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CTRF Leadership Meeting

CTRF Leadership Meeting. Cancer Genomics and Development. of Diagnostic Tools and Therapies. December 9, 2002. Institutional Partners. V C U. G M U. I N O V A. 11/04/02. Minutes. Corrections Approval.

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CTRF Leadership Meeting

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  1. CTRF Leadership Meeting Cancer Genomics and Development of Diagnostic Tools and Therapies December 9, 2002 Institutional Partners V C U G M U I N O V A

  2. 11/04/02 Minutes Corrections Approval

  3. Develop Infrastructure and Intellectual Property that Enhances the Competitiveness of the Partners for Clinical and Extramural Funds Principal Objective

  4. 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

  5. Funding for CTRF

  6. 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

  7. Account Balances as of 12/03/02

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

  9. Cost Share Expenses • 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. (http://www.vcu.edu/finance/ In-kind%20Cost%20Sharing%Certification.pdf)

  10. Cost Sharing Report

  11. 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.

  12. Reminder Cost Share Form (VCU)

  13. Website Update • Website still incomplete; information regarding focus group activities is needed www.ctrf-cagenomics.vcu.edu

  14. SPIN Research • 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: http://www.InfoEd.org/default.stm

  15. Focus Groups Tissue Bank Clinical & Pathology Laboratory Data Database Design QA/QC Data Analysis Chip Fabrication

  16. Focus Group Leaders

  17. VCU Tissue Bank

  18. INOVA – CTRF – Tissue Bank • 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

  19. 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 (8-20-02) • Update of Database for Histopathologic parameters of existing cases needed - Completed

  20. Diagnosis Distribution - Breast

  21. Diagnosis Distribution - Ovary **Data incomplete for this tissue type

  22. Diagnosis Distribution - Hematopoietic Neoplasia **Data incomplete for this tissue type

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

  24. 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.

  25. CTRF CA GENOMICS TISSUE UTILIZATION - PLAN QA Program

  26. Choice of the RNA Extraction Procedure for Best Microarray Results (I) Starting material: 10 mm OCT sections of snap-frozen tissue (in liquid N2) • TRIZOL (Invitrogen) Or • TRIZOL (Invitrogen) + RNeasy cleanup (QIAGEN)

  27. RNA extraction ds cDNA synthesis TRIZOL + RNeasy cleanup 28S/18S ratio: 1.9 1,500 bp ~ 50 bp 28S/18S ratio: 1.8 TRIZOL

  28. Results (I) • By using TRIZOL 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 TRIZOL with the RNeasy cleanup protocol, we obtained cDNA molecules of greater size, with a max. peak at ~1,500 bp.

  29. Choice of the RNA Extraction Procedure for Best Microarray Results (II) Starting material: Snap-frozen tissue (in liquid N2), 10 mm OCT sections dumped in a solution containing guanidinium thiocyanate (RNAse inhibitor): • TRIZOL (Invitrogen) + RNeasy cleanup (QIAGEN) or • RLT from RNeasy - Solution D (Chomczynski P and Sacchi N)

  30. RNA extraction ds cDNA synthesis TRIZOL + RNeasy cleanup 28S/18S ratio: 1.9 1,500 bp 28S/18S ratio: 0.2 500 bp RNeasy Isolation

  31. Results (II) • By usingthe RNeasyRNA 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 TRIZOL followed by the RNeasy cleanup protocol, to ensure cDNA molecules of greater size, (max. peak at ~1,500 bp).

  32. Congratulations to…. Dr. Catherine Dumur 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

  33. Devitalization of Tissue Dr. Nasim and Dr. Grant

  34. Tissue Devitalization • 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

  35. Cy3Cy5 Pool 1 1 2 2 3 3 4 A B C D E 4 5 5 GMU - Quality Control Protocol for Custom Spotted Arrays (Process for Single Run) A. Labeling B. Chip Fabrication 2 X 5 Labeling Reactions • cDNA • – 5000 Probes • Probe Excess • - Includes Control Genes and Lambda Slides A thru E

  36. Quality Control Protocol for Custom Spotted Arrays (Process for Single Run) C. Hybridization Slide/Hyb Chamber Variability = between slide variability - pooled rxns Slide A Slide B Slide C Slide D Slide E Hybridization Oven Chamber 1 Chamber 2 Chamber 3 Chamber 4 Chamber 5 Hybrid Chambers

  37. Quality Control Protocol for Custom Spotted Arrays (Process for Single Run) Slide Carrier D. Washing B C A E. Scanning S C A N

  38. Measures of Experimental Variances

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

  40. Comparison of the variability between different days and differentchambers Same day, different chambers Same chamber, different days

  41. Filtering of the data based on the value of negative controls Ratio between Cy5 and Cy3 Normalization with the median

  42. Frequency distribution of Cy5/Cy3 ratio

  43. Frequency distribution of the % error of Cy5/Cy3 ratio

  44. Percent of genes detected in Ch1, Ch2, and both channels. Total genes: 5297

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

  46. Communication Amongst Members and Focus Groups 8 - 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

  47. 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 - 4 - 8 million in D.C. from CTRF CG Project • 5/21/02 - 1 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

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

  49. Updates for Annual Report Needed NOW!!!

  50. Next Leadership Meeting Monday January 13, 2002 9:30am

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