. The NRL Array Biosensor has been developed with the goal of rapid and sensitive detection of multiple targets from multiple samples analyzed simultaneously. A key characteristic of this system is its two-dimensional configuration, which allows controls and standards to be analyzed in parallel with unknowns. Although the majority of our work has focused on instrument automation and immunoassay development, we have recently initiated efforts to utilize alternative recognition molecules, such as peptides and sugars, for detection of a wider variety of targets. The array biosensor has demonstrated utility for a variety of applications, including food safety, disease diagnosis, monitoring immune response, and homeland security, and is presently being transitioned to the commercial sector for manufacturing.

Botulinum neurotoxin serotype A (BoNT/A), one of seven serotypes of botulinum neurotoxin, is taken up by neurons of the peripheral nervous system. Within the neurons it catalyzes cleavage of the synaptosomal-associated protein having a mass of 25kDa, SNAP-25, thereby blocking neurotransmission. BoNT/A has been shown to interact with SV2, as well as gangliosides that are often found in lipid rafts. Lipid rafts are microdomains that can be found on the outer leaflet of the plasma membrane and are enriched in cholesterol and glycosphingolipids. To determine whether lipid rafts are needed for BoNT/A activity, those associated with the plasma membranes of murine N2a neuroblastoma cells were disrupted using either methyl-beta-cyclodextrin or filipin. Disruption of cholesterol-containing lipid rafts by either reagent did not prevent the action of BoNT/A on N2a cells, in fact activity was enhanced. While our results indicate that disruption of lipid rafts enhances BoNT/A activity, disruption of clathrin-dependent endocytosis appeared to be inhibitory.

The research described here investigates the hypothesis that nanoarchitecture contained in a nanowire array is capable of attenuating the adverse host response generated when medical devices are implanted in the body. This adverse host response, or biofouling, generates an avascular fibrous mass transfer barrier between the device and the analyte of interest, disabling the implant if it is a sensor. Numerous studies have indicated that surface chemistry and architecture modulate the host response. These findings led us to hypothesize that nanostructured surfaces will inhibit the formation of an avascular fibrous capsule significantly. We are investigating whether arrays of oscillating magnetostrictive nanowires can prevent protein adsorption. Magnetostrictive nanowires were fabricated by electroplating a ferromagnetic metal alloy into the pores of a nanoporous alumina template. The ferromagnetic nanowires are made to oscillate by oscillating the magnetic field surrounding the wires. Radiolabeled bovine serum albumin, enzyme-linked immunosorbent assay (ELISA), and other protein assays were used to study protein adhesion on the nanowire arrays. These results display a reduced protein adsorption per surface area of static nanowires. Comparing the surfaces, 14-30% of the protein that absorbed on the flat surface adsorbed on the nanowires. Our contact angle measurements indicate that the attenuation of protein on the nanowire surface might be due to the increased hydrophilicity of the nanostructured surface compared to a flat surface of the same material. We oscillated the magnetostrictive wires by placing them in a 38 G 10 Hz oscillating magnetic field. The oscillating nanowires show a further reduction in protein adhesion where only 7-67% of the protein on the static wires was measured on the oscillating nanowires. By varying the viscosity of the fluid the nanowires are oscillated in, we determined that protein detachment is shear-stress modulated. We created a high shearing fluid with dextran, which reduced protein adsorption on the oscillating nanowires by 70% over nanowires oscillating in baseline viscosity fluid. Our preliminary studies strongly suggest that the architecture in the static nanowire arrays and the shear created by oscillating the nanowire arrays would attenuate the biofouling response in vivo.

Hanson Technologies has developed the automated OmniFresh 1000 system to sample large volumes of produce wash water, collect the pathogens, and detect their presence. By collecting a continuous sidestream of wash water, the OmniFresh uses a sample that represent the entire lot of produce being washed. The OmniFresh does not require bacterial culture or enrichment, and it detects both live and dead bacteria in the collected sample using an in-line sensor. Detection occurs in an array biosensor capable of handling large samples with complex matrices. Additionally, sample can be sent for traditional confirming tests after the screening performed by the OmniFresh.

The research described here investigates the hypothesis that nanoarchitecture contained in a nanowire array is capable of attenuating the adverse host response, biofouling, generated when medical devices, such as sensors, are implanted in the body. This adverse host response generates an avascular fibrous mass transfer barrier between the device and the analyte of interest, disabling the sensor. Numerous studies have indicated that surface chemistry and architecture modulated the host response. These findings lead us to hypothesize that nanostructured surfaces will significantly inhibit the formation of an avascular fibrous capsule. We are investigating whether vibrating magnetostrictive nanowires, formed in nanowire arrays, can prevent protein and cell adhesion. Magnetostrictive nanowires are fabricated by electroplating a ferromagnetic metal alloy into the pores of a nanoporous alumina template. The ferromagnetic nanowires are made to vibrate by altering the magnetic field surrounding the wires. Enzyme-linked immunosorbent assay (ELISA) and other protein assays were used to study protein adhesion on the nanowire arrays. These results display a reduced protein adhesion per surface area of static nanowires. The vibrating nanowires show a further reduction in protein adhesion, compared to static wires. Studies were also preformed to investigate the effects of nanoarchitecture have on cell adhesion. These studies were performed with both static and vibrating nanowires. Preliminary protein adhesion studies have shown that a nanowire arrays modulate protein adhesion in vitro.