© 2002 VCU CTRF Leadership Meeting December 9, 2002 Institutional Partners V C U G M U I N O V A

© 2002 VCU 11/04/02 Minutes Corrections Approval

© 2002 VCU Develop Infrastructure and Intellectual Property that Enhances the Competitiveness of the Partners for Clinical and Extramural Funds Principal Objective

© 2002 VCU  Evaluate gene expression (and genetic changes) in human brain, ovarian, breast and hematopoetic cancers  Link gene expression (and genetic changes) to clinical findings and clinical laboratory findings (including histopathological diagnoses) in a common database  Evaluate linked data using bioinformatics Research Objective

© 2002 VCU Funding for CTRF

© 2002 VCU FY02 Funds Pending State has approved a transfer of $500,000 to VCU for the new CTRF accounts Allocation of money into the accounts is pending action by VCU Grants and Contracts

© 2002 VCU Account Balances as of 12/03/02

© 2002 VCU CTRF YR02 Initial Budget Allocation * Year 2 Modified Budget distribution based on State allocation of $500,000 as of 11/2002

© 2002 VCU  Cost share expenditures not paid from cost share linked accounts must be documented using ‘In Kind/3rd Party Cost Share form’ obtained from Margie Booker’s office. ( In-kind%20Cost%20Sharing%Certification.pdf) Cost Share Expenses

© 2002 VCU Cost Sharing Report

© 2002 VCU Cost Sharing Report * GMU cost share documented on report signed by GMU PI 10/2/02 VCU cost sharing must be documented in the correct cost sharing accounts.

© 2002 VCU Reminder Cost Share Form (VCU)

© 2002 VCU  Website still incomplete; information regarding focus group activities is needed Website Update

© 2002 VCU  Jo Ann Breaux receiving daily notices of grant opportunities  Compiling weekly document of relevant findings  Monthly SMART documents currently on the CTRF website Training is available: SPIN Research

© 2002 VCU Focus Groups Tissue Bank Clinical & Pathology Laboratory Data Database Design Chip Fabrication QA/QC Data Analysis

© 2002 VCU Focus Group Leaders

© 2002 VCU VCU Tissue Bank

© 2002 VCU IRB has approved tissue acquisition system for INOVA Dr. Dorriane Watts to replace Marianne Smith as Interim Director of Research Renee Brenner to be collecting specimens for INOVA currently; a new permanent coordinator to be hired INOVA – CTRF – Tissue Bank

© 2002 VCU Access Database –Computer has been installed at INOVA –Database has been installed on machine at VCU –INOVA connected to database at VCU using PC Anywhere ( ) Update of Database for Histopathologic parameters of existing cases needed - Completed Tissue Acquisition Database

© 2002 VCU

** Data incomplete for this tissue type

© 2002 VCU ** Data incomplete for this tissue type

Study ID Tissue ID Sample ID Sub-sample ID Study ID SSN Clinical Data Model Clinical Data Model (VCU) - Primary: Data Collection Secondary: Queries, Data Reduction, Anonymization Tertiary: Analysis & Hypothesis Testing AFFY TISSBK & 1 o CLINICAL & Consent CERNER PathShadw REGISTRY CLAIMS Clinical Data Repository SPOTTED Gene Expression Non-genetic predictors Treatments Outcomes MRN SSN ACCSN SEQ MRN SSN PAN MRN SSN Path Accsn Study ID Lab ID Tissue ID Run ID CEL file data Spot data Experimental (Metadata) Reg Shadw GeneX Clinical Risk Factors Treatments Outcomes Histopath Risk Factors Path Dx Clin Lab Expanded GeneX Table: Consent Info Tables: Demogrphs Risk Factrs Nutirtion Comorbidty etc Tables: Extract Info StorageInfo Usage Info etc Tables: Histopath parameters Path Dxs SNOMED Text Repts Tables: Tumor info Treatment Follow-up etc Tables: Surg Tx Medical Tx Radiatin Tx other dxs

© 2002 VCU GMU Informatics Update Create or Identify existing databases into which expression microarray data can be stored in electronic format in real time at this juncture. –Identified GeneX as candidate microarray database. –Worked with GeneX developers and UVA to modify GeneX to accept both cDNA and Affymetrix gene expression data –Instantiated new version of GeneX –Defined new LIMS schema for data management Create or Identify existing databases into which clinical, laboratory, tissue bank information, and expression microarray can be stored in electronic format in real time at this juncture. –Examined several available clinical databases and found none to be sufficient in terms of performance and flexibility. –Used CGO as starting basis to generate new clinical schema. –Currently implementing clinical databases. Create ODBC links between separate databases containing clinical, laboratory, and tissue bank data. –In progress.

© 2002 VCU CTRF CA GENOMICS TISSUE UTILIZATION - PLAN

© 2002 VCU Choice of the RNA Extraction Procedure for Best Microarray Results (I) Starting material: 10  m OCT sections of snap- frozen tissue (in liquid N 2 ) T RIZOL (Invitrogen) Or T RIZOL (Invitrogen) + RNeasy cleanup (QIAGEN)

© 2002 VCU T RIZOL + RNeasy cleanup T RIZOL RNA extraction ds cDNA synthesis 1,500 bp ~ 50 bp 28S/18S ratio: S/18S ratio: 1.8

© 2002 VCU Results (I) By using T RIZOL we obtained undegraded RNA (28S/18S >1.5) but the cDNA synthesis was inhibited (accumulation of short, ~50 bp, molecules). By cleaning up the RNA isolated using T RIZOL with the RNeasy cleanup protocol, we obtained cDNA molecules of greater size, with a max. peak at ~1,500 bp.

© 2002 VCU Choice of the RNA Extraction Procedure for Best Microarray Results (II) Starting material: Snap-frozen tissue (in liquid N 2 ), 10  m OCT sections dumped in a solution containing guanidinium thiocyanate (RNAse inhibitor): T RIZOL (Invitrogen) + RNeasy cleanup (QIAGEN) or RLT from RNeasy - Solution D ( Chomczynski P and Sacchi N)

© 2002 VCU T RIZOL + RNeasy cleanup RNeasy Isolation 28S/18S ratio: S/18S ratio: 0.2 RNA extraction ds cDNA synthesis 1,500 bp 500 bp

© 2002 VCU Results (II) By using the RNeasy RNA isolation protocol from breast tissue sections, we obtained total RNA with 28S/18S ratios << 1.5, and the cDNA molecules were shorter than expected (max. peak at ~500 bp). Therefore, we decided to isolate the RNA using T RIZOL followed by the RNeasy cleanup protocol, to ensure cDNA molecules of greater size, (max. peak at ~1,500 bp).

© 2002 VCU Congratulations to…. Young Investigator Award QUALITY CONTROL AND QUALITY ASSURANCE IN MICROARRAY DATA ANALYSIS Dumur CI(1), Best A(2), Garrett CT(1), Nasim S(1), Wilkinson DS(1) and Ferreira-Gonzalez A(1). (1)Department of Pathology, (2)Department of Biostatistics, VCU, Richmond, VA 23298

© 2002 VCU Devitalization of Tissue Dr. Nasim and Dr. Grant

© 2002 VCU Breast samples collected VCU –Tissue to be snap frozen over a time series (15, 30, 60, 120 minutes) Sections cut and placed directly in TRIZOL –Problem – Different blocks of tissue differed significantly in amount and viability of cancer cells (Pathologist review) –Outcome – repeat study with new cancer specimen Tissue Devitalization

GMU - Quality Control Protocol for Custom Spotted Arrays (Process for Single Run) Cy3 Cy5 Pool cDNA – 5000 Probes - Probe Excess - Includes Control Genes and Lambda 2 X 5 Labeling Reactions BCADE Slides A thru E

Quality Control Protocol for Custom Spotted Arrays (Process for Single Run) Slide ASlide BSlide CSlide DSlide E Hybrid Chambers Chamber 1 Chamber 2 Chamber 3 Chamber 4 Chamber 5 Hybridization Oven

Quality Control Protocol for Custom Spotted Arrays (Process for Single Run) A BC S C A N

Measures of Experimental Variances VarianceComparison Labeling ReactionPooled Reactions vs. Individual Reactions Slide VariationChanges between individual slides for pooled reactions (factoring in effect of different hybridization chambers over separate runs) Hybridization Chamber Differences Changes of mean between chamber hybridizations for multiple runs (factoring in effect of between slide variation). Run to Run Variability factors: Wash solutions hybrid oven temp handling – other Between run comparisons of gene expression intensities controlled for hybridization chamber over multiple runs

© 2002 VCU Human reference RNA (aRNA) 5 labeling reactions with Cy3 5 labeling reactions with Cy5 Pool of Cy3 5 independent hybridizations: same time & temp

© 2002 VCU Comparison of the variability between different days and different chambers Same day, different chambers Same chamber, different days

© 2002 VCU Normalization with the median Filtering of the data based on the value of negative controls Ratio between Cy5 and Cy3

© 2002 VCU Frequency distribution of Cy5/Cy3 ratio

© 2002 VCU Frequency distribution of the % error of Cy5/Cy3 ratio

© 2002 VCU Percent of genes detected in Ch1, Ch2, and both channels. Total genes: 5297

© 2002 VCU  Establish Standing Weekly or Biweekly Meeting Dates and Times  Complete the Milestone Updates  Document Discussions and Progress Using Listservs CTRF – Promoting Focus Group Activity

© 2002 VCU  CG-TISBK: Tissue Bank  CG-CLNDT: Clinical and Pathology Data  CG-DBDSN: Database Design  CG-ANLDT: Analyze Data (Data Analysis)  CG-QAQC: QAQC  CG-LDRPI: Focus Group Leaders and PIs  CG-MEMBS: All Members  CG-FBCHP: Chip Fabrication Communication Amongst Members and Focus Groups

 5/21/ million (1yr) submission to VTSF (Penberthy-PI)  “Early Clinical Trials of Imaging Agents” –contract to permit the VCU Molecular Imaging Center to respond to subsequent specific RFPs for development of new imaging agents.  10/01/02 – 1.5 Million – WT1 As A Determinate of Ovarian Cancer Cell Genotype  10/02 – $500,000 - “Genomics and Other Risk Factors for Oral Cancer Outcomes” (Penberthy)  1/1/02 – $42,000 – Gleevec/Novartis – “Phase I Label Study of Combination of Gleevec with Cisplatin and Etopside for Previously Untreated Extensive Stage Small Lung Cancer” (Nasim)  10/1/02 - $200,000 – “Digestion Chain Reaction (DCR) to identify differentially expressed Genes” (Ping Xu)  Any other discoveries  Federal money leveraged  Private research money leveraged  Advancement of technology and economic development in VA CTRF - Specific Reportables - - Reminder - -  Intellectual property reporting - licenses, patents, etc  Publications  New applications  CTRF Administrative office  will search for new funding opportunities (SPIN)  will collect CVs, other support, facilities, interest documents  goal million in D.C. from CTRF CG Project

© 2002 VCU  Old Business  New Business  Annual Report due December 31, 2002 – Infrastructure created  Samples collected  Samples extracted & arrayed  Samples analyzed  Publications  Grants submitted/awarded

© 2002 VCU Updates for Annual Report Needed NOW!!!

Monday January 13, :30am Next Leadership Meeting

© 2002 VCU