Life SciencesShared Resources

Cancer.Dartmouth.eduMarch 2012

2012

02-1

920

1202

-19

SHARED RESOURCES MANAGEMENT

MANAGEMENT TEAM:

Mark Israel, MDDirector, Norris Cotton Cancer Center

Bob Gerlach, MPAAssociate Director, Norris Cotton Cancer Center

Craig Tomlinson, PhDAssociate Director for Shared Resources, Norris Cotton Cancer Center

Stephen Bobin, MBAAdministrative Coordinator for Shared Resources, Norris Cotton Cancer Center

Norris Cotton Cancer Center organizes and manages 12 Shared Resources to provide Dartmouth investigators access to instruments, technologies, services, and expert consultation that can advance their research. The rapid pace of technological change requires continuing and significant investment in advanced instrumentation and highly skilled scientists and technologists to optimize its use. Norris Cotton Cancer Center is supported by an NCI Cancer Center Support Grant that provides financial support for these Shared Resources. Every five years this grant is re-competed, and a critical, external evaluation of the Shared Resources is performed. Our ability to serve the cancer research community, and the importance of the research that is facilitated by our Shared Resources, are key criteria for continued funding and for approval of the grant itself. Our leadership in this area relies heavily on the engagement, feedback, and vision of our investigators, and I am hopeful the Cancer Center can be a forum that supports frequent conversations to identify the best possible support, as well as serving as a means to provide that support.

Mark Israel, MD

WHAT ARE SHARED RESOURCES?Norris Cotton Cancer Center (NCCC) recognizes the need to provide its 150 biomedical investigators access to instruments, technologies, services, and expert consultation that can advance their research, which now garners nearly $50 million annually in peer-reviewed direct costs. Consolida-tion of core research services into shared resources provides a wider range of faculty with: •Increasedavailabilityanduseoftechnology •Dedicated,highly-trainedstaff •Improvedresearchefficiencyandproductivity •CoordinationofmultipleservicesConsolidationcreatesamoreefficientenvironment,promotesgreaterresourceutilization,andprovidesanimpetustodevelopadditionalresearchprojects. With the designation of Shared Resources as a key component of the Center, NCCC has established a common platform (CCOPs) to support scheduling, order/charge entry, and reporting.

Our quarterly meeting of Shared Resource Directors maintains a forum where common considerations, such as quality control and data processing, canbeaddressedsystematically.Wealsohavefacilitatedtheefficientintroductionoffledglingnewservicesthroughtheirincorporationintotheestablished operation of the most relevant existing Shared Resources.

CONNECTED RESEARCHERS WITH RESOURCESIn addition to providing the latest technologies and resources to the Dartmouth research community to facilitate basic and clinical research, a major goal of the Shared Resources is to provide a communication pipeline among DMS, DHMC, NCCC, Dartmouth, basic and clinical science departments, andregionalinstitutionsofhigherlearning.ThepipelineallowsanefficienttransferofinformationfromResourceManagementtoandfrominvestigators: •Feedbackontheneedsandconcernsofinvestigators/users •NewSharedResources •Theadditionofnewinstrumentationandsharedinstruments •Newexpertiseandmethods •Informationonnewtechnologies,databases,andservices •Workshops,seminars,visitingscientists

HOW TO USE THIS BINDERIn the interest of providing NCCC investigators with high quality services, we have consolidated an overview on each Shared Resource in this guide. Our goal is to insure that all investigators know: •Whatsharedresourcesareavailable •Theavailableexpertise •Wheretheresourcesarelocated •Theavailableservices •Whothepersonnelare •Howtocontacttheresource •Howtosubmitasampletotheresource •Thepricesarefairandreasonable •Theserviceisofthehighestquality

The Shared Resources will work with investigators to become a trusted partner in the process of scientific investigation by responding to needs with the highest quality service at the lowest possible price. Please reach out to any of us to learn more.

THE BENEFITS OF SHARED RESOURCES:• Cost-efficiency• Qualitycontrol• Stability• Methodology• Consultationandsupport• Increasedproductivity• Scientificinteraction

BIOINFORMATICS RESOURCE

LOCATION:706 Rubin BuildingDartmouth-Hitchcock Norris Cotton Cancer CenterOne Medical Center Drive | Lebanon, NH 03756 | (603) 653-9939 bisr.org

Jason H. Moore, PhD, Scientific Director Jason.H.Moore@Dartmouth.eduDr. Moore is the Third Century Professor of Genetics and Professor of Community and FamilyMedicineandDirectoroftheInstituteforQuantitativeBiomedicalSciencesatDartmouthMedical School. Dr. Moore serves as Associate Director for Bioinformatics at Norris Cotton Cancer Center, and leads a successful NIH-funded bioinformatics research program at the Norris Cotton Cancer Center. Dr. Moore’s Computational Genetics Laboratory web site at epistasis.org provides more information about Dr. Moore and his research interests. His Epistatis Blog is available at compgen.blogspot.com.

Walter Taylor, PhD, Managing DirectorWalter.Taylor@Dartmouth.eduDr. Taylor is Managing Director of the Bioinformatics Shared Resource (BISR) at Norris Cotton Cancer Center. Dr. Taylor works closely with BISR personnel to assist Cancer Center investigators with their bioinformatics needs, and provides bioinformatics collaboration and expertise in support of high-throughput DNA sequencingservicesofferedthroughtheCancerCenter’s Genomics Shared Resource. His training includes over 10 years of software engineering experience for first- and second-generation DNA sequencing platforms at Life Technologies, 454 Life Sciences, and Helicos Biosciences.

To support the implementation of bioinformatics resources for cancer research at Dartmouth. Our goal is to provide expert consultation and collaboration for research projects of NCCC members. The Bioinformatics Shared Resource also strives to educate members of the community in different aspects of computational biology by providing regular workshops and seminars.

We provide a wide range of different services including applied bioinformatics and data mining, computer programming and software engineering, database development and programming, and high-performance computing and systems administration. We look forward to helping you plan, execute, analyze, and interpret your next biomedical research study.

OUTLINE OF SERVICES:

Applied Bioinformatics and Data Miningi. TheBISRoffersawidevarietyofdifferentappliedbioinformaticsanalysisservicesforgenetic,genomic,andproteomicstudies.Thisincludes

use of state-of-the-art software such as the Bioconductor package in R to normalize, transform, and analyze your gene expression microarray datausingbothsupervised(e.g.SAM)andunsupervised(clusteranalysis)methods.Foranexample,seetherecentpaperfromMarkIsrael’slabthat includes a bootstrapped hierarchical cluster analysis of microRNA results (Gaur et al. 2007).

ii. The Cancer Center’s Genomics core, with which we work closely, is an important generator of array and high-throughput sequencing data for the Bioinformatics core.

iii. Wealsoofferanumberofadvancedmachinelearninganddataminingmethodsforidentifyingcomplexpatternsofgenetic,genomic,andproteomicbiomarkersassociatedwithdiscreteorcontinuousbiomedicalendpoints.Foranexample,seetherecentpaperfromAngelineAndrew and Margaret Karagas that used data mining methods to identify combinations of genetic and environmental risk factors for bladder cancer (Andrew et al. 2006).

iv. Inadditiontobioinformaticsanalysis,theBISRalsooffersservicestoassistwiththeinterpretationof‘omics’results.WeofferourownExplor-atory Visual Analysis (EVA) database and software for exploring analytical results in the context of pathways and Gene Ontology, for example, andweofferaccesstotwodifferentcommercialpathwayanalysisproductstoassistyouwithmakingconnectionsbetweenyourresultsandspecificpathways,ortocreateyourownnovelpathways.TheseincludePathwayStudioandIPA,eachwithparticularstrengths.Foranexample, see the recent paper by Angeline Andrew that used both of these tools to interpret gene expression microarray results (Andrew et al. 2008). We also have expertise in the use of publicly-accessible web sites for bioinformatics data analysis, including NCBI, Galaxy, UCSC genome browser, and many more.

Computer Programming and Software EngineeringTheBISRofferscomputerprogrammingsupportusingawidevarietyofcomputerlanguagesandsoftwarepackagesincludingC,C++,Java,Perl,Python, R, HTML, PHP, Visual Basic, Objective-C, LabView, etc.

Database Development and AdministrationDatabasedevelopment,programmingandadministrationservicesareofferedusingOracleandmySQL.ComprehensiveOraclesupportismadepossible through a Dartmouth site license. Thus, there are no chargebacks necessary for the Oracle license. The BISR maintains its own database solution called Metatable Database Development System (MDDS) that was specifically designed and implemented in Oracle for integrating research data and clinical data. This system has already been modified and expanded for several projects, including the NCCC billing and financial database forthesharedresources.TheBISRcurrentlysupportsandmakesavailabletheGeneTrafficmicroarraydatabase.

High-Performance Computing and Systems AdministrationAccesstoa1440-processorparallelcomputerisofferedasaBISRservice.Facultymembersareabletobuyaccesstothesystembypurchasingoneor more nodes at $5000 per 16-cpu node. This price includes hardware and personnel support for four years along with software and maintenance costs. Details of the hardware architecture and other information can be found at discovery.dartmouth.edu. We also provide computer program-ming support and/or training to assist you with developing parallel applications.

TheBISRemploysatalentedteamofbioinformaticspersonneleachwithawiderangeofcomputationalskills.Eachofthestaffareavailabletohelpyou with your bioinformatics needs. Please contact Walter Taylor or Craig Tomlinson if you are considering a sequencing or array-based project.

PUBLICATIONS:Gaur A, Jewell DA, Liang Y, Ridzon D, Moore JH, Chen C, Ambros VR, Israel MA. Characterization of microRNA expression levels and their biological correlates in human cancer cell lines. Cancer Res. 2007. Mar 15;67(6):2456-68.

Andrew AS, Nelson HH, Kelsey KT, Moore JH, Meng AC, Casella DP, Tosteson TD, Schned AR, Karagas MR. Concordance of multiple analytical ap proaches demonstrates a complex relationship between DNA repair gene SNPs, smoking and bladder cancer susceptibility. Carcinogenesis. 2006. May;27(5):1030-7. Epub 2005 Nov 25.

BIOSTATISTICS

LOCATION:Rubin Room 875Dartmouth-Hitchcock Norris Cotton Cancer CenterOne Medical Center Drive | Lebanon, NH 03756 | (603) 653-3677email: Biostatistics.Shared.Resource@dartmouth.edu.synergy.dartmouth.edu/research_design_request_form.php

Tor D. Tosteson, ScDTor.Tosteson@Dartmouth.eduProfessorofCommunityandFamilyMedicineand Professor of The Dartmouth Institute.Dr. Tosteson’s research interests revolve around challenging methodological and statistical problems in medicine. Major projects include the Center for Comparative EffectivenessResearchinAdvancedImagingin Cancer and the Breast PROSPR Research Center and related statistical methods for comparativeeffectivenessresearch.

Zhongze Li, MS, Research ManagerZhongze.Li@Dartmouth.eduMA Economics, Stony Brook 1999MSFinancialEngineering,U.Michigan2003MS Biostatistics, U. Michigan 2005Member, American Statistical Association

Expertise includes clinical trial design, longitudinal data, statistical methods for genomics and managing data, measurement error methods, nonlinear dose response modeling, quality of life data, decision sciences, cost effectiveness analysis, and diagnostic test assessment. Resource maintains an extensive statistical software library providing access to all major statistical analysis platforms. All initial consultations are free.

OUTLINE OF SERVICES:• Studydesign/development• Statisticalhypothesisformulation• Analysisplans• Samplesizecalculations• Randomization/sampling• Statisticalanalysis/interpretation

• Protocoldevelopment,reviewandinterimmonitoring• Researchproposaldevelopment• Statisticaldatamanagement• Statisticaleducation• NovelstatisticalmethodsforNCCCstudies

PUBLICATIONS:OnegaT,MacKenzieT,WeissJ,GoodrichM,Titus-ErnstoffL.ScreeningMammographyIntervals

Among Post-Menopausal Hormone Therapy Users and Non-Users. Cancer Causes & Control.2010. 21(1):147-52.

GunturuK,MeehanK,MacKenzieT,FisherJ,ErnstoffM.ACytokineWorkingGroupStudyofLymphodepletingChemotherapy,IL-2andGM-CSFinMetastatic Melanoma Patients: Clinical Outcomes and Peripheral Blood Cell Recovery. Journal of Clinical Oncology. 2010. 28(7):1196-202.

MacKenzie T, Zens MS, errara A, Schned A,Karagas MR. Diabetes and Risk of Bladder Cancer: Evidence from a Case-Control Study in New England. Cancer.2011. 117(7):1552-6 [21425156].

Dietrich K, Demidenko E, Schned A, Zens MS, Heaney J, Karagas MR. Parity, early menopause and the incidence of bladder cancer in women: A case-control study and meta-analysis. European Journal of Cancer. Volume: 47 Issue: 4 Pages: 592-599 DOI: 10.1016/j.ejca.2010.10.007

Published: MAR 2011.

Robinson CM, Cassells AN, Greene MA, Beach ML, Tobin JN, Dietrich AJ. Barriers to colorectal cancer screening among publicly insured urban women: no knowledge of tests and no clinician recommendation. J Natl Med Assoc. 2011;103(8):746-53.

Gui J, Moore JH, Kelsey KT, Marsit CJ, Karagas MR, Andrew AS. A novel survival multifactor dimensionality reduction method for detecting gene-gene interactions with application to bladder cancer prognosis. Human Genetics. 129(1):101-10.

FadulCE,FisherJL,HamptonTH,LallanaEC,LiZ,GuiJ,SzczepiorkowskiZM,TostesonTD,RhodesCH,WishartHA,LewisLD,ErnstoffMS.Immuneresponse in patients with newly diagnosed glioblastoma multiforme treated with intranodal autologous tumor lysate-dendritic cell vaccination after radiation chemotherapy. J Immunother. 2011 May;34(4):382-9.

SAMPLING OF CURRENT PROJECTS:

Effects of a Palliative Care Intervention on Clinical Outcomes in Patients With Advanced Cancer: The Project ENABLE II Randomized Controlled Trial Cancer Control Research Program

NCCC PIs: Marie Bakitas; Kathleen Doyle Lyons; Mark T. Hegel;

Scientific Focus: Dr. Bakitas led the ENABLE II trial of an innovative palliative care intervention. This randomized trial was among the first to establish quality of life advantages for a palliative care intervention. A detailed randomized, longitudinal design was developed with the help of the Biostatistics Shared Resource (BSR). Several innovative approaches were used to compare theinterventionandcontrolgroups.First,alongitudinalanalysiswasconductedwithtimemeasured from the baseline evaluations, showing significant advantages for the intervention. Then a longitudinal analysis was performed using only data for patients who died, with time orientedbackwardsfromtimeofdeath.Finally,asurvivalanalysiswasperformedtocomparethe two intervention groups. A post-hoc analysis shows survival advantages during the first year when quality was generally higher in both groups.

BSR Role: BSR members created the statistical design of the study. Dr. Tosteson and Z. Li designed and performed the analyses.

BakitasM,LyonsKD,HegelMT,BalanS,BrokawFC,SevilleJ,HullJ,LiZ,TostesonTD,ByockIR,AhlesTA.EffectsofaPalliativeCareInterventiononClinicalOutcomesinPatientsWith Advanced Cancer: The Project ENABLE II Randomized Controlled Trial. JAMA.2009. 302(7):741-749. PMID: 19690306.

OFFICE OF CLINICAL RESEARCH

LOCATION:Rubin8thFloorDartmouth-Hitchcock Norris Cotton Cancer CenterOne Medical Center Drive | Lebanon, NH 03756 | (603) 653-6023

STAFF:• RebeccaCarsonRogers RegulatoryandComplianceOfficer Rebecca.C.Rogers@Dartmouth.edu• SaraSimeone Research Nurse Manager - Sara.A.Simeone@Hitchcock.org

Clinical Research Coordinators (CRCs) are all members of either the Association for Clinical Research professionals (ACRP) or Society of Clinical Research Associates (SOCRA). Over 50% are certified CRCs.

Research Nurses are experienced nurses with oncology training.

Regulatory specialists receive specialized training on CPHS submissions.

James R. Rigas, MDJames.R.Rigas@Dartmouth.eduMedical Director Area of Research Interest: Thoracic oncology

Nancy J. RollingsNancy.J.Rollings@Dartmouth.edu Administrative Director

The mission of the Office of Clinical Research is to assist with the planning, conduct, and compliance of clinical trials involving cancer treatment medications and devices.

SERVICES:· Provide research support to clinical investigators for the

management of clinical trials including investigator initiated, federally funded, and industry trials

· Assist Principal Investigators in the activation and administration of studies

· Prepare records for internal and external compliance and quality monitoring and audits

· Screening and enrolling patients for clinical trials · Coordinate the completion of participant-specific study

requirements · Provide data management and regulatory support for

clinical trials · Provide multi-center project management services for

investigator-initiated trials· Provideeducationonclinicaltrialmanagementtostaffand

new investigators · Lab has -20 and -80 freezer for short term storage; 2 centrifuges (one refrigerated, one ambient) · Procure, process, and ship blood samples same day as required. Can short-term freeze samples

SERVICE PROCESS:

1. Notify OCR, disease specific, Clinical Research Coordinator or Administrative Director (OCR general number 603-653-9071) when a new clinical trial is being proposed.

2. Assistance is available for all steps of opening a clinical trial including investigator initiated, federal, and industry sponsored trials. Confidentialityagreements(CDA),contracts,andbudgetsarenegotiatedthroughtheClinicalTrialsOffice(CTO).

3. CRCs and regulatory specialists provide complete services (prior to opening a study through close out of a study) including IRB submission, submission and maintenance of regulatory documents and participant data, coordination with sponsors for site initiation, monitoring, and investigationalpharmacyvisits.OCRstaffworkwithCTO,CPHS,andsponsorsonallaspectsofatrial.

4. Research Nurses serve as the clinical liaison to assist with informed consent, symptom management, and clinical conduct of the study.

STRUCTURE AND SUPPORT:

• Clinical Cancer Review Committee (CCRC) Provides required peer review of scientific merit prior to IRB review. Reviews clinical research protocols for treatment, prevention, control or intervention of cancer and protocol amendments• NCCC Data, Safety Monitoring, and Accrual Committee (DSMAC) Provides required peer review of clinical research protocols for which a Dartmouth investigator is both investigator and sponsor of the protocol. Reviews toxicity patterns, data integrity, protocol adherence, and progress toward study objectives. Reviews study accrual. • Clinical Trials Investigational Order Set Committee (CTIOSC) Provides required review of drug order sets for clinical trials prior to study activation• Cancer Clinical Research Quality Improvement Committee (QIC) FocusesoncontinuousclinicalresearchprocessimprovementattheNorrisCottonCancerCenter.Analyzestrendsinthequalityofresearchby quarterly reviews of protocol deviation reports, local serious adverse event reports, ongoing corrective and preventative actions plans, reportsfromresearchancillarysupportservices,andreportsfromPIs,NPs,ClinicalResearchCoordinators,ResearchNurses,Regulatorystaff, and administrators.

CAPABILITIES:

Studies include investigator-initiated, cooperative group, working group, and industry-supported clinical trials. Services include regulatory work, clinical research coordination, and research nursing.

TECHNOLOGY:

NCCC utilizes an automated IT environment (Velos eResearch) as its central repository for study- and patient-related data. Velos eResearch is a comprehensive, web-based suite of software that addresses many research-related tasks, including Study Management, Patient Management, Document Management, Patient Calendars, and Centralized Reporting.

GENOMICS RESOURCE

LOCATION:Rubin Room 634Dartmouth-Hitchcock Norris Cotton Cancer CenterOne Medical Center Drive | Lebanon, NH 03756 | (603) 653-9978dms.dartmouth.edu/tomlinson/

Craig R. Tomlinson, PhD, DirectorCraig.R.Tomlinson@Dartmouth.eduDepartments of Medicine and Pharmacology & Toxicology

Heidi Trask, BSHeidi.W.Trask@Dartmouth.eduMicroarray Manager

Joanna Hamilton, PhDJoanna.S.Hamilton@Dartmouth.eduDeep Sequencing Manager

Carol Ringelberg, BSCarol.S.Ringelberg@Dartmouth.eduMicroarray Data Analyst

Walter Taylor, PhDWalter.Taylor@Dartmouth.eduHigh-Throughput Data Analyst

Christian LytleChristian.H.Lytle@Dartmouth.eduManager, Molecular Biology Shared Resource, Deep Sequencing Specialist

The Genomics Shared Resource provides technologies to Cancer Center and Dartmouth community investigators that enable profiling of gene expression, miRNA, GpC Island, and CGH on a whole-genome scale. The long-term goal of the Genomics Shared Resource is to provide an efficient and affordable fee-for-service operation that will provide high quality genomics and microarray data for the growing number of Cancer Center investigators who require this service.

INSTRUMENTATION:•Illumina-Beads,fiberoptics•Affymetrix-Photolithographicarrays.Shortoligos(25nt)•Agilent-Spottedarrays(inkjet).cDNAorlongoligos(60nt)•IlluminaGenomeAnalyzer IIx (pictured)

PUBLICATIONS:Hoffmannetal.(2012).ImplicationofthemiR-184andmiR-204CompetitiveRNANetworkinControlofMouseSecondaryCataract.Molec. Med. (in press).

Kerley-Hamiltonetal.(2012).InherentandBenzo[a]pyrene-InducedDifferentialArylHydrocarbonReceptorSignalingGreatlyAffectsLifespan,Atherosclerosis,CardiacGene Expression, and Body and Heart Growth in Mice. Toxicol. Sci. (in press).

Thornley et akl. (2012). SMAD4-dependent polysome RNA recruitment in human pancreatic cancer cells. Molec. Carcinogenesis (in press).

Thornleyetal.(2011).DifferentialregulationofpolysomemRNAlevelsinmousehepa-1c1c7cellsexposedtodioxin. Toxicol. In Vitro 45: 1457-1467.

Suetsugu-Maki et al. (2011). A complement receptor C5a antagonist regulates epithelial to mesenchymal transition and crystallin expression after lens cataract surgery in mice. Molec. Vis. 17: 949-64.

Nakamura et al. (2010). miRNAs in newt lens regeneration: specific control of proliferation and evidence for miRNA networking. Plos One 5: e12058.

Trask et al. (2009). Microarray analysis of cytoplasmic versus whole cell RNA reveals a considerable number of missed and false positive mRNAs. RNA 15: 1917-1928.

SERVICES:Phase 1: EXPERIMENTAL DESIGN•Biologicalquestions•Biologicalreplicates•Maximizestatistcalpower•Feasibility•Budget

QUALITY CONTROL FOR RNA AND DNA:•NanoDrop.Fiberopticsystem.Nocuvettes–Veryaccuratedeterminationof

concentration and purity on A260/280 readings•AgilentBioanalyzer–VeryaccuratedeterminationofRNAquality(intactness)

MICROARRAYS AND DEEP SEQUENCING:MICROARRAYS – •mRNAlevels•smallRNAlevels•Genomicin-delsfromfrozenandFFPEsamples•GenomicsDNAmethylationlevels•Genotyping

HOW TO PREPARE YOUR SAMPLE:Clean intact RNA or DNA •TheRNAandDNAintissuesstoreindefinitelyinRNAlater(Ambion)•TotalRNA.Noneedtoisolatepoly(A)-RNA•RecommendTRizolorTRIReagent.miRNAnotlost,betteryield•RNeasycolumnalsoworks(Qiagen)•Suspendinwater•Or,wecanisolatetheRNA

DEEP SEQUENCING – •DNASequencing–Single-readandlong-(mate-pair)and

short-insert (paired-end) reads for small whole-genome sequencing and re-sequncing, de novo sequencing, structural variation, and copy number analysis

•Transcriptomeanalysis–CharacterizemRNAsplicevariants,mRNAs, nuclear RNA, coding SNPSs, and relative expression of transcripts in one experiment; profile rare and discover novel RNAs absolute quantitative transcript data

•GeneRegulation&ControlAnalysis–Studyepigenticmodifications and DNA-protein interactions across the entire genome and obtain a comprehensive view of the epigenome

Phase 2: DATA GENERATION•RNA/DNAisolation•RNA/DNAQC•MicroarrayPlatforms •Agilent •Affymetrix •Illuminabead•DeepSequencing •IlluminaGAIIX •Ion Torrent

Phase 3: DATA ANALYSIS•Statistics,Clustering•PCRvalidation•Biologicalmeaning•Database

GEOSPATIAL RESOURCE

LOCATION:Rubin Room 837Dartmouth-Hitchcock Norris Cotton Cancer CenterOne Medical Center Drive | Lebanon, NH 03756 | (603) 653-6023

AFFILIATED FACULTY:• EthanBerke,AssociateProfessorCFMED,TDI• EugeneDemidenko,ResearchProfessorCFMED• TracyOnega,AssistantProfessorCFMED• DavidGoodman,ProfessorofPediatrics

STAFF:• HeatherCarlos• NancyMarth

James D. Sargent, MD, DirectorJames.Sargent@Dartmouth.eduProfessor of PediatricsDr. Sargent is the Co-Director of the Cancer Control Program and the Director of the Dartmouth Media Research Laboratory, which studies adolescent risk behaviors, mediainfluences,tobaccocontrolandearlyonset alcohol use.

Heather Carlos, ManagerHeather.Carlos@Dartmouth.eduSpatial Analyst

Supports geospatial analysis by providing expert consultation and collaboration for research projects in behavior, epidemiology, health services, and other disciplines.

OUTLINE OF SERVICES:• Dataacquisitionandanalysis• Geocoding• Preliminarydataanalysis/exploration• ProjectDesign• SpatialAnalysis• Traveltimesanddistances,catchmentareas• Densitysurfaces• Clusteranalysisandidentifyingpatterns• Geospatialregressionanalysis• Geographicdistributions• Spatial/temporaltrends• Mapping/Datavisualization

PUBLICATIONS:CarlosHA,ShiX,SargentJ,TanskiS,BerkeEM.Densityestimationandadaptivebandwidths:aprimerforpublichealthpractitioners. Int J Health Geogr. 2010;9:39.

BerkeEM,TanskiSE,DemidenkoE,Alford-TeasterJ,ShiX,SargentJD.Alcoholretaildensityanddemographicpredictorsofhealthdisparities:ageographic analysis. Am J Public Health. 2010 Oct;100(10):1967-71.

Donohue J, Morden N, Gellad W, Bynum J, Zhou W, Hanlon J, Skinner. Is regional variation in Medicare Part D spending driven by drug choice or by prescription volume? J. N Engl J Med. 2012; 366:530-538.

Predictors of Tobacco Outlet Density Nationwide: A Geographic Analysis. Int J Health Geogr.

Rodriguez D, Carlos HA, Adachi-Mejia AM, Sargent JD. Is regional variation in Medicare Part D spending driven by drug choice or by prescription volume? Int J Health Geogr. Under review.

Adachi-MejiaAM,CarlosHA,BerkeEM,TanskiSE,SargentJD.Acomparisonofindividualversuscommunityinfluencesonyouthsmokingbehaviors. Under review.

SAMPLING OF CURRENT PROJECTS:• RuralTownWalkability• OrganTransplantReferralRegions• CountyHealthRankings• MediaInfluencesonAdolescentSmokingBehavior• PrimaryCareServiceAreas• AgingandtheBuiltEnvironment• KickTobaccoCampaign• AdolescentAccesstoTanningFacilities• DistancetoCare:OvarianCancerSurvival• Community-basedUtilizationofBreastImagingTechnologies

Cartographic comparison of density estimation of alcohol outlets near San Antonio, Texas. A-D show San Antonio in the center and Austin in the upper right with more rural areas to the south and west, E-G are zoomed in to show just San Antonio. A and E illustrate KDE using a static bandwidth of ~10km. B and F illustrate KDE using an adaptive bandwidth with an expected population of 1,000 people and a maximum distance of ~25 km. C shows a LandScan™ dataset where each pixel represents a population count. D shows point data representing alcohol outlets. G is a map of census tracts showing the percentage of families below the poverty level.

IMMUNOASSAYS AND FLOW CYTOMETRY CORE FACILITY

LOCATION:Rubin Room 710 (Immunoassays)BorwellBuilding342W(FlowCytometry)Dartmouth-Hitchcock Norris Cotton Cancer CenterOne Medical Center Drive | Lebanon, NH 03756 | (603) 653-9913cancer.dartmouth.edu/res/immune_monitoring.html

Jacqueline Channon Smith, PhD Jacqueline.Smith@Dartmouth.eduFacultyDirectorResearch Assistant Professor Dept of Microbiology and ImmunologyDr. Smith’s interests lie in monitoring human immunology, particularly multiple sclerosis.

Alan Bergeron, BS AJB@Dartmouth.eduImmunoassays Laboratory ManagerAffiliations:The Association of Biomolecular ResourceFacilities(ABRF)North East Regional Life Science Core Directors (NERLSCD)

Gary Ward, BS Gary.Ward@Dartmouth.eduFlowCytometryLaboratoryManagerAffiliations:The Association of Biomolecular ResourceFacilities(ABRF)North East Regional Life Science Core Directors (NERLSCD)

SERVICES OFFERED:• Wholebloodprocessing• Customimmunoassaysforclinicaltrials• Cellsubsetenrichment• Leukocytephenotyping• ELISPOTassay• CFSElymphoproliferationassay• Upto42-plexcytokineassay• Mousemelanomatumormodel• FACS(cellsorting)• Upto10-colorflowcytometry• Flowcytometryanalysis

Provides various services such as isolation and cryopreservation of PBMCs and plasma or serum for clinical trials, as well as carrying out a spectrum of immunoassays and developing customized immunoassays; also provides dedicated equipment such as cell sorters, flow cytometers, an ELISPOT reader, a Bio-Plex array reader, an autoMACS and a Robosep.

QUALITY CONTROL:DartLab technologists carry out Proficiency Testing annually for ELISPOTs,intracellularstaining,tetramerstaining,andflowcytometryanalysis. Each immunoassay has a Standard Operation Procedure that is strictlyadheredtoandthatcontainsmultipleQCcheckpoints.All instruments are calibrated daily and variance over time is followed using Levy-Jennings plots.

TURNAROUND TIME:Flowcytometryandcell-sorting–acquisitionfilesavailablesamedayMultiplexedcytokineassays–analyzedresultsnextdayWholebloodprocessing–samedayELISPOTassays–analyzedresultsnextdayOtherimmunoassays–asarranged

DATA ANALYSIS AND INTERPRETATION:ComputersareavailableinBorwell340WforflowcytometryanalysisusingthelatestFlowJo,Modfit,Gemstone,andGraphpadPrismsoftware.Core personnel are available to discuss experiments and to help with data analysis and interpretation. You are strongly encouraged to discuss multi-colorflowcytometryexperimentswithJacquelineSmithorDanMielcarzBEFOREHAND.

PUBLICATIONS:Lyori M, Zhang T, Pantel H, Gagne BA, Sentman CL. TRAIL/DR5 plays a critical role in NK cell-mediated negative regulation of dendritic cell cross-priming of T cells. J Immunol.

2011 187(6):3087-95. PubMed PMID: 21832159.

BarberA,SentmanCL.NKG2DreceptorregulateshumaneffectorT-cellcytokineproduction.Blood. 2011 117(24):6571-81. PubMed PMID: 21518928.

Pino-LagosK,GuoY,BrownC,AlexanderMP,ElguetaR,BennettKA,DeVriesV,NowakE,BlomhoffR,SockanathanS,ChandraratnaRA,DmitrovskyE,NoelleRJ.Aretinoicacid-dependentcheckpointinthedevelopmentofCD4+Tcell-mediatedimmunity.J Exp Med. 208(9):1767-75. PubMed PMID: 21859847.

BarthRJJr,FisherDA,WallacePK,ChannonJY,NoelleRJ,GuiJ,ErnstoffMS.ArandomizedtrialofexvivoCD40Lactivationofadendriticcellvaccineincolorectalcancerpatients:tumor-specific immune responses are associated with improved survival. Clin Cancer Res. 2010 16(22):5548-56. PubMed PMID: 20884622.

MolloyMJ,ZhangW,UsherwoodEJ.SuppressiveCD8+TcellsariseintheabsenceofCD4helpandcompromisecontrolofpersistentvirus.J Immunol. 2011 186(11):6218-26. PubMed PMID: 21531895.

EXAMPLE OF PUBLISHED WORK:FromNesbethetal.,CCL5-mediatedendogenousantitumorimmunityelicited by adoptively transferred lymphocytes and dendritic cell depletion. Cancer Research. 2009. 69:6331-8.

Figure 5. Expanded Tcells secrete CCL5 and activate host immune cells.A. Supernatants from naive splenic T cells primed for 7 days against ID8-Defb29/Vegf-a tumor cell antigens were examined by Luminex assays for cytokine production. B. Top, phenotypic analysis of host (CD45.2+CD3+) T cells accumulated at the tumor site 3 d after ACT; bottom, expression profile of host CD11c+ (DCs) at tumor sites 3 d after treatment.Values represent mean percentages + SEM.

OUTLINE OF SERVICES:• FACScan,FACSCaliburandFACSCantocytometersareavailableforoperator-unassistedusebycardaccess24/7inBorwell342W.Sign-upusing

CCOPS. • MACSQuantandGallioscytometersareavailableforoperator-assisteduseinRubin710andRemsen5from9amthrough6pm–prearrange

with Alan Bergeron. • FACSAriacellsortinginBorwell336Wisavailable9amthrough5pm–pre-arrangewithGaryWard.• Cytokineassaysarepre-arrangedwithKathySmithinRubin710.• Allotherimmunoassaysarepre-arrangedwithAlanBergeronafterdiscussionwithJacquelineSmith.

IRRADIATION AND SMALL ANIMAL IMAGING

LOCATION:Irradiators–SecondlevelofBorwellandRubinbuildingsImagingfacilities–ThirdlevelofBorwellintheAnimalResourceCenterDartmouth-Hitchcock Norris Cotton Cancer CenterOne Medical Center Drive | Lebanon, NH 03756

P. Jack Hoopes DVM, PhDP.Jack.Hoopes@Dartmouth.edu (603) 650-5031DirectorResearch Interest: Experimental cancer therapeutics including radiation, chemotherapy, hyperthermia, photodynamic therapy and nanotechnology (iron oxide nanoparticle hyperthermia cancer treatment), animal pathology, animal models, experimental surgical technology.

Rendy Strawbridge, BS Rendy.Strawbridge@Dartmouth.edu (603) 650-8548Irradiation

Karen Moodie, DVM, MSKaren.Moodie@Dartmouth.edu (603) 650-5685Small-Animal Imaging

The mission of the NCCC’s Irradiation and Small Animal Imaging Shared Resource:• Assisting investigators in radiation treatment

planning and delivery of ionizing irradiation to cells, rodents, large animals, and spontaneous animal tumors.

• Provide non-invasive, whole animal imaging of rodents used in preclinical research studies.

OUTLINE OF SERVICES:IRRADIATION –The resource currently operates multiple experimental and clinical irradiators, including a Cs -gamma source (9,000 Curie, Cs-137), an orthovoltage x-ray source (Pantak multiple energy, 300 KV x-irradiator), a 6-18 MeV linear accelerator (which produces both photons and electrons) and an Ir-192 high-dose rate after-loader.

SMALL ANIMAL IMAGING – • VarianDirect-Drive9.4TMRIResearchScanner.PrimaryUse:pre-clinicalrodentimagingstudies• PhilipsAchieva3.0TMRIClinicalScanner.PrimaryUse:pre-clinicalrodentandlarge-animalimagingstudiesandMRI• VisualSonicsVevoHighFrequencyUltrasound.PrimaryUse:highdetailreal-timeultrasoundstudies,cardiacoutputandtumor volume estimates • XenogenVivoVisionIVISBioluminescentandFluorescentImager.PrimaryUse:imagingbioluminescentandfluorescentreportersbothin-vivo and in-vitro. • PhilipsMOSAICmicro-PETScanner.PrimaryUse:small-animalradioisotopebasedmolecularimaging• GEeXploreLocusIn-Vivomicro-CTScanner.PrimaryUse:highlydetailedin-vivoimagingstudies• GEeXploreLocusSpecimenmicro-CTScanner.PrimaryUse:highlydetailedex-vivoimagingstudies• GELightspeedCTClinicalScanner.PrimaryUse:largeandsmall-animalimagingstudies

TURNAROUND TIME AND QUALITY –Qualityassurance-SpecificQA/QCprocedures,foreachoftheirradiatorservicesandinstruments.

Irradiators operated in compliance with licensing requirements stipulated by the State of New Hampshire’s Department of Health and Human Services Radiation Division, which regulates such sources on behalf of the U.S. Nuclear Regulatory Commission (NRC).

Phantom resolution studies on MRI and CT scanners.

PUBLICATIONS:KM,BakSP,AlonsoA,BerwinB.,PhenotypicandfunctionaldelineationofmurineCX(3)CR1monocyte-derivedcellsinovariancancer.Neoplasia. 2009 Jun;11(6):564-73.Garner KM, Eastman A., Variations in Mre11/Rad50/Nbs1 status and DNA damage-induced S-phase arrest in the cell lines of the NCI60 panel. BMC Cancer. 2011 May 27;11:206:1-13.CôtéAL,ZhangP,O’SullivanJA,JacobsVL,ClemisCR,SakaguchiS,Guevara-PatiñoJA,TurkMJ.,Stimulationoftheglucocorticoid-inducedTNF receptor family-related receptor on CD8 T cells induces protective and high-avidity T cell responses to tumor-specific antigens. J Immunol. 2011 Jan 1;186(1):275-83.AllieSR,ZhangW,FuseS,UsherwoodEJ.,Programmeddeath1regulatesdevelopmentofcentralmemoryCD8Tcellsafteracuteviralinfection. J Immunol. 2011 Jun 1;186(11):6280-6.WangL,RubinsteinR,LinesJL,WasiukA,AhonenC,GuoY,LuLF,GondekD,WangY,FavaRA,FiserA,AlmoS,NoelleRJ.,VISTA,anovelmouseIg superfamily ligand that negatively regulates T cell responses. J Exp Med. 2011 Mar 14;208(3):577-92.ZagorchevL,OsesP,ZhuangZW,MoodieK,Mulligan-KehoeMJ,SimonsM,CouffinhalT.,Microcomputedtomographyforvascularexploration. J Angiogenes Res. 2010 Mar 5;2:7.Zagorchev L, Mulligan-Kehoe MJ., Molecular imaging of vessels in mouse models of disease. Eur J Radiol. 2009 May;70(2):305-11.Enelow-R01A1069360-TNFprocessinginPulmonaryImmunopathology.Ernstoff-R01CA095648-ImmunotherapyforRenalCellCarcinoma.Green - R01CA050157 - The Pathogenesis of MAIDS and Specific T Cell Responses.Noelle - R01CA123079 - Synergy of the Innate and Acquired Immune Responses in Tumor Immunology.Sentman - R01CA130911 - Chimeric NKG2D Receptors in Ovarian Cancer Immunotherapy.Swartz-U19A1067733-InVivoEPR:After-The-FactMeasurementofDose.Turk - R01CA120777 - Mechanisms of Concomitant Tumor Immunity.Usherwood - R01CA103642 - Immune Surveillance in Murine Gammaherpesvirus Infection.

MOLECULAR BIOLOGY CORE FACILITY

LOCATION:Remsen Building, Room 243Dartmouth Medical School Hanover, NH 03755 | (603) 650-6546cancer.dartmouth.edu/res/molecular_biology.htmlmbcore@dartmouth.edu

YolandaSanchez,PhD,FacultyDirectorYolanda.Sanchez@Dartmouth.eduAssociate Professor, Dept. of Pharmacology and ToxicologyDr. Sanchez’s laboratory focuses on checkpoint signaling events triggered by DNA damage or replication interference.

Christian Lytle, Lead Tech/ManagerChristian.Lytle@Dartmouth.eduAmerican Society for Clinical PathologyBoard of Certification CG(ASCP)CMThe Association of Bimolecular Resource Facilities(ABRF)North East Regional Life Science Core Directors (NERLSCD)

OUTLINE OF SERVICES:• SamplepreparationforSangerDNAsequencing• SangerDNAsequencing• DNAfragmentanalysis• Datareview,troubleshootingandanalysisresources

Applied Biosystems Model 3730 Genetic analyzer

Provides molecular biology products, services, and support to help investigators solve their basic, translational, and clinical research problems.It is our goal to offer the highest quality products and services possible in the most cost effective manner.

CAPACITY OF SERVICE:The current instrumentation has a capacity 96,000 Sanger sequencing reactions/yr.

Supported applications: The system-capable applications:•Denovosequencing •AFLP®•Resequencing(mutationalprofiling) •BACFingerprinting•Microsatelliteanalysis •Methylation•SNPgenotyping •LOH(lossofheterozygosity)

Sequencing Analysis software—Automates basecalling, and assigns quality values. Allows the option to visualize, edit, print and re-basecall sequence data using the KB basecaller.

SeqScape®software—Offersthemostcompletesolutionforvariantdetectionprojects.ThesoftwarereadsGenbankfilestocreateareferencesequenceandannotate sequence features such as protein coding sequence, introns, and exons. In addition, data from dbSNP can be automatically imported to provide accurate nucleotide variants information.

GeneMapper®software—Enablesconfigurable,automatedallelecalling,avaluableassetforhigh-throughputgenotyping.Thesoftwarecanprocessover100,000genotypesperhour,andsignificantlyreducethetimeandeffortinthegenotypingprocess.

ACCESSIBILITY OF SERVICES:Drop-offsitesaccessible24/7,samplesarerefrigerated.Weekday pickup of samples at the Borwell Loading Dock. DatafromFridaysubmissionsarepostedonSaturday(mostweekends).

DATA INTERPRETATION:The MB Core personnel are always happy to review data with our customers. We encourage you to stop by anytime for help. We will have recommendations and suggestions regarding sample prep, software, and interpretation you’re your data.

SERVICE PROCESS:Sample RequirmentsSamples should be delivered in 1.5 ml mircocentrifuge tubes. DNA template and primer concentrations can vary depending on the sample type. PCR products require20to50ngoftemplatewhilelargerplasmidsneed300to500ng.Primerconcentrationsusuallyworkwellinthe3to5pmolerange.Formorespecificsample information contact the MB Core.

Finalsamplevolumesshouldbe20microliters.Iftheyarelower,addMilli-Qwatertobringthemtovolume.TheCoreFacilityperformstheextensionreactionsand sample cleanup operations.

There are special pricing and submission requirements for full 96 well-plate submissions. Specific guidelines must be followed in setting up a full 96 well-plate. Please contact the Core facility for assistance in meeting the guidelines.

PUBLICATIONS:Stitham J, Arehart EJ, Gleim S, Douville KL, MacKenzie T, Hwa J. Arginine (CGC) codon targeting in the human prostacyclin receptor gene (PTGIR) and G-protein

coupled receptors (GPCR). Gene. 2007.396(1):180-187.Stitham J, Arehart EJ, Gleim SR, Li N, Douville K, Hwa J. New insights into human prostacyclin receptor function through natural and synthetic mutations of

transmembrane charged residues. British Journal of Pharmacology. 2007.152(4):513-22. ArehartE,StithamJ,AsselbergsF,DouvilleK,MacKenzieT,FetalveroK,GleimS,KaszaZ,RaoY,MartelL,SegelS,RobbJ,KaplanA,SimonsM,PowellR,Moore

J,RimmEB,MartinK,HwaJ.Accelerationofcardiovasculardiseasebyadysfunctionalprostacyclinreceptormutation,potentialimplicationsforCOX-2inhibition. Circulation Research. 2008.102:986-993 (Cover of Issue & Editors Pick).

PatrignaniP,DiFebboC,TacconelliS,DouvilleK,GuglielmiMD,HorvathR,DingM,KentS,StithamJ,GleimS,BaccanteG,MorettaV,DiFrancescoL,CaponeML,PorrecaE,HwaJ.Differentialassociationbetweenhumanprostacyclinreceptorpolymorphismsandthedevelopmentofvenousthrombosisandintimalhyperplasia: a clinical biomarker study. Pharmacogenetics & Genomics. 2008.18(7):611-620.

GleimS,StojanovicA,ArehartE,DouvilleK,ByingtonD,HwaJ.Conservedrhodopsinintradiscalstructuralmotifsmediateproteindestabilizingeffectsoftracemetals: Implications for Retinitis Pigmentosa Biochemistry. In press.

OPTICAL CELLULAR IMAGING

LOCATION:Borwell 338 WestDartmouth-Hitchcock Norris Cotton Cancer CenterOne Medical Center Drive | Lebanon, NH 03756 | (603) 650-7661

CraigTomlinson,PhD,FacultyDirectorCraig.R.Tomlinson@Dartmouth.edudms.dartmouth.edu/tomlinson/Assistant Professor of Medicine, Assistant Professor of Pharmacology & Toxicology

KenOrndorffMS,ManagerKenneth.A.Orndorff@Dartmouth.edu

The Optical Cellular Imaging Shared Resource strives to provide reliable and affordable access to point scanning confocal microscopy, conventional bright field and fluorescence light microscopy, and image analysis resources. The facility provides individual and group training for operation of the light microscopes. Training is intended to provide an understanding of the basic light microscopy and digital imaging principles involved. Training for our microscope use and imaging consultation is open to all members of Dartmouth College, DMS, DHMC, and outside users.

SIGN-UP SEQUENCE FOR THE OPTICAL MICRO-SCOPES AND CONFOCAL IN THE OPTICAL CELLULAR IMAGING SHARED RESOURCE:Only fully trained and authorized users will be able to see confocal schedule. All microscopes accessed 24/7 for trained users. OCI facility open M-F9:00am-5:30pmforoperatorassistedimagingbypriorarrangement.Contact resource for fee schedule. Kenneth.A.Orndorff@Dartmouth.edu

1) https://freedom7.dartmouth.edu/login2) Enter your CCOPs login name and password3) select: Cancer Center Core Operations4) select: NCCC User5) select: Additional Instruments and Services6) select: Requests/Orders7) select: Shared Instruments

Phot

o Cred

it: Ka

tie Fr

anca

, Bry

an W

ikart’

s lab

.

Zeiss LSM 510 META confocal system:7 laser lines from four different lasers

•405nm(DAPI,Hoechst,PPIX,Filipin,paGFP,FluoroGold)•TunableArgon458/477/488/514(GFP,CFP,FITC,Alexa488,YFP)•Hene543nm(Rhodamine,TxRed,Alexa555,PropidiumIodide)•Hene633nm(Alexa647,CY5,APC)

•2PMTlightdetectors(photomultipliertubes)•1Metadetector(spectraldetector,canalsobeusedasthirdPMT)•Photo-diodedetectorfortransmitted-lightimaging.•Axiovert200Widefieldinvertedfluorescencemicroscope.

Objectives available:

• 5XPlan-Apochromat/0.16NA• 10XPlan-Apo/0.45NA• 20XPlan-Apo/0.75NAwithDICcapability• 40XPlan-Neo/1.3NAOilwithDICcapability• 40XC-Apo/1.2NAWaterImmersion• 63XPlan-Apo/1.4NAOilwithDICcapability

•Pstageinsertfor37Cwork

•Objectiveheaterbandfor63XPlan-Apoobjective

Zeiss M2Bio Stereomicroscope:

This stereomicroscope system provides low magnification long working distance(stereo)imagingwithan11Xzoom.Fieldwidthsaslargeas2.5cmcanbeimaged.Themicroscopeconvertstomonocularimagingwith10Xor20XLWDobjectivesandthe11Xzoom.TransorreflectedBrightfieldandfluorescenceimagingispossible.ImagesarerecordedwitheitheraB/Worcolor digital camera using Zeiss Axiovision software.

Objectives available:•0.6X•1.0X•1.6X•10X0.45NAf=200•20X0.6NAf=200

FluorescenceCubesavailable:•Qdot705ex460SPUVv2;bs475DCXRU;emD705/20•CoumarinexD405/10,bs425DCLP;emD460/50•cGFPexD436/20;bs455DCLP;emD480/40•GFPexHQ470/40;bsQ495LP;emHQ525/50•yGFPexHQ500/20;bsQ515LP;emHQ535/30•TRITCexD540/25;bs565DCLP;emD605/55•Cy5exHQ620/60;bsQ660LP;emHQ700/75

Olympus BX 50 Fluorescence Microscope:

TheOlympusBX50uprightfluorescencemicroscopehasaUPlanFL10X/0.3,UPlanFL20X/0.5,andUPlanFL40X/0.75objective;aZeissAchroplan100X/1,25oilcanbeplacedintotheturretonrequest.FluorescencefiltercubesforDAPI(ex-BP360-370,DM400,em-BA420),FITC(ex-460-490,DM505,em-BA515IF),andRhodamine(ex-BP520-550,DM565,em-BA580IF)areavailable.

Zeiss IM-35 Inverted Microscope:

TheZeissIM-35invertedfluorescencemicroscopehasatwoposi-tionsliderthathasfiltersforPEandFITC(ex470/40_em515LP).RoutineobjectivesontheturretincludeaPlan2.5X/NA0.08,Planapo4.0X/0.16,Neofluar6.3X/0.2,Neofluar16X/0.4Ph2,F-LD20X/0.25Ph1,andaF-LD32X/0.4Ph1.Highermagnificationoilobjectives are available on request. This microscope is used primar-ilyforcheckingGFPtransfectedcelllinesandprovidingphaseimaging of cultured cells.

PUBLICATIONS:LovewellRR,CollinsRM,AckerJL,O’TooleGA,WargoMJ,BBerwin.Step-WiseLossofBacterialFlagellarTorsionConfersProgressivePhagocyticEvasion.PLoS Pathogens. 2011.

7(9):e1002253. Pasieka T, Collins L, O’Connor MA, Chen Y, Parker Z, Berwin B, Piwnica-Worms DR, DA Leib. Bioluminescent imaging reveals divergent viral pathogenesis in two strains of Stat1-

deficient mice, and in interferon receptor deficient mice. PLoS One. 2011. 6(9):e24018.Mollmark J, Ravi S, Sun B, Shipman S, Buitendijk M, Simons M, Mulligan-Kehoe MJ. Antiangiogenic Activity of rPAI-123 Promotes Vasa Vasorum Regression in Hypercholesterolemic Mice Through a Plasmin-Dependent Mechanism. Circ Res. 2011. 108:1419-1428.Ren B, Deng Y, Mukhopadhyay A, Lanahan AA, Zhuang ZW, Moodie KL, Mulligan-Kehoe MJ, Byzova TV, Peterson RT, Simons M. ERK1/2-Akt1 crosstalk regulates arteriogenesis in mice and zebrafish. J Clin Invest. 2010. Apr;120(4):1217-28. doi: 10.1172/JCI39837. Epub 2010 Mar 8.Drinane MC, Mollmark JI, Zagorchev L, Sun B, Moodie KL, Hall AE, Shipman SL, Morganelli P, Simons M, Mulligan-Kehoe MJ. The anti-angiogenic activity of rPAI-123 inhibits vasa vasorum and growth of atherosclerotic plaque. Circulation Res. 2009. 104:337-345.ParkA,WangT,MollmarkJ,ShipmanS,SimonsM,Mulligan-KehoeMJ.FGF-2GuidanceofAngiogenicVasaVasoruminAtheroscleroticMice.J. Amer. Pathology. In review.

High molecular weight FITC-dextran infusion of coronary circulation in an excised heart from an eight week old control mouse (Chittenden et al. Dev. Cell. 2006 Jun;10(6):783-95). The image was made by Thomas Chittenden, Department of Medicine, using a Zeiss M2Bio fluorescence stereomicroscope in the Optical Cellular Imaging shared resource. Scale bar 0.5 mm.

PATHOLOGY TRANSLATIONAL RESEARCH PROGRAM

LOCATION:Borwell Building 510W and 650EDartmouth-Hitchcock Norris Cotton Cancer CenterOne Medical Center Drive | Lebanon, NH 03756 | (603) 653-6023

STAFF:• RebeccaO’Meara,HT–LeadHistotechnologist Responsible for all histology requests, including special staining, immunohistochemistry, TMA \ production, and routine histology• MaryC.Schwab,BS–LeadMolecularTechnologist Responsible for Genomic applications including nucleic acid extraction/banking, real time PCR, SNP genotyping, gene expression, and miRNA expression.

Wendy A. Wells, MD, MSc, Co-DirectorWendy.A.Wells@hitchcock.orgProfessor and Chair in the Department of Pathology Medical DirectorClinical Laboratory (CAP-accredited and CLIA-certified), DHMC Board-certified in surgical and cyto-pathology with specialist expertise in breast cancer.Responsible for oversight and quality control management of:•Histology,includingspecialstains•Immunohistochemistry•TissueMicroarray(TMA)creation•Quantitativeimageprocessingandanalysis•Tissueprocurement,complexprotocols

Gregory J. Tsongalis, PhD, HCLDCo-DirectorGregory.J.Tsongalis@Dartmouth.eduProfessor of PathologyDirector, Molecular PathologyBoard certified in Molecular DiagnosticsResponsible for:•Experimentalplanningandimplementation

support•Dailyoperations•Platformacquisition•Externalclinicaltrials,evaluationsand funding resources•Totalqualitymanagement(PTtesting,

accreditation/inspection)

Carol R. Hart, Program AdministratorCarol.R.Hart@Hitchcock.org(603) 650-6821Program AdministratorPathology Translational Research Program

Our mission is to facilitate project planning, clinical validation and implementation of novel translational technology and research in the fields of molecular diagnostics, molecular therapeutics, pharmacogenomics, quantitative morphologic image analysis and immunohistochemistry (IHC) in a CLIA-certified, CAP-accredited laboratory ensuring optimal clinical quality assurance. This past year, the TRL expanded its physical footprint by moving all of the histology services to a 5th floor research module in order to reorganize and expand the molecular biology and imaging services in the 6th floor lab.

OUTLINE OF SERVICES:Animal studies Tissue processing/block creation Histology Unstained section Hematoxylin and Eosin (H&E) Stained section Special stains

Human studies Tissue processing/block creation Histology Unstained section Hematoxylin and Eosin (H&E) Stained section Special stains Tissue procurement Freshtissue,customized (protocol-based) Tissue bank retrieval (fresh frozen)

Immunohistochemistry Routine Antibodies Testing of Antibodies (protocol-based) Antibody purchase Quantitativeimageprocessingand analysis (protocol-based) Tissue microarrays creation and use (protocol-based) Standard Customized Molecular pathology: molecular diagnostic testing development and validation, SNP genotyping, gene expression, miRNA expression (protocol-based) Clinical pathology laboratory testing (protocol-based) Apheresis collection (protocol-based)

Clinical trial studies: Tissue processing (protocol-based) Histology (protocol-based)

Administrative requests (handling) Off-siteblockand/orslideretrieval LIS review (protocol-based) CIS review (protocol-based)

PUBLICATIONS:Black CC, Bentley HA, Davis TH, Tsongalis GJ. Use of a linear array for the detection of human papillomavirus genotypes in head and neck cancer. Arch Pathol Lab Med. 2010. Dec

134(12):1813-1817.DragnevKH,MaT,CyrusJ,GalimbertiF,MemoliV,BuschAM,TsongalisGJ,SeltzerM,JohnstoneD,ErkmenCP,NugentW,RigasJR,LiuX,FreemantleS,KurieJM,WaxmanS,

Dmitrovsky E. Bexarotene Plus Erlotinib Suppress Lung Carcinogenesis Independent of KRAS Mutations in Two Clinical Trials and Transgenic Models. Can Prev Res. 2011. Jun 4(6):818-828.

FuldAD,SpeckME,HarrisBT,SimmonsNE,CorlessCL,TsongalisGJ,PastelDA,HartfordAC,ErnstoffMS.PrimaryMelanomaoftheSpinalCord:Acasereport,molecularfootprintand review of the literature. J Clin Oncol. Jun. 2011 10;29(17):e499-502.

ChenH,LeffertsJA,SchwabMC,SuriawinataAA,TsongalisGJ.Correlationofpolypoidcolorectaladenocarcinomawithpre-existingadenomatouspolypsandKRASmutation.Cancer Genet, Apr 204:245-251, 2011.

RimmDL,NielsenTO,JewellS,RohrerDC,BroadwaterG,WaldmanF,MitchellK,SinghB,TsongalisGJ,FrankelWL,MaglioccoAM,LaraJF,HisED,BleiweissIJ,BadveS,ChenB,Ravdin PM, Schilsky RL, Thor A, Berry DA. CALGB Pathology Committee Guidelines for tissue Microarray Construction Representing Multi-Center Prospective Clinical Trial Tissues. J Clin Oncol, Jun 29(16):2282-2290; 2011.

MarottiJD,SchwabMC,McNultyNJ,RigasJR,DeLongPA,MemoliVA,TsongalisGJ,PadmanabhanV.CytomorphologicFeaturesofAdvancedLungAdenocarcinomasTestedforEGFRandKRASMutations:ARetrospectiveReviewof50Cases.Diagn Cytopathol J, Jun 16, 2011. 10.1002/dc.21749. [Epub ahead of print]

PetrasML,LeffertsJA,SuriawinataAA,TsongalisGJ.KRASDetectioninColonicTumorsViaDNAExtractionfromFTAPaper:TheMolecularTouch-Prep.Diagn Molec Pathol, Dec 20(4):189–193,2011.

Petty WJ, Voelzke WR, Urbanic JJ, Varela VA, Waller LL, Swift CB, Graham RM, Memoli VA, Dragnev KH. High cyclin D3 expression confers erlotinib resistance in aerodigestive tract cancer. Lung Cancer. 2011 Dec;74(3):384-91. Epub 2011 May 8.

PakalniskisMG,WellsWA,SchwabMC,FroehlichHM,JiangS,LiZ,TostesonTD,PoplackSP,KaufmanPA,PogueBW,PaulsenKD.Tumorangiogenesischangeestimatedbyusingdiffuseopticalspectroscopictomography:demonstratedcorrelationinwomenundergoingneoadjuvantchemotherapyforinvasivebreastcancer?Radiology. 2011 May;259(2):365-74. Epub 2011 Mar 15.

KuemmerleNB,RysmanE,LombardoPS,FlanaganAJ,LipeBC,WellsWA,PettusJR,FroehlichHM,MemoliVA,MorganelliPM,SwinnenJV,TimmermanLA,ChaychiL,FricanoCJ,Eisenberg BL, Coleman WB, Kinlaw WB. Lipoprotein lipase links dietary fat to solid tumor cell proliferation. Mol Cancer Ther. 2011 Mar;10(3):427-36. Epub 2011 Jan 31.

TURNAROUND TIME:Turnaround time is dependent on scale of project.

DROP OFF:Drop-offsiteisavailable24/7inthehistologylabintheBorwellBuildingonlevel4,aslongasrefrigerationisnotneeded.Ifsamplesneedtoberefrigerated please contact program administrator and set up a time, during business hours, to deliver samples.

SAMPLE REQUIREMENTS:Samples for routine histology must be in cassettes and placed in a container with 10% formalin. Samples requiring alternative handling must be discussed/scheduled with the program administrator.

DATA INTERPRETATION HELP IF APPLICABLE: The PTRP Core personnel are available to assist with data review. Please contact the program administrator to schedule a meeting date/time.

SPEED CONGENICS/DARTMOUSE

LOCATION:610W BorwellDartmouth-Hitchcock Norris Cotton Cancer CenterOne Medical Center Drive | Lebanon, NH 03756 | (603) 650-8686Dartmouse@Dartmouth.edu

James Gorham, MD, PhDJames.D.Gorham@Dartmouth.eduProfessor of Pathology, Professor of Microbiology and Immunology, Head of MD/PhD ProgramResearch Interest: The principle research goal in the Gorham laboratory is to understand the biological mechanisms regulating T helper cells, immune tolerance, and autoimmunity in the liver.

Matthew RansonMatthew.S.Ranson@Dartmouth.eduLaboratory Manager

OUTLINE OF SERVICES:Mouse 1449 SNP; Genome-wide Genetic Background AnalysisMouse 1449 SNP;2-Strain Genetic AnalysisMouse 1449 SNP;3-Strain Genetic AnalysisMouse genomic DNA Extraction (tail)NearFuture:PCRbasedTG/KOgenotyping

Facilitates the development of congenic mice in support of pre-clinical projects; provides expert advice on mouse speed congenic development, mouse genetic background analysis, and mouse genetic mapping.

EQUIPMENT CAPABILITIES AND OTHER PROCESSES:Illumina BeadArray Reader Workhorse instrument for our SNP analysis Capable of scanning up to three BeadChips at one timePromega Maxwell Automated DNA extraction in 45 minutes Capable of extracting DNA from up to 16 samples concurrentlyQiagen QIA-Xcel Advanced Capillary Electropheresis of PCR amplicons Automated loading Capable of finer resolution than agarose gel Results in ~15 min

PUBLICATIONS:Aktan I, Chant A, Borg ZD, Damby DE, Leenstra PC, Lilley GW, Petty J, Suratt BT, Teuscher C, Wakeland EK, Poynter ME, Boyson JE. Slam haplotypes

modulate the response to lipopolysaccharide in vivo through control of NKT cell number and function. J Immunology. 2010. 185:144-56.

Pasieka T, Collins L, O’Connor MA, Chen Y, Parker ZM, Berwin B, Piwnica-Worms DR, Leib DA. Bioluminescent imaging reveals divergent viral patho-genesis in two strains of Stat1-deficient mice, and in αβγ interferon receptor-deficient mice. PLOS One. 2011. 6(9): e24018.

Gogliotti RG, Lutz C, Jorgensen M, Huebsch K, Koh S, DiDonato CJ. Characterization of a commonly used mouse model of SMA reveals increased seizuresusceptibilityandheightenedfearresponseinFVB/Nmice.Neurobiology of Disease. 2011. 43:142-151.

SERVICE PROCESS, STEPS :Mouse SNP Genotyping Servicei. The first step in use of the DartMouse SNP genotyping services is to visit our website (www.dartmouse.org) and download the latest copy of our

accessionform.Fillthisformoutcompletelyandreturnittothelabthroughemail.ii. Once this form has been received, the lab manager will review the project and contact the client to discuss the project. This usually happens within

one business day of receiving the form.iii. Once the form has been reviewed and okayed by the DartMouse lab, users submit tail clippings directly to our lab for each mouse they would like

analyzed. These tail clippings should be between 0.5cm and 1.0cm in length, and placed in well labeled containers, such as microcentrifuge tubes. Tissue samples are preferred to previously extracted DNA.

iv. Once the DartMouse lab has received these samples, we will analyze each sample fully, and return chromosome maps and a formal written report to the client within two weeks.

v. Billing will take place following completion of the project and following receipt of the results by the client. DNA extractioni. At the present time, DartMouse can only extract DNA from mouse tissue.ii. ForDNAextractionservices,preparealistofthesamplesyouplanonsubmittingforextraction.Ifyouareplanningtobringmorethan20samples

for extraction at one time, email the DartMouse lab to let us know.iii. Place each tissue sample into a well labeled microcentrifuge tube.iv. Bringthesamplestothelab,andmakesureyoudropthemoffwithoneofouremployees.v. The extracted DNA, along with a sheet with the DNA concentrations and purity will be ready for pick-up the next day.

This is an example of a chromosome map that would be returned with any given project’s report.

TRANSGENIC AND GENETIC CONSTRUCT

LOCATION:Rubin 622Dartmouth-Hitchcock Norris Cotton Cancer CenterOne Medical Center Drive | Lebanon, NH 03756 | (603) 653-6023

StevenFiering,PhDFiering@Dartmouth.edu(603) 653-9966Our lab studies the cancer immunology of ovarian and melanoma cancers. We currently are using treatments with bacteria or nano-particles to decrease or eliminate tumors

JenniferFields,BSJennifer.L.Fields@Dartmouth.eduJennifer is a veterinary technologist/research assistant. She has extensive skills in creating transgenic mice and performing mouse surgeries. She has been with this core since 1997.

We support the generation and utilization of genetically modified mice by members of the Dartmouth research community.

We offer a wide variety of services that assist researchers in construct design and creation of novel transgenic mice. We also offer mice with a human immune system that allows our investigators a better way to study virus and other human infections.

PUBLICATIONS:UcianeK,ScarlettUL,RutkowskiMR,RauwerdinkAM,FieldsJ,Escovar-FadulX,BairdJ,Cubillos-RuizJR,JacobsAC,GonzalezJL,WeaverJ, FieringS,Conejo-GarciaJR.OvarianCancerProgressionisControlledbyPhenotypicChangesinDendriticCells.Journal of Experimental Medicine.

2012.

OchielDO,RossollRM,SchaefeTM,WiraCR.Effectofoestradiolandpathogen-associatedmolecularpatternsonclassII-mediatedantigenpresentation and immunomodulatory molecule expression in the mouse female reproductive tract. Immunology.2012.135:51–62.doi:10.1111/j.1365-2567.2011.03512.x

FreemantleSJ,DmitrovskyE.CyclinEtransgenicmice:discoverytoolsforlungcancerbiology,therapy,andprevention.Cancer Prev Res. 2010;3:1513–8.

CoonCI,FieringS,GaudetJ,WyattCA,BrinckerhoffCE.Sitecontrolledtransgenicmicevalidatingincreasedexpressionfromhumanmatrixmetalloproteinase (MMP-1) promoter due to a naturally occurring SNP. Matrix Biology. Volume 28, Issue 7, September 2009, Pages 425-431, ISSN0945-053X,10.1016/j.matbio.2009.06.003.

OUTLINE OF SERVICES:• Provideconsultationontransgenicmiceconstructdesignandproduction,overallexperimentaldesign,utilization,maintenance,breeding,and

preservation• GeneticconstructgenerationUserecombineeringinE.colioryeasttogenerateconstructsforexperimentaluse• Oocyteinjectiontransgenicproduction.Generatetransgenicfoundersbyinjectionofplasmids,BAC,orlentivirusintooocytesandtransplantthe

oocytes in foster mothers• EScellcultureforgenetargetingorotherpurposes• ProvideavarietyofEScelllines,transfectwithKOconstructs,selectcloneswithdrugselection,freezeandprovidelysatesforgenotyping,

expand and refreeze targeted clones for mouse generation, karyotype clones to select best clones• GenerationofchimericmicefromEScells.InjectEScellsintoblastocyststogenerategermlinechimericmice• Establishlinesfromfrozenembryosfromotherinstitutionsorrederiveapathogeninfectedtransgeniclinebyembryotransfer• Genotyping.PurifyDNAfromESclones(toscreenfortargetedclones)ortailbiopsies(toscreenforfoundersortransgenicoffspring).Establisha

genotype assay if not already established.Genotype by PCR or Southern blotCryopreservation of embryos and sperm to preserve & protect your valuable transgenic mouse lines

• Humanizedmice.Creatingspecializedmicewithahumanimmunesystemandhumanlivers.CurrentlyofferingtwodifferenttechniquesusingNod/SCIDIL2gammareceptorXOmice

• Otherservices.Tailbiopsies,tailveininjections,ovariectomies,vasectomies,embryotransfer,blooddrawing,timedpregnancies,superovulations,maintaintransgeniccolonies(husbandry),maintainingFlpandcreexpressingmouselines