Jason R. Banfelder

Whole-Exome Sequencing of Metastatic Cancer and Biomarkers of Treatment Response

IMPORTANCEnUnderstanding molecular mechanisms of response and resistance to anticancer therapies requires prospective patient follow-up and clinical and functional validation of both common and low-frequency mutations. We describe a whole-exome sequencing (WES) precision medicine trial focused on patients with advanced cancer.nnnOBJECTIVEnTo understand how WES data affect therapeutic decision making in patients with advanced cancer and to identify novel biomarkers of response.nnnDESIGN, SETTING, AND PATIENTSnPatients with metastatic and treatment-resistant cancer were prospectively enrolled at a single academic center for paired metastatic tumor and normal tissue WES during a 19-month period (February 2013 through September 2014). A comprehensive computational pipeline was used to detect point mutations, indels, and copy number alterations. Mutations were categorized as category 1, 2, or 3 on the basis of actionability; clinical reports were generated and discussed in precision tumor board. Patients were observed for 7 to 25 months for correlation of molecular information with clinical response.nnnMAIN OUTCOMES AND MEASURESnFeasibility, use of WES for decision making, and identification of novel biomarkers.nnnRESULTSnA total of 154 tumor-normal pairs from 97 patients with a range of metastatic cancers were sequenced, with a mean coverage of 95X and 16 somatic alterations detected per patient. In total, 16 mutations were category 1 (targeted therapy available), 98 were category 2 (biologically relevant), and 1474 were category 3 (unknown significance). Overall, WES provided informative results in 91 cases (94%), including alterations for which there is an approved drug, there are therapies in clinical or preclinical development, or they are considered drivers and potentially actionable (category 1-2); however, treatment was guided in only 5 patients (5%) on the basis of these recommendations because of access to clinical trials and/or off-label use of drugs. Among unexpected findings, a patient with prostate cancer with exceptional response to treatment was identified who harbored a somatic hemizygous deletion of the DNA repair gene FANCA and putative partial loss of function of the second allele through germline missense variant. Follow-up experiments established that loss of FANCA function was associated with platinum hypersensitivity both in vitro and in patient-derived xenografts, thus providing biologic rationale and functional evidence for his extreme clinical response.nnnCONCLUSIONS AND RELEVANCEnThe majority of advanced, treatment-resistant tumors across tumor types harbor biologically informative alterations. The establishment of a clinical trial for WES of metastatic tumors with prospective follow-up of patients can help identify candidate predictive biomarkers of response.

The Clathrin Adaptor AP-1A Mediates Basolateral Polarity

Clathrin and the epithelial-specific clathrin adaptor AP-1B mediate basolateral trafficking in epithelia. However, several epithelia lack AP-1B, and mice knocked out for AP-1B are viable, suggesting the existence of additional mechanisms that control basolateral polarity. Here, we demonstrate a distinct role of the ubiquitous clathrin adaptor AP-1A in basolateral protein sorting. Knockdown of AP-1A causes missorting of basolateral proteins in MDCK cells, but only after knockdown of AP-1B, suggesting that AP-1B can compensate for lack of AP-1A. AP-1A localizes predominantly to the TGN, and its knockdown promotes spillover of basolateral proteins into common recycling endosomes, the site of function of AP-1B, suggesting complementary roles of both adaptors in basolateral sorting. Yeast two-hybrid assays detect interactions between the basolateral signal of transferrin receptor and the medium subunits of both AP-1A and AP-1B. The basolateral sorting function of AP-1A reported here establishes AP-1 as a major regulator of epithelial polarity.

High-Resolution 3D Reconstruction Reveals Intra-Synaptic Amyloid Fibrils

β-Amyloid (Aβ) accumulation and aggregation are hallmarks of Alzheimers disease (AD). High-resolution three-dimensional (HR-3D) volumetric imaging allows for better analysis of fluorescence confocal microscopy and 3D visualization of Aβ pathology in brain. Early intraneuronal Aβ pathology was studied in AD transgenic mouse brains by HR-3D volumetric imaging. To better visualize and analyze the development of Aβ pathology, thioflavin S staining and immunofluorescence using antibodies against Aβ, fibrillar Aβ, and structural and synaptic neuronal proteins were performed in the brain tissue of Tg19959, wild-type, and Tg19959-YFP mice at different ages. Images obtained by confocal microscopy were reconstructed into three-dimensional volumetric datasets. Such volumetric imaging of CA1 hippocampus of AD transgenic mice showed intraneuronal onset of Aβ42 accumulation and fibrillization within cell bodies, neurites, and synapses before plaque formation. Notably, early fibrillar Aβ was evident within individual synaptic compartments, where it was associated with abnormal morphology. In dendrites, increasing intraneuronal thioflavin S correlated with decreases in neurofilament marker SMI32. Fibrillar Aβ aggregates could be seen piercing the cell membrane. These data support that Aβ fibrillization begins within AD vulnerable neurons, leading to disruption of cytoarchitecture and degeneration of spines and neurites. Thus, HR-3D volumetric image analysis allows for better visualization of intraneuronal Aβ pathology and provides new insights into plaque formation in AD.

The fast-recycling receptor Megalin defines the apical recycling pathway of epithelial cells

The basolateral recycling and transcytotic pathways of epithelial cells were previously defined using markers such as transferrin (TfR) and polymeric IgA (pIgR) receptors. In contrast, our knowledge of the apical recycling pathway remains fragmentary. Here we utilize quantitative live-imaging and mathematical modelling to outline the recycling pathway of Megalin (LRP-2), an apical receptor with key developmental and renal functions, in MDCK cells. We show that, like TfR, Megalin is a long-lived and fast-recycling receptor. Megalin enters polarized MDCK cells through segregated apical sorting endosomes and subsequently intersects the TfR and pIgR pathways at a perinuclear Rab11-negative compartment termed common recycling endosomes (CRE). Whereas TfR recycles to the basolateral membrane from CRE, Megalin, like pIgR, traffics to subapical Rab11-positive apical recycling endosomes (ARE) and reaches the apical membrane in a microtubule- and Rab11-dependent manner. Hence, Megalin defines the apical recycling pathway of epithelia, with CRE as its apical sorting station.

Three-Dimensional Reconstruction and Analysis of Vitreomacular Traction: Quantification of Cyst Volume and Vitreoretinal Interface Area

Author Affiliations: LuEsther T. Mertz Retinal Research Center, Manhattan Eye, Ear, and Throat Hospital and Vitreous Retina Macula Consultants of New York (Drs Vance and Freund), Department of Ophthalmology, New York University Medical Center (Dr Wald), and Department of Optometry, State University of New York College of Optometry (Dr Sherman), New York. Correspondence: Dr Freund, Vitreous Retina Macula Consultants of New York, 460 Park Ave, Fifth Floor, New York, NY 10022 (kbfnyf@aol.com). Financial Disclosure: None reported. Funding/Support: This study was supported by the Macula Foundation, Inc, New York, New York.

Automatic Control of Solvent Density in Grand Canonical Ensemble Monte Carlo Simulations

We present automated methods for determining the value of Adams B parameter corresponding to a target solvent density in grand canonical ensemble Monte Carlo simulations. The method found to work best employs a proportional-integral control equation commonly used in industrial process control applications. We show here that simulations employing this method rapidly converge to the desired target density. We further show that this method is robust over a wide range of system sizes. This advance reduces the overall CPU time and user effort in determining the equilibrium excess chemical potential in these systems.

Virtual operating room for team training in surgery

BACKGROUNDnWe proposed to develop a novel virtual reality (VR) team training system. The objective of this study was to determine the feasibility of creating a VR operating room to simulate a surgical crisis scenario and evaluate the simulator for construct and face validity.nnnMETHODSnWe modified ICE STORM (Integrated Clinical Environment; Systems, Training, Operations, Research, Methods), a VR-based system capable of modeling a variety of health care personnel and environments. ICE STORM was used to simulate a standardized surgical crisis scenario, whereby participants needed to correct 4 elements responsible for loss of laparoscopic visualization. The construct and face validity of the environment were measured.nnnRESULTSnThirty-three participants completed the VR simulation. Attendings completed the simulation in less time than trainees (271 vs 201 seconds, P = .032). Participants felt the training environment was realistic and had a favorable impression of the simulation. All participants felt the workload of the simulation was low.nnnCONCLUSIONSnCreation of a VR-based operating room for team training in surgery is feasible and can afford a realistic team training environment.

Automatic Determination of Stepsize Parameters in Monte Carlo Simulation Tested on a Bromodomain-Binding Octapeptide

Abstract: The proportional integral controller, commonly used in engineering applicationsfor process control, has been implemented for the tuning of the stepsizes in Metropolis MonteCarlo simulations. Similarly to the recent application for tuning the chemical potential param-eter in grand-canonical ensemble simulation, the process-control approach was found to workwell for the problem of selecting the stepsize for each torsion angle that results in a targetedacceptance rate during the simulation of an octapeptide in aqueous environment.Keywords: Monte Carlo, acceptance rate, stepsize, process control.1. IntroductionThe Metropolis Monte Carlo method [1] is the first computational procedure that was shown capableof sampling an ensemble of configurations suitable for statistical-mechanical treatment. While currentsimulation work in macromolecular systems generally employs the molecular dynamics approach withsignificant success, there are applications where Monte Carlo was found to be more efficient [2]. Also,the ease of restricting the sampling to a limited number of degrees of freedom makes Monte Carloan attractive choice [3]. However, it is important to recognize that the Metropolis method actually

Volumetric three-dimensional reconstruction and segmentation of spectral-domain OCT.

Despite advances in optical coherence tomography (OCT), three-dimensional (3D) renderings of OCT images remain limited to scanning consecutive two-dimensional (2D) OCT slices. The authors describe a method of reconstructing 2D OCT data for 3D retinal analysis and visualization in a Computer Assisted Virtual Environment (CAVE). Using customized signal processing software, raw data from 2D slice-based spectral-domain OCT images were rendered into high-resolution 3D images for segmentation and quantification analysis. Reconstructed OCT images were projected onto a four-walled space and viewed through stereoscopic glasses, resulting in a virtual reality perception of the retina. These 3D retinal renderings offer a novel method for segmentation and isolation of volumetric images. The ability to manipulate the images in a virtual reality environment allows visualization of complex spatial relationships that may aid our understanding of retinal pathology. More importantly, these 3D retinal renderings can be viewed, manipulated, and analyzed on traditional 2D monitors independent of the CAVE.

Contemporary PBSB: Modular Graduate Education in Cells, Systems, and Quantitative Methods

Physiology and biophysics contributes to both graduate (Ph.D.) and medical (M.D.) education. The goals and curricula differ, but faculty, facilities, and educational tools are shared.Last year we introduced (Gardner et al, Biophys.J. 102:210a,2012) Membranes, Ions, and Signals, our new modular biophysics unit for first-year medical students. We now report a new year-long modular course to prepare first-year Ph.D. students for twenty-first century research in the function, analysis, modeling, and understanding of living systems: Contemporary Physiology, Biophysics, and Systems Biology (CPBSB). With Departmental support, two dozen faculty were able to shape design within three months, toward introduction in September 2012.Multiscale and translational examples develop conceptual skills necessary to design meaningful experiments, derive insight from journal reports, work within research groups, and communicate findings. Quantitative and computational methods are central, integrated, and rigorous; structural and developmental concepts are covered as they illuminate function.Organization is modular: six semi-independent multi-week modules that form a coherent whole. Typical weeks include two in-depth lecture-conferences combining core material with student participation, and one computational analysis, model, or journal-club paper.Initial modules cover essentials:CPBSB1: Membranes and cellsCPBSB2: Protein function signaling and synthesisCPBSB3: Control and communication in bodies and brainsTwo modules build on fundamentals: one focuses on an organ system; another on an informative set of computational tools:CPBSB4: Action and mechanical work from biochemical energy,CPBSB5: Introduction to computational systems biology.The concluding module:CPBSB6: Physiology of Systems and Diseases, integrates lecture/conferences, problem-based learning, and journal clubs toward problem solving. Some weeks offer translational correlates of ideas and tools from the five preceding modules; others follow an investigative thread toward relating questions and techniques.Assessments and student feedback are obtained following each module.