©2002 VCU
CTRF Leadership MeetingCTRF Leadership Meeting
December 9, 2002
Institutional Partners
V C UV C U G M UG M U I N O V AI 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 ObjectivePrincipal 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 ObjectiveResearch Objective
©2002 VCU
ChandhokeGrant
ChristensenFryxell
Jamison
Torr (Central Admin)GinderGarrettBuck
Guiseppe-ElieAbraham
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
Funding for CTRFFunding for CTRF
©2002 VCU
FY02 Funds PendingFY02 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 12/03/02Account Balances as of 12/03/02
Account # PI BudgetYTD Exp11-26-02
Account Balance % of Balance
535282 Central 43,839 43,145.43 693.57 1.10%
535283 Garrett 169,597 152,582.34 17,014.66 27.06%
535284 Buck 124,684 152,528.05 (27,844.05) -44.28%
535285 Ginder 93,396 101,052.17 (7,656.17) -12.18%
535286 Guiseppi-Eli 150,484 154,477.78 (3,993.78) -6.35%
535287 INOVA 93,000 14,556.66 78,443.34 124.75%
535288 GMU 325,000 318,777.12 6,222.88 9.90%
Total: 1,000,000 937,119.55 62,880.45 100%
% of Total 100% 93.71% 6.29%
Residual Year 1 Account Balances as of 12-3-02
©2002 VCU
CTRF YR02 Initial Budget AllocationCTRF YR02 Initial Budget Allocation
Account # PI Budget
535412 Central 21,902
535413 Garrett 87,487
535499 Buck 65,607
535417 Ginder 64,675
535443 Guiseppi-Eli 49,425
535414 INOVA 48,405
535415 GMU 162,500
Total: 500,001
* 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.
(http://www.vcu.edu/finance/In-kind%20Cost%20Sharing
%Certification.pdf)
Cost Share ExpensesCost Share Expenses
©2002 VCU
Matching
PI Acct
MatchingRequirement
9/30/2002FRS Actual In-Kind
Total Matching (Surplus) Shortage
Garrett 290000 97,500 117,493 - 117,493 (19,993)Garrett 412310 326,595 73,246 273,553 346,799 (20,204)Buck 130139 138,144 - 150,854 150,854 (12,710)Ginder 412320 98,750 98,750 - 98,750 0Guiseppi-Eli 137100 217,950 148,679 - 148,679 69,271
878,939 438,168 424,407 862,575 16,365
GMU 345,651 - 385,472 385,472 (39,821)INOVA 364,536 - 9,936 9,936 354,600
710,187 - 395,408 395,408 314,779
Total 1,589,126 438,168 819,815 1,257,983 331,144
Cost Sharing ReportCost Sharing Report
©2002 VCU
Cost Sharing ReportCost 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.
CTRF Grant
Acct #
Cost Share
Acct # PI
Matching
Req.
YTD FRS
Exp
6-30-02
YTD FRS Exp
12-6-02
Total Cost
Share
Expenses In-Kind
Surplus Shortage
535282 2-90000 Central 146,875 91,384.00 46,786.23 138,170.23 (8,704.77)
535283 4-12310 Garrett 384,240 45,663.00 36,531.90 82,194.90 273,553 (28,492.10)
535284 1-30139 Buck 170,654 0.00 0.00 0.00 150,854 (19,800.00)
535285 4-12320 Ginder 199,122 0.00 0.00 0.00 (199,122.00)
535286 1-37100 Guiseppi-Elie 276,825 0.00 13,313.00 13,313.00 (263,512.00)
535287 - INOVA 314,105 0.00 0.00 0.00 (314,105.00)
535288 - GMU * 272,936 0.00 385,472.00 385,472.00 112,536.00
Total 1,764,757 137,047 482,103.13 619,150.13 424,407 (721,199.87)
©2002 VCU
Reminder Cost Share Form (VCU)Reminder Cost Share Form (VCU)
©2002 VCU
Website still incomplete; information regarding focus group activities is needed
Website UpdateWebsite 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: http://www.InfoEd.org/default.stm
SPIN ResearchSPIN Research
©2002 VCU
Focus GroupsFocus Groups
Tissue Bank
Clinical & Pathology Laboratory Data
Database Design
Chip Fabrication
QA/QC
Data Analysis
©2002 VCU
Focus Group Leaders
G MU
G e rald in e G ran t ( G M U)S u ha il N as im ( VC U)B a rr ie C o o k ( I n o va)
Tissue Bank
L yn ne P en b e rth y (V C U)S u ha il N as im ( VC U)
J a m e s C o op e r ( I n o va)
ClinP ath
C u rtis J am is on ( G M U)L yn ne P en b e rth y (V C U)
G re g M ille r (V C U)M ike S he ride n ( I no va)
D B D esign
V ika s C h an d h oke ( G M U)G re g B u ck ( V C U)
D ataAnalysis
A la n C hris ten s en ( G M U)A n d re a Fe rre ira - G o n zale z (V C U)
S u ha il N as im ( VC U)G e rald ine G ra n t
Q A/ LQ C
Anthony Guiseppi-E lieA lan Christensen
Chip Fabrication
VCU I nova
Focus Group LeadersFocus Group Leaders
©2002 VCU
Organ Number of Specimens
Breast 32
Bone Marrow 95
Ovary 12
Head & Neck 2
Lymph Node 6
VCU Tissue BankVCU 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 BankINOVA – 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 (8-20-02)
• Update of Database for Histopathologic parameters of existing cases needed - Completed
Tissue Acquisition DatabaseTissue Acquisition Database
©2002 VCU
ICD9_Code Description
Patient Total
Patient w/ one or more
Grade 1/2 Samples
Patients w/ one or more
Grade 3 Samples
Patients w/one or
more Lymph Node
Dissected Samples
Patients w/ one or more Lymph Node
Positive Samples
Patients w/ one or more
Estrogen Recep
Positive Samples
Patients w/ one or more
Her2Neu Positive Samples
Patients w/ one or
more Stage I/II
Samples
Patients w/ one or
more Stage III/IV
Samples
85203 Adenocarcinoma Lobular 2 1 0 2 0 1 0 2 0
85213
Carcinoma Ductular
Infiltrating 19 9 10 19 11 5 5 13 6
85223
Carcinoma Duct Infiltrating &
Lobular 2 0 1 2 2 1 1 0 2
89803 Carcinosarcoma NOS 1 0 1 1 0 0 0 1 0
©2002 VCU
ICD9_Code Description
Patient
Total
Patient w/
one or more
Grade 1/2
Samples
Patient w/
one or more
Grade 3
Samples
Patient w/
one or more
Path Stage
I/II Samples
Patient w/
one or more
Path Stage
III/IV
Samples
Patient w/ one
or more Path
Invasion
Samples
84603 Adenocarcinoma Papillary Serous 3 1 1 0 0 0
84413 Adenocarcinoma Serous NOS 1 0 1 0 0 0
84423 Cystadenoma Serous Borderline Ma 1 0 0 0 1 0
**Data incomplete for this tissue type
©2002 VCU
ICD9_Code Description
Patient
Total
Patient w/one or
more Diagnostic
Samples
Patient w/one or
more Remission
Samples
Patient w/one or
more Remiss
Post BMT
Samples
Patient w/one
or more
Relapseon
Samples
Patient w/one
or more
Unknown
Samples
203.0 Multiple myeloma 3 2 1 0 0 0
204.0 Lymphoid leukemia, acute 1 1 1 0 0 0
204.1 Lymphoid leukemia, chronic 3 3 0 0 0 0
205.0 Myeloid leukemia, acute 14 5 9 0 0 4
205.1 Myeloid leukemia, chronic 7 3 4 0 0 0
206 Monocytic leukemia 1 2 1 0 0 0
206.0 Monocytic leukemia, acute 2 2 2 0 0 0
285.9 Anemia, unspecified 2 1 0 0 0 0
285.0 Sideroblastic anemia 1 1 0 0 0 0
202.8 Other lymphomas 7 0 1 0 0 0
288.3 Eosinophilia 1 0 0 0 0 0
**Data incomplete for this tissue type
Study IDTissue IDSample ID
Sub-sample ID
Study IDSSN
Clinical Data ModelClinical Data Model (VCU) - Primary: Data Collection
Secondary: Queries, Data Reduction, Anonymization
Tertiary: Analysis & Hypothesis Testing
AFFYTISSBK & 1o CLINICAL & Consent
CERNER
PathShadw
REGISTRY CLAIMS
Clinical Data Repository
SPOTTED
Gene Expressio
n
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 dataSpot data
Experimental(Metadata)
Reg Shadw
GeneX
Clinical Risk Factors
Treatments
Outcomes
Histopath Risk Factors
Path Dx
Clin Lab
ExpandedGeneX
Table: Consent
Info
Tables: Demogrph
s Risk Factrs
Nutirtion Comorbidt
y 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 UpdateGMU 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 CTRF CA GENOMICS TISSUE UTILIZATION - PLANUTILIZATION - PLAN
©2002 VCU
Choice of the RNA Extraction Procedure for Choice of the RNA Extraction Procedure for Best Microarray Results (I)Best Microarray Results (I)
Starting material: 10 m OCT sections of snap-frozen tissue (in liquid N2)
•TRIZOL (Invitrogen)Or
•TRIZOL (Invitrogen) + RNeasy cleanup (QIAGEN)
©2002 VCU
TRIZOL + RNeasy cleanup
TRIZOL
RNA extractionRNA extraction ds cDNA synthesisds cDNA synthesis
Fluo
resc
ence
Migration Time
1,500 bp~ 50 bp
28S/18S ratio: 1.9
28S/18S ratio: 1.8
©2002 VCU
Results (I)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.
©2002 VCU
Choice of the RNA Extraction Procedure Choice of the RNA Extraction Procedure for Best Microarray Results (II)for Best Microarray Results (II)
Starting material: Snap-frozen tissue (in liquid N2), 10 m 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)
©2002 VCU
Fluo
resc
ence
Migration Time
TRIZOL + RNeasy cleanup
RNeasy Isolation
28S/18S ratio: 1.9
28S/18S ratio: 0.2
RNA extractionRNA extraction ds cDNA synthesisds cDNA synthesis
1,500 bp
500 bp
©2002 VCU
Results (II)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 TRIZOL followed by the RNeasy cleanup protocol, to ensure cDNA molecules of greater size, (max. peak at ~1,500 bp).
©2002 VCU
Congratulations Congratulations to….to….
Young Investigator AwardYoung Investigator Award
QUALITY CONTROL AND QUALITY ASSURANCE IN MICROARRAY DATA
ANALYSISDumur 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 TissueDevitalization of Tissue
Dr. Nasim and Dr. Nasim and Dr. GrantDr. 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 DevitalizationTissue Devitalization
GMU - Quality Control Protocol for Custom GMU - Quality Control Protocol for Custom Spotted Arrays (Process for Single Run)Spotted Arrays (Process for Single Run)
Cy3 Cy5
Pool
1
2
3
4
5 5
4
3
2
1
cDNA
– 5000 Probes
- Probe Excess
- Includes Control Genes and Lambda
2 X 5 Labeling Reactions
B CA D E
Slides A thru E
Quality Control Protocol for Custom Spotted Quality Control Protocol for Custom Spotted Arrays (Process for Single Run)Arrays (Process for Single Run)
Slide A Slide B Slide C Slide D Slide E
Hybrid Chambers
Chamber
1Chamber
2Chamber
3Chamber
4Chamber
5
Hybridization Oven
Quality Control Protocol for Custom Spotted Quality Control Protocol for Custom Spotted Arrays (Process for Single Run)Arrays (Process for Single Run)
A B C
S C A N
Measures of Experimental VariancesMeasures of Experimental Variances
Variance Comparison
Labeling Reaction Pooled Reactions vs. Individual Reactions
Slide Variation Changes 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 betweendifferent 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
0.5 < ratio < 2 Day 1 96.3 % Day 2 97.8 % Day 3 99.2 % Day 4 99.6 %
day 2
0.0 0.5 1.0 1.5 2.0 2.50
250
500
750
ratio
n.ge
nes
day 3
0.0 0.5 1.0 1.5 2.0 2.50
250
500
750
ratio
n.ge
nes
day 4
0.0 0.5 1.0 1.5 2.0 2.50
250
500
750
ratio
n.ge
nes
day 1
0.0 0.5 1.0 1.5 2.0 2.50
250
500
750
ratio
n.ge
nes
Frequency distribution of Cy5/Cy3 ratio
©2002 VCU
0 10 20 30 400
500
1000
1500
% error
n.g
enesFrequency distribution of the % error of Cy5/Cy3 ratio
% error Day 1 (n=5) 14.2 Day 2 (n=5) 10.1 Day 3 (n=5) 5.5 Day 4 (n=5) 7.4 All slides (n=20) 6.8
©2002 VCU
Ch1 Ch2 Both
Day4 Slide 1 65.1 79.6 64.7 Slide 2 54.3 52.6 50.1 Slide 3 45.2 46.7 42.9 Slide 4 52.6 64.3 52.2 Slide 5 61.4 62.2 57.8 All slides 54.0
Ch1 Ch2 Both
Day2 Slide 1 32.1 39.7 31.6 Slide 2 36.6 29.2 26.5 Slide 3 39.9 35.7 33.2 Slide 4 31.4 36.2 29.5 Slide 5 40.3 37.4 35.2 All slides 31.0
Ch1 Ch2 Both
Day3 Slide 1 40.9 59.4 40.9 Slide 2 46.1 54.6 45.6 Slide 3 17.9 40.8 17.7 Slide 4 45.5 58.2 45.3 Slide 5 56.8 58.6 53.4 All slides 46.0
Ch1 Ch2 Both
Day1 Slide 1 45.3 49.7 42.3 Slide 2 37.1 53.4 36.0 Slide 3 46.0 24.5 24.0 Slide 4 42.8 52.7 40.2 Slide 5 28.6 44.7 28.0 All slides 34.0
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 CTRF – Promoting Focus Group ActivityActivity
©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 Communication Amongst Members and Focus GroupsMembers and Focus Groups
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 discoveriesFederal money leveraged
Private research money leveraged
Advancement of technology and economic development in VA
CTRF - Specific Reportables - - Reminder - -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
©2002 VCU
Old BusinessOld BusinessNew BusinessNew Business
Annual Report due December 31, 2002 – Infrastructure created Samples collectedSamples extracted & arrayedSamples analyzedPublicationsGrants submitted/awarded
©2002 VCU
Updates for Annual Updates for Annual Report Needed Report Needed
NOWNOW!!!!!!
Monday
January 13, 2002
9:30am
Next Leadership Meeting
©2002 VCU