Analyses of non-coding somatic drivers in 2,658 cancer whole genomes.

The discovery of drivers of cancer has traditionally focused on protein-coding genes1-4. Here we present analyses of driver point mutations and structural variants in non-coding regions across 2,658 genomes from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium5 of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). For point mutations, we developed a statistically rigorous strategy for combining significance levels from multiple methods of driver discovery that overcomes the limitations of individual methods. For structural variants, we present two methods of driver discovery, and identify regions that are significantly affected by recurrent breakpoints and recurrent somatic juxtapositions. Our analyses confirm previously reported drivers6,7, raise doubts about others and identify novel candidates, including point mutations in the 5' region of TP53, in the 3' untranslated regions of NFKBIZ and TOB1, focal deletions in BRD4 and rearrangements in the loci of AKR1C genes. We show that although point mutations and structural variants that drive cancer are less frequent in non-coding genes and regulatory sequences than in protein-coding genes, additional examples of these drivers will be found as more cancer genomes become available.

Multicenter evaluation of the BIOFIRE Joint Infection Panel for the detection of bacteria, yeast, and AMR genes in synovial fluid samples.

The bioMérieux BIOFIRE Joint Infection (JI) Panel is a multiplex in vitro diagnostic test for the simultaneous and rapid (~1 h) detection of 39 potential pathogens and antimicrobial resistance (AMR) genes directly from synovial fluid (SF) samples. Thirty-one species or groups of microorganisms are included in the kit, as well as several AMR genes. This study, performed to evaluate the BIOFIRE JI Panel for regulatory clearance, provides data from a multicenter evaluation of 1,544 prospectively collected residual SF samples with performance compared to standard-of-care (SOC) culture for organisms or polymerase chain reaction (PCR) and sequencing for AMR genes. The BIOFIRE JI Panel demonstrated a sensitivity of 90.9% or greater for all but six organisms and a positive percent agreement (PPA) of 100% for all AMR genes. The BIOFIRE JI Panel demonstrated a specificity of 98.5% or greater for detection of all organisms and a negative percent agreement (NPA) of 95.7% or greater for all AMR genes. The BIOFIRE JI Panel provides an improvement over SOC culture, with a substantially shorter time to result for both organisms and AMR genes with excellent sensitivity/PPA and specificity/NPA, and is anticipated to provide timely and actionable diagnostic information for joint infections in a variety of clinical scenarios.

Adaptable microfluidic system for single-cell pathogen classification and antimicrobial susceptibility testing.

Infectious diseases caused by bacterial pathogens remain one of the most common causes of morbidity and mortality worldwide. Rapid microbiological analysis is required for prompt treatment of bacterial infections and to facilitate antibiotic stewardship. This study reports an adaptable microfluidic system for rapid pathogen classification and antimicrobial susceptibility testing (AST) at the single-cell level. By incorporating tunable microfluidic valves along with real-time optical detection, bacteria can be trapped and classified according to their physical shape and size for pathogen classification. By monitoring their growth in the presence of antibiotics at the single-cell level, antimicrobial susceptibility of the bacteria can be determined in as little as 30 minutes compared with days required for standard procedures. The microfluidic system is able to detect bacterial pathogens in urine, blood cultures, and whole blood and can analyze polymicrobial samples. We pilot a study of 25 clinical urine samples to demonstrate the clinical applicability of the microfluidic system. The platform demonstrated a sensitivity of 100% and specificity of 83.33% for pathogen classification and achieved 100% concordance for AST.

Simulation-assisted machine learning.

MOTIVATION: In a predictive modeling setting, if sufficient details of the system behavior are known, one can build and use a simulation for making predictions. When sufficient system details are not known, one typically turns to machine learning, which builds a black-box model of the system using a large dataset of input sample features and outputs. We consider a setting which is between these two extremes: some details of the system mechanics are known but not enough for creating simulations that can be used to make high quality predictions. In this context we propose using approximate simulations to build a kernel for use in kernelized machine learning methods, such as support vector machines. The results of multiple simulations (under various uncertainty scenarios) are used to compute similarity measures between every pair of samples: sample pairs are given a high similarity score if they behave similarly under a wide range of simulation parameters. These similarity values, rather than the original high dimensional feature data, are used to build the kernel. RESULTS: We demonstrate and explore the simulation-based kernel (SimKern) concept using four synthetic complex systems-three biologically inspired models and one network flow optimization model. We show that, when the number of training samples is small compared to the number of features, the SimKern approach dominates over no-prior-knowledge methods. This approach should be applicable in all disciplines where predictive models are sought and informative yet approximate simulations are available. AVAILABILITY AND IMPLEMENTATION: The Python SimKern software, the demonstration models (in MATLAB, R), and the datasets are available at https://github.com/davidcraft/SimKern. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Campylobacter culture fails to correctly detect Campylobacter in 30% of positive patient stool specimens compared to non-cultural methods.

Campylobacter diagnosis is hampered because many laboratories continue to use traditional stool culture, which is slow and suffers false-negative results. This large multi-site study used a composite reference method consisting of a new FDA-cleared immunoassay and four molecular techniques to compare to culture. Prospectively collected patient fecal specimens (1552) were first preliminarily categorized as positive or negative by traditional culture. All specimens were also tested by EIA, and any EIA-positive or culture-discrepant results were further characterized by 16S rRNA qPCR, eight species-specific PCR assays, bidirectional sequencing, and an FDA-cleared multiplex PCR panel. The five non-culture methods showed complete agreement on all positive and discrepant specimens which were then assigned as true-positive or true-negative specimens. Among 47 true-positive specimens, culture incorrectly identified 13 (28%) as negative, and 1 true-negative specimen as positive, for a sensitivity of 72.3%. Unexpectedly, among the true-positive specimens, 4 (8%) were the pathogenic species C. upsaliensis. Culture had a 30% false result rate compared to immunoassay and molecular methods. More accurate results lead to better diagnosis and treatment of suspected campylobacteriosis.

Nanotube assisted microwave electroporation for single cell pathogen identification and antimicrobial susceptibility testing.

A nanotube assisted microwave electroporation (NAME) technique is demonstrated for delivering molecular biosensors into viable bacteria for multiplex single cell pathogen identification to advance rapid diagnostics in clinical microbiology. Due to the small volume of a bacterial cell (~femtoliter), the intracellular concentration of the target molecule is high, which results in a strong signal for single cell detection without amplification. The NAME procedure can be completed in as little as 30 minutes and can achieve over 90% transformation efficiency. We demonstrate the feasibility of NAME for identifying clinical isolates of bloodborne and uropathogenic pathogens and detecting bacterial pathogens directly from patient's samples. In conjunction with a microfluidic single cell trapping technique, NAME allows single cell pathogen identification and antimicrobial susceptibility testing concurrently. Using this approach, the time for microbiological analysis reduces from days to hours, which will have a significant impact on the clinical management of bacterial infections.

The value of prior knowledge in machine learning of complex network systems.

MOTIVATION: Our overall goal is to develop machine-learning approaches based on genomics and other relevant accessible information for use in predicting how a patient will respond to a given proposed drug or treatment. Given the complexity of this problem, we begin by developing, testing and analyzing learning methods using data from simulated systems, which allows us access to a known ground truth. We examine the benefits of using prior system knowledge and investigate how learning accuracy depends on various system parameters as well as the amount of training data available. RESULTS: The simulations are based on Boolean networks-directed graphs with 0/1 node states and logical node update rules-which are the simplest computational systems that can mimic the dynamic behavior of cellular systems. Boolean networks can be generated and simulated at scale, have complex yet cyclical dynamics and as such provide a useful framework for developing machine-learning algorithms for modular and hierarchical networks such as biological systems in general and cancer in particular. We demonstrate that utilizing prior knowledge (in the form of network connectivity information), without detailed state equations, greatly increases the power of machine-learning algorithms to predict network steady-state node values ('phenotypes') and perturbation responses ('drug effects'). AVAILABILITY AND IMPLEMENTATION: Links to codes and datasets here: https://gray.mgh.harvard.edu/people-directory/71-david-craft-phd. CONTACT: dcraft@broadinstitute.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Antibiotic-tolerant Staphylococcus aureus Biofilm Persists on Arthroplasty Materials.

BACKGROUND: The continued presence of biofilm may be one cause of the high risk of failure observed with irrigation and débridement with component retention in acute periprosthetic joint infection (PJI). There is a poor understanding of the role of biofilm antibiotic tolerance in PJI. QUESTIONS/PURPOSES: (1) Do increasing doses of cefazolin result in decreased viable biofilm mass on arthroplasty materials? (2) Is cefazolin resistance phenotypic or genotypic? (3) Is biofilm viability a function of biofilm depth after treatment with cefazolin? (4) Is the toxin-antitoxin system, yoeB expression, associated with antibiotic stress? METHODS: Methicillin-sensitive Staphylococcus aureus biofilm was cultured on total knee arthroplasty (TKA) materials and exposed to increasing doses of cefazolin (control, 0.5, 1.0, 10.0, 100.0 µg/mL). Quantitative confocal microscopy and quantitative culture were used to measure viable biofilm cell density. To determine if cefazolin resistance was phenotypic or genotypic, we measured minimum inhibitory concentration (MIC) after exposure to different cefazolin concentrations; changes in MIC would suggest genotypic features, whereas unchanged MIC would suggest phenotypic behavior. Finally, quantitative reverse transcription-polymerase chain reaction was used to quantify expression of yoeB levels between biofilm and planktonic bacteria after exposure to 1 µg/mL cefazolin for 3 hours. RESULTS: Although live biofilm mass was reduced by exposure to cefazolin when compared with biofilm mass in controls (39.2 × 10(3) ± 26.4 × 10(3) pixels), where the level after 0.5 µg/mL exposure also showed reduced mass (20.3 × 10(3) ± 11.9 × 10(3) pixels), no further reduction was seen after higher doses (mass at 1.0 µg/mL: 5.0 × 10(3) pixels ± 1.1 × 10(3) pixels; at 10.0 µg/mL: 6.4 × 10(3) ± 9.6 × 10(3) pixels; at 100.0 µg/mL: 6.4 × 10(3) ± 3.9 × 10(3)). At the highest concentration tested (100 µg/mL), residual viable biofilm was present on all three materials, and there were no differences in percent biofilm survival among cobalt-chromium (18.5% ± 15.1%), polymethylmethacrylate (22.8% ± 20.2%), and polyethylene (14.7% ± 10.4%). We found that tolerance was a phenotypic phenomenon, because increasing cefazolin exposure did not result in changes in MIC as compared with controls (MIC in controls: 0.13 ± 0.02; at 0.5 µg/mL: 0.13 ± 0.001, p = 0.96; at 1.0 µg/m: 0.14 ± 0.04, p = 0.95; at 10.0 µg/m: 0.11 ± 0.016, p = 0.47; at 100.0 µg/m: 0.94 ± 0.047, p = 0.47). Expression of yoeB after 1 µg/mL cefazolin for 3 hours in biofilm cells was greater in biofilm but not in planktonic cells (biofilm: 62.3-fold change, planktonic cells: -78.8-fold change, p < 0.001). CONCLUSIONS: Antibiotics are inadequate at complete removal of the biofilm from the surface of TKA materials. Results suggest that bacterial persisters are responsible for this phenotypic behavior allowing biofilm high tolerance to antibiotics. CLINICAL RELEVANCE: Antibiotic-tolerant biofilm suggests a mechanism behind the poor results in irrigation and débridement for acute TKA PJI.

Volumetric-modulated arc therapy using multicriteria optimization for body and extremity sarcoma.

This study evaluates the implementation of volumetric-modulated arc therapy (VMAT) using multicriteria optimization (MCO) in the RayStation treatment planning system (TPS) for complex sites, namely extremity and body sarcoma. The VMAT-MCO algorithm implemented in RayStation is newly developed and requires an integrated, comprehensive analysis of plan generation, delivery, and treatment efficiency. Ten patients previously treated by intensity-modulated radiation therapy (IMRT) with MCO were randomly selected and replanned using VMAT-MCO. The plan quality was compared using homogeneity index (HI) and conformity index (CI) of the planning target volume (PTV) and dose sparing of organs at risk (OARs). Given the diversity of the tumor location, the 10 plans did not have a common OAR except for skin. The skin D50 and Dmean was directly compared between VMAT-MCO and IMRT-MCO. Additional OAR dose points were compared on a plan-by-plan basis. The treatment efficiency was compared using plan monitor units (MU) and net beam-on time. Plan quality assurance was performed using the Sun Nuclear ArcCHECK phantom and a gamma criteria of 3%/3 mm. No statistically significant differences were found between VMAT- and IMRT-MCO for HI and CI of the PTV or D50 and Dmean to the skin. The VMAT-MCO plans showed general improvements in sparing to OARs. The VMAT-MCO plan set showed statistically significant improvements over the IMRT-MCO set in treatment efficiency per plan MU (p < 0.05) and net beam-on time (p < 0.01). The VMAT-MCO plan deliverability was validated. Similar gamma passing rates were observed for the two modalities. This study verifies the suitability of VMAT-MCO for sarcoma cancer and highlighted the comparability in plan quality and improve-ment in treatment efficiency offered by VMAT-MCO as compared to IMRT-MCO.

Antimicrobial resistance determinants in Acinetobacter baumannii isolates taken from military treatment facilities.

Multidrug-resistant (MDR) Acinetobacter baumannii infections are of particular concern within medical treatment facilities, yet the gene assemblages that give rise to this phenotype remain poorly characterized. In this study, we tested 97 clinical A. baumannii isolates collected from military treatment facilities (MTFs) from 2003 to 2009 by using a molecular epidemiological approach that enabled for the simultaneous screening of 236 antimicrobial resistance genes. Overall, 80% of the isolates were found to be MDR, each strain harbored between one and 17 resistant determinants, and a total of 52 unique resistance determinants or gene families were detected which are known to confer resistance to ß-lactam (e.g., blaGES-11, blaTEM, blaOXA-58), aminoglycoside (e.g., aphA1, aacC1, armA), macrolide (msrA, msrB), tetracycline [e.g., tet(A), tet(B), tet(39)], phenicol (e.g., cmlA4, catA1, cat4), quaternary amine (qacE, qacEΔ1), streptothricin (sat2), sulfonamide (sul1, sul2), and diaminopyrimidine (dfrA1, dfrA7, dfrA19) antimicrobial compounds. Importantly, 91% of the isolates harbored blaOXA-51-like carbapenemase genes (including six new variants), 40% harbored the blaOXA-23 carbapenemase gene, and 89% contained a variety of aminoglycoside resistance determinants with up to six unique determinants identified per strain. Many of the resistance determinants were found in potentially mobile gene cassettes; 45% and 7% of the isolates contained class 1 and class 2 integrons, respectively. Combined, the results demonstrate a facile approach that supports a more complete understanding of the genetic underpinnings of antimicrobial resistance to better assess the load, transmission, and evolution of MDR in MTF-associated A. baumannii.

Bioterrorism: a laboratory who does it?

In October 2001, the first disseminated biological warfare attack was perpetrated on American soil. Initially, a few clinical microbiology laboratories were testing specimens from acutely ill patients and also being asked to test nasal swabs from the potentially exposed. Soon after, a significant number of clinical microbiology and public health laboratories received similar requests to test the worried well or evaluate potentially contaminated mail or environmental materials, sometimes from their own break rooms. The role of the clinical and public health microbiology laboratory in response to a select agent event or act of bioterrorism is reviewed.

Pulse lavage is inadequate at removal of biofilm from the surface of total knee arthroplasty materials.

In acute periprosthetic infection, irrigation and debridement with component retention has a high failure rate in some studies. We hypothesize that pulse lavage irrigation is ineffective at removing biofilm from total knee arthroplasty (TKA) components. Staphylococcus aureus biofilm mass and location was directly visualized on arthroplasty materials with a photon collection camera and laser scanning confocal microscopy. There was a substantial reduction in biofilm signal intensity, but the reduction was less than a ten-fold decrease. This suggests that irrigation needs to be further improved for the removal of biofilm mass below the necessary bioburden level to prevent recurrence of acute infection in total knee arthroplasty.

Genome sequences of four divergent multidrug-resistant Acinetobacter baumannii strains isolated from patients with sepsis or osteomyelitis.

Acinetobacter baumannii is a Gram-negative bacterium that causes nosocomial infections worldwide, with recent prevalence and higher frequency in wounded military personnel. Four A. baumannii strains from the Walter Reed Army Medical Center (WRAMC) isolated between 2008 and 2009 were sequenced, representing diverse, multidrug-resistant isolates from osteomyelitis or septic patients.

Antibacterial activities of iron chelators against common nosocomial pathogens.

The activities of iron chelators (deferoxamine, deferiprone, Apo6619, and VK28) were evaluated against type strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli. Deferiprone, Apo6619, and VK28 each inhibited growth in standard and RPMI tissue culture medium, while deferoxamine had no effect. Additionally, time-kill assays revealed that VK28 had a bacteriostatic effect against S. aureus. Therefore, these newly developed iron chelators might provide a nontraditional approach for treatment of bacterial infections.

Multicriteria VMAT optimization.

PURPOSE: To make the planning of volumetric modulated arc therapy (VMAT) faster and to explore the tradeoffs between planning objectives and delivery efficiency. METHODS: A convex multicriteria dose optimization problem is solved for an angular grid of 180 equi-spaced beams. This allows the planner to navigate the ideal dose distribution Pareto surface and select a plan of desired target coverage versus organ at risk sparing. The selected plan is then made VMAT deliverable by a fluence map merging and sequencing algorithm, which combines neighboring fluence maps based on a similarity score and then delivers the merged maps together, simplifying delivery. Successive merges are made as long as the dose distribution quality is maintained. The complete algorithm is called VMERGE. RESULTS: VMERGE is applied to three cases: a prostate, a pancreas, and a brain. In each case, the selected Pareto-optimal plan is matched almost exactly with the VMAT merging routine, resulting in a high quality plan delivered with a single arc in less than 5 min on average. CONCLUSIONS: VMERGE offers significant improvements over existing VMAT algorithms. The first is the multicriteria planning aspect, which greatly speeds up planning time and allows the user to select the plan, which represents the most desirable compromise between target coverage and organ at risk sparing. The second is the user-chosen epsilon-optimality guarantee of the final VMAT plan. Finally, the user can explore the tradeoff between delivery time and plan quality, which is a fundamental aspect of VMAT that cannot be easily investigated with current commercial planning systems.

Probabilistic definition of the clinical target volume-implications for tumor control probability modeling and optimization.

Evidence has been presented that moving beyond the binary definition of clinical target volume (CTV) towards a probabilistic CTV can result in better treatment plans. The probabilistic CTV takes the likelihood of disease spread outside of the gross tumor into account. An open question is: how to optimize tumor control probability (TCP) based on the probabilistic CTV. We derive expressions for TCP under the assumptions of voxel independence and dependence. For the dependent case, we make the assumption that tumors grow outward from the gross tumor volume. We maximize the (non-convex) TCP under convex dose constraints for all models. For small numbers of voxels, and when a dose-influence matrix is not used, we use exhaustive search or Lagrange multiplier theory to compute optimal dose distributions. For larger cases we present (1) a multi-start strategy using linear programming with a random cost vector to provide random feasible starting solutions, followed by a local search, and (2) a heuristic strategy that greedily selects which subvolumes to dose, and then for each subvolume assignment runs a convex approximation of the optimization problem. The optimal dose distributions are in general different for the independent and dependent models even though the probabilities of each voxel being tumorous are set to the same in both cases. We observe phase transitions, where a subvolume is either dosed to a high level, or it gets 'sacrificed' by not dosing it at all. The greedy strategy often yields solutions indistinguishable from the multi-start solutions, but for the 2D case involving organs-at-risk and the dependent TCP model, discrepancies of around 5% (absolute) for TCP are observed. For realistic geometries, although correlated voxels is a more reasonable assumption, the correlation function is in general unknown. We demonstrate a tractable heuristic that works very well for the independent models and reasonably well for the dependent models. All data are provided.

Genetic Modification of Sodalis Species by DNA Transduction.

Bacteriophages (phages) are ubiquitous in nature. These viruses play a number of central roles in microbial ecology and evolution by, for instance, promoting horizontal gene transfer (HGT) among bacterial species. The ability of phages to mediate HGT through transduction has been widely exploited as an experimental tool for the genetic study of bacteria. As such, bacteriophage P1 represents a prototypical generalized transducing phage with a broad host range that has been extensively employed in the genetic manipulation of Escherichia coli and a number of other model bacterial species. Here we demonstrate that P1 is capable of infecting, lysogenizing, and promoting transduction in members of the bacterial genus Sodalis, including the maternally inherited insect endosymbiont Sodalis glossinidius While establishing new tools for the genetic study of these bacterial species, our results suggest that P1 may be used to deliver DNA to many Gram-negative endosymbionts in their insect host, thereby circumventing a culturing requirement to genetically manipulate these organisms.IMPORTANCE A large number of economically important insects maintain intimate associations with maternally inherited endosymbiotic bacteria. Due to the inherent nature of these associations, insect endosymbionts cannot be usually isolated in pure culture or genetically manipulated. Here we use a broad-host-range bacteriophage to deliver exogenous DNA to an insect endosymbiont and a closely related free-living species. Our results suggest that broad-host-range bacteriophages can be used to genetically alter insect endosymbionts in their insect host and, as a result, bypass a culturing requirement to genetically alter these bacteria.

Targeting the spliceosome through RBM39 degradation results in exceptional responses in high-risk neuroblastoma models.

Aberrant alternative pre-mRNA splicing plays a critical role in MYC-driven cancers and therefore may represent a therapeutic vulnerability. Here, we show that neuroblastoma, a MYC-driven cancer characterized by splicing dysregulation and spliceosomal dependency, requires the splicing factor RBM39 for survival. Indisulam, a "molecular glue" that selectively recruits RBM39 to the CRL4-DCAF15 E3 ubiquitin ligase for proteasomal degradation, is highly efficacious against neuroblastoma, leading to significant responses in multiple high-risk disease models, without overt toxicity. Genetic depletion or indisulam-mediated degradation of RBM39 induces significant genome-wide splicing anomalies and cell death. Mechanistically, the dependency on RBM39 and high-level expression of DCAF15 determine the exquisite sensitivity of neuroblastoma to indisulam. Our data indicate that targeting the dysregulated spliceosome by precisely inhibiting RBM39, a vulnerability in neuroblastoma, is a valid therapeutic strategy.

Simultaneous navigation of multiple Pareto surfaces, with an application to multicriteria IMRT planning with multiple beam angle configurations.

PURPOSE: To introduce a method to simultaneously explore a collection of Pareto surfaces. The method will allow radiotherapy treatment planners to interactively explore treatment plans for different beam angle configurations as well as different treatment modalities. METHODS: The authors assume a convex optimization setting and represent the Pareto surface for each modality or given beam set by a set of discrete points on the surface. Weighted averages of these discrete points produce a continuous representation of each Pareto surface. The authors calculate a set of Pareto surfaces and use linear programming to navigate across the individual surfaces, allowing switches between surfaces. The switches are organized such that the plan profits in the requested way, while trying to keep the change in dose as small as possible. RESULTS: The system is demonstrated on a phantom pancreas IMRT case using 100 different five beam configurations and a multicriteria formulation with six objectives. The system has intuitive behavior and is easy to control. Also, because the underlying linear programs are small, the system is fast enough to offer real-time exploration for the Pareto surfaces of the given beam configurations. CONCLUSIONS: The system presented offers a sound starting point for building clinical systems for multicriteria exploration of different modalities and offers a controllable way to explore hundreds of beam angle configurations in IMRT planning, allowing the users to focus their attention on the dose distribution and treatment planning objectives instead of spending excessive time on the technicalities of delivery.