CTRF Leadership Meeting

©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


Estrogen Recep

Positive Samples


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


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


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


Ch1 Ch2 Both


Ch1 Ch2 Both


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

Documents

CTRF Leadership Meeting