CTRF Leadership Meeting September 9, 2002 Institutional Partners V C U G M U I N O V A 8/05/02 Minutes Corrections Approval Develop Infrastructure and Intellectual Property that Enhances the Competitiveness of the Partners for Clinical and Extramural Funds Principle Objective 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 Funding From CTRF New Account Numbers for CTRF New Account Numbers for FY Torr Central Garrett INOVA Ginder GMU Guiseppi-Eli FY2002 Account Balances Accounts will be closed as of September 30, 2002 Reallocation of CTRF Cancer Genomics Fiscal Yr. 1 Funds Inova & GMU Invoice for August Expenditures List of non-personnel expenditures that have not been invoiced List of non-personnel invoices through September 14 th List of personnel expenses September, October, November Reallocation of CTRF Cancer Genomics Fiscal Yr. 1 Funds NARF (Buck) Personnel expenses September, October, November (Balance and Commitments to be determined from FRS) Reallocation of CTRF Cancer Genomics Fiscal Yr. 1 Funds Total Accounts balance less than $ 200,000 by September 30, 2002 Request new allocation October 1, 2000 Cost share expenditures not paid from cost share linked accounts must be documented using In Kind/3rd Party Cost Share form obtained from Margie Bookers office. Cost Share Expenses (http://www.vcu.edu/finance/ In-kind%20Cost%20Sharing%Certification.pdf) Reminder - Cost Share Form (VCU) Website Update Website has been completely redesigned Information regarding focus groups, news, or updates are welcome SPIN Research Jo Ann Breaux receiving daily notices of grant opportunities Compiling weekly document of relevant findings Will be distributed over the CTRF LISTSERV SMART documents currently on the CTRF website Training is available: Tissue Bank Clinical and Pathology Laboratory Data Database Design Data Analysis Quality Assurance in Microarray Analysis Chip Fabrication Focus Group Focus Group Leaders INOVA -CTRF - Tissue Bank IRB has approved Tissue Acquisition at INOVA Consent form resubmitted to the IRB with changes to reflect the request that the consent would be signed after the biopsy was performed Marianne Smiths group has identified Eileen Kelley as coordinator for tissue acquisition project Study coordinator to obtain informed consent and acquisition VCU Tissue Bank Manual Form for Tissue Acquisition Histopathologic Parameters Tissue Acquisition Database 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 CTRF Ca Genomics Project Tissue Utilization Group Project Pis Garrett Buck Ginder Guiseppi-Eli Cooper Chandhoke Tissue Guardians Nasim Grant Cook Clinical Data Leadership Penberthy Smith Quality Assessment Leadership Ferreira-Gonzales Christensen Taylor Issues QA Program Pre-Analytical Tissue Handling Storage Conditions (Freezer Monitoring, etc) Manner in which tissue is supplied Storage and availability of data Devitalization of Tissue Dr Nasim and Dr Grant Tissue Devitalization Ovarian samples collected VCU Tissue to be snap frozen over a time series Immediate snap freezing (time 0) Freeze extra pieces at 15, 30 and 60 and 120 minutes (from time 0). Frozen sections will be performed to monitor for purity of tumor. Sections cut and placed directly in TRIZOL and distributed. RNA extracted and analyzed by both VCU and GMU teams for integrity over the period of experimentation Temporary Storage (If appropriate) -80C freezer monitored 24hr X 7 days with daily recording of temperature; or, -80C freezer monitored 5 days/wk with week day (including holiday) daily recording of temperatures and with liquid nitrogen back-up capable of maintaining -40C for 72hr. (Note: liquid nitrogen back-up must include written quality assurance plan to insure that liquid N 2 maintained at satisfactory levels). Permanent Storage -80C freezer maintained on an emergency power line (ie, line with back-up generator in case of system wide power loss) monitored 5 days/wk with week-day (including holiday) daily recording of temperatures and with liquid nitrogen back-up capable of maintaining -40C for 72hr. (Note: liquid N 2 is maintained at satisfactory levels). Laboratory must have a plan for emergency storage of tissue specimens in the event of freezer failure including persons responsible to respond in the event of freezer failure and identification of alternative freezer space. Tissue Storage Amount of Tissue Based on current accessioning practices for solid tumors (ie, breast, ovary [no VCU experience yet with brain]) it is anticipated that each specimen will consist of approximately 200mg total weight and will contain 0.5 2.5 g total RNA per mg of tissue for a total of g total RNA per sample. GMU requires ~20 g per run. VCU requires ~10 g per run for the Affymetirx platform. Supplying Tissue to Project PI Labs Preparation of Tissue Samples In order to minimize the chances for RNA degradation and to monitor percent tumor cellularity as well as other potential confounding morphological changes (ie, inflammation, necrosis, desmoplasia, etc), tissue will be handled as follows: blocks of tissue will be maintained frozen at all times. blocks of tissue will be sectioned (5 thick) on a cryostat. no more than sections will be taken from any one sample without removal of a section for frozen section histology. the tissue sections will be placed directly into cold Trizol, dispersed by vigorous shaking at the time of sectioning and maintained at -80 o C for storage and -37 o C (dry ice temp) for transportation. (Note: participating laboratories may request transportation solution other than Trizol based on their specific needs and experience.) the standard sample will consist of Trizol with 40 section of tissue or the equivalent amount of total RNA. Institutional Tissue Utilization Committee Formulate criteria for who is eligible to obtain human residual samples at the institution. Faculty status, IRB approval, ?scientific validity ?minimum QA requirements ?minimum data access requirements Review requests for human tissue utilization. Formulate criteria for the degree of clinical information which can be provided with the samples. Assess resources required to fill request and whether PI is prepared to provide them. Tissue Utilization Summary VCU Tissue Committee Inova Tissue Committee CTRF Tissue Utilization Group Analyze Samples CTRF CA GENOMICS TISSUE UTILIZATION - PLAN Monitoring RNA Quality Purity contaminants - DNA, Protein, phenol DNAdetect DNA via fluorescence Protein260/280 ratio phenol270 absorbency DNA Affy ribosomal genes (see Excel file: uChipControlGenes.xls) Integrity 18S/28S ratio PAGE, LabChip-electropherogram housekeeping genes (affy)GAPDH, -actin (ratio3'/5' = 1) low abundance genes (affy)ISGF-3A (ratio 3'/5' 3) Labeling Process cRNAyield (= g/cRNA/g total RNA) size range cRNA Fragmentationsize range Labeling Efficiencyspotted array -~1kbp DNA fragment of Lambda A DNA on slides + include Lambda A polyA RNA in labeling reactions affy arrays - see gene list uChipControlGenes.xls RNA Degradation and Gene Expression Goal To assess the impact of sample RNA degradation on the gene expression data from microarrays Protocol Sample Type - Total RNA from the Ovary Sample (OV T) Degradation Experiment different degrees of degradation (to be determined) RNA Degradation and Gene Expression Analysis of the sample RNA degradation reflected by rRNA 28S/18S ratio (Agilent Bioanalyzer, Denaturing Gel, etc) For Example : Agilent Ratio Sample Sample 21 Sample 30.5 Sample 40.1 RNA Degradation and Gene Expression Microarray Data Analysis: Find a common parameter to compare data across platforms (Ratio between Control genes for all the platforms against 1 Control Gene within the linear range of the system) Check for changes in 35 ratios for Housekeeping genes Low and high abundance genes Check for changes in gene calls (P A) Data Analysis Control Genes (GMU & VCU) Within Platform Performance Assessment Each PI will perform quality assessment designed to measure the degree of variation due to each of the critical steps in the generation of results. PI Laboratories will use the Stratagene Human Reference mRNA (Product #: ) ( lot number can be obtained from A. Christensen or A. Ferreira-Gonzalez) Written report summarizing findings will be prepared by PI laboratories Copy of report/manuscript will be provided to the Project Directors Office Data for study to be treated as Project Data Example Within Platform Quality Control Study for Affymetrix GeneChip System All chips hybridized to cRNA prepared from Stratagene Human Reference mRNA is Product Number (lot number is ) Database Design/Clinical Info Clinical Data Elements (Update for October Meeting) Define minimal set of common clinical data elements; Initial choice to be the elements required to be sent to state cancer registry Data elements should include MIAME (Minimum Information about a Microarray Experiment) for holding Expression Array Data GeneX Database Initial choice for storing project data GeneX Database (Update) Version 1.5 UVA (Jae K. Lee) Version 2.0 GMU-VT (J. Weller) Developer meeting planned in October Database Design/Data Analysis Establish Standing Weekly or Biweekly Meeting Dates and Times Dont Forget! Complete the Milestone Updates Document Discussions and Progress Using Listservs 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 Address messages to: Unsubscribe to the listserv by submitting a message with the words SIGNOFF listname to: Subscribe to the listserv by asking the PI with whom you work to submit your name andaddress to the Program Director (C.Garrett) USE the listserv(s) to inform members of your activity or to seek advice from the members. LISTSERVS Old Business New Business 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. 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 Monday October 7, :30AM