Transcript CTRF Leadership Meeting

CTRF Leadership Meeting
September 9, 2002
Institutional Partners
VCU G M U
INOVA
Minutes
8/05/02
Corrections
Approval
Principle Objective
Develop Infrastructure and
Intellectual Property that
Enhances the
Competitiveness of the
Partners for Clinical and
Extramural Funds
Research Objective
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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
Funding From CTRF
Chandhoke
Grant
Christensen
Fryxell
Jamison
Torr (Central Admin)
Ginder
Garrett
Buck
Guiseppe-Elie
Abraham
Cooper
Year 1
Year 1
Year 1
$325,000
$582,000
$93,000
Total (3 yrs)
Total (3 yrs)
Total (3 yrs)
$975,000
$1,734,603
$290,397
Year 2
Year 2
Year 2
$325,000
$578,191
$96,809
New Account Numbers for CTRF
New Account Numbers for FY2003
535411
Torr
535412
Central
535413
Garrett
535414
INOVA
535415
Ginder
535417
GMU
535443
Guiseppi-Eli
FY2002 Account Balances
FY2002 Account Balances as of 8/31/02
Account #
PI
535282
Torr
535283
Budget
Rollover June 30
YTD Exp
Personnel
Commitments
Operating
Commitments Account Balance
43,839.00
-95.00
42,954.89
0.00
884.11
0.00
Garrett
169,597.00
150,231.00
135,260.81
19,279.19
7,872.51
7,184.49
535284
Buck
124,684.00
119,684.00
46,412.53
0.00
0.00
78,271.47
535285
Ginder
93,396.00
-11,515.00
89,806.00
0.00
0.00
3,590.00
535286
Guiseppi-Eli
150,484.00
26,694.00
140,505.04
10,790.54
3,712.33
-4,523.91
535287
INOVA
93,000.00
78,434.00
14,556.66
0.00
0.00
78,443.34
535288
GMU
325,000.00
231,875.00
156,565.70
0.00
37,683.94
130,750.36
1,000,000.00
595,308.00
626,061.63
30,069.73
50,152.89
293,715.75
Total:
% of Total
100%
62.61%
3.01%
5.02%
29.37%
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 14th
– 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 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.
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(http://www.vcu.edu/finance/
In-kind%20Cost%20Sharing%Certification.pdf)
Reminder - Cost Share Form (VCU)
Website Update
www.ctrf-cagenomics.vcu.edu
 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: http://www.InfoEd.org/default.stm
Focus Group
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Tissue Bank
Clinical and Pathology Laboratory Data
Database Design
Data Analysis
Quality Assurance in Microarray Analysis
Chip Fabrication
Focus Group Leaders
Focus Group Leaders
GMU
VCU
Inova
Tissue Bank
ClinPath
DB Design
DataAnalysis
QA/LQC
Chip Fabrication
Geraldine Grant (GMU)
Suhail Nasim (VCU)
Barrie Cook (Inova)
Lynne Penberthy (VCU)
Suhail Nasim (VCU)
James Cooper (Inova)
Curtis Jamison (GMU)
Lynne Penberthy (VCU)
Greg Mil er (VCU)
Mike Sheriden (Inova)
Vikas Chandhoke (GMU)
Greg Buck (VCU)
Alan Christensen (GMU)
Andrea Ferreira-Gonzalez (VCU)
Suhail Nasim (VCU)
Geraldine Grant
Anthony Guiseppi-Elie
Alan Christensen
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 Smith’s group has identified Eileen Kelley as
coordinator for tissue acquisition project
– Study coordinator to obtain informed consent and
acquisition
VCU Tissue Bank
Organ
Number of
Specimens
Breast
19
Bone Marrow
57
Ovary
6
Brain
0
Lymph Node
3
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 (8-20-02)
• 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
Tissue Storage
 Temporary Storage (If appropriate)
 -80°C freezer monitored 24hr X 7 days with daily recording of
temperature; or, -80°C freezer monitored 5 days/wk with week day
(including holiday) daily recording of temperatures and with liquid
nitrogen back-up capable of maintaining -40°C for 72hr. (Note: liquid
nitrogen back-up must include written quality assurance plan to
insure that liquid N2 maintained at satisfactory levels).
 Permanent Storage
 -80°C 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
-40°C for 72hr. (Note: liquid N2 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.
Supplying Tissue to Project PI
Labs
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 100-500 g total RNA per sample. GMU
requires ~20g per run. VCU requires ~10g per run
for the Affymetirx platform.
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 μ thick) on a cryostat.
 no more than 40 - 5 μ 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 -80oC for
storage and -37oC (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.
ο Faculty status, IRB approval, ?scientific validity
ο ?minimum QA requirements
ο ?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
DNA
Protein
phenol
detect DNA via fluorescence
260/280 ratio
270 absorbency
DNA
Affy ribosomal genes (see Excel file:
uChipControlGenes.xls)
Integrity
18S/28S ratio PAGE, LabChip®-electropherogram
housekeeping genes (affy)
GAPDH, β-actin (ratio3'/5' = 1)
low abundance genes (affy)
ISGF-3A (ratio 3'/5'  3)
Labeling Process
• cRNA
• cRNA Fragmentation
• Labeling Efficiency
yield (= μg/cRNA/μg total RNA)
size range
size range
spotted 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-169-001T)
– 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 1
Sample 2
Sample 3
Sample 4
2.0-1.5
1
0.5
0.1
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 3’5’ ratios for Housekeeping
genes
– Low and high abundance genes
– Check for changes in gene calls (P – A)
Data Analysis
Control Genes
(GMU & VCU)
Clone Name
actin, beta
tubulin alpha
ribosomal protein L19
vimentin
glucose-6-phosphate dehydrogenase
phosphofructokinase, platelet
mitochondrial ribosomal protein L19
lactate dehydrogenase A
angio-associated, migratory cell protein
ribosomal protein S27a
phosphoglycerate mutase 1 (brain)
ribosomal protein L11
non-POU-domain-containing, octamer-binding
Rho GDP dissociation inhibitor (GDI) alpha
asparagine synthetase
aldolase A, fructose-bisphosphate
beta-2-microglobulin
heat shock 90kD protein 1, alpha
ribosomal protein L29
ribosomal protein S3
ribosomal protein L19
phosphoglycerate kinase 1
tubulin alpha
heat shock 90kD protein 1, alpha
Rho GDP dissociation inhibitor (GDI) alpha
Gene ID
ACTB
K-ALPHA-1
RPL19
VIM
G6PD
PFKP
MRPL19
LDHA
AAMP
RPS27A
PGAM1
RPL11
NONO
ARHGDIA
ASNS
ALDOA
B2M
HSPCA
RPL29
RPS3
RPL19
PGK1
K-ALPHA-1
HSPCA
ARHGDIA
Accession #
AA031770
R53480
AA083485
AA486321
AA424938
AA608558
AA521243
AA497029
AA452848
AA625632
AA676970
AA680244
AA056465
AA453756
AA894927
AA775241
AA670408
N62400
AI018613
AA046713
AA707531
AA426516
R53480
AA199881
AA459400
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 #: 740000-41) ( lot number 1000207 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
Director’s 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 740000-41 (lot
number is 1000207)
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
Database Design/Data Analysis
GeneX Database (Update)
1.5 UVA (Jae K. Lee)
 Version 2.0 GMU-VT (J. Weller)
 Version
Developer meeting planned in
October
 Establish Standing Weekly or
Biweekly Meeting Dates and
Times
Document Discussions
and Progress Using
Listservs
Don’t
Forget!
Complete the Milestone
Updates
Communication Amongst
Members and Focus Groups
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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
LISTSERVS
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Address messages to:
[email protected]
Unsubscribe to the listserv by submitting a
message with the words SIGNOFF listname
to:
[email protected]
Subscribe to the listserv by asking the PI
with whom you work to submit your name
and E-mail address to the Program Director
(C.Garrett)
USE the listserv(s) to inform members of
your activity or to seek advice from the
members.
Old
Business
New
Business
CTRF - Specific Reportables - - Reminder - 
Intellectual property reporting - licenses, patents, etc
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Publications
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New applications
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CTRF Administrative office
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will search for new funding opportunities (SPIN)
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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.
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Any other discoveries
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Federal money leveraged
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Private research money leveraged
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Advancement of technology and economic development in VA