Transcript December 2002 - Cancer Genomics

CTRF Leadership Meeting
December 9, 2002
Institutional Partners
VCU
©2002 VCU
G M U
INOVA
Minutes
11/04/02
Corrections
Approval
©2002 VCU
Principal Objective
Develop Infrastructure and
Intellectual Property that
Enhances the
Competitiveness of the
Partners for Clinical and
Extramural Funds
©2002 VCU
Research 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
Funding for CTRF
©2002 VCU
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
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
©2002 VCU
Account Balances as of 12/03/02
Residual Year 1 Account Balances as of 12-3-02
PI
Budget
YTD Exp
11-26-02
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%
1,000,000
937,119.55
62,880.45
100%
100%
93.71%
6.29%
Account #
Total:
% of Total
©2002 VCU
Account
Balance
% of Balance
CTRF YR02 Initial Budget Allocation
* Year 2 Modified Budget distribution based on State allocation
of $500,000 as of 11/2002
Account #
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
Total:
©2002 VCU
PI
162,500
500,001
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)
©2002 VCU
Cost Sharing Report
Matching
PI
Acct
Garrett
Garrett
Buck
Ginder
Guiseppi-Eli
290000
412310
130139
412320
137100
GMU
INOVA
Total
©2002 VCU
Matching
Requirement
9/30/2002
FRS Actual
In-Kind
Total
Matching
(Surplus) Shortage
97,500
326,595
138,144
98,750
217,950
117,493
73,246
98,750
148,679
273,553
150,854
-
117,493
346,799
150,854
98,750
148,679
(19,993)
(20,204)
(12,710)
0
69,271
878,939
438,168
424,407
862,575
16,365
345,651
364,536
-
385,472
9,936
385,472
9,936
(39,821)
354,600
710,187
-
395,408
395,408
314,779
819,815
1,257,983
331,144
1,589,126
438,168
Cost Sharing Report
YTD FRS
CTRF Grant Cost Share
Total Cost
Matching
Exp
YTD FRS Exp
Share
Req.
6-30-02
12-6-02
Expenses
Surplus
Shortage
Acct #
Acct #
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
137,047
482,103.13
619,150.13
Total
PI
1,764,757
In-Kind
424,407 (721,199.87)
* 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.
©2002 VCU
Reminder Cost Share Form (VCU)
©2002 VCU
Website Update

Website still incomplete; information
regarding focus group activities is needed
©2002 VCU
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
©2002 VCU
Focus Groups
Tissue Bank
Clinical & Pathology
Laboratory Data
Database Design
QA/QC
Data Analysis
Chip Fabrication
©2002 VCU
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 Miller (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
©2002 VCU
VCU Tissue Bank
Organ
Number of Specimens
Breast
32
Bone Marrow
95
Ovary
12
Head & Neck
2
Lymph Node
6
©2002 VCU
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
©2002 VCU
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
©2002 VCU
IC D 9 _
C o de
D e s c rip t io n
85203 Adenocarcinoma Lobular
P a t ie n t w/
o ne o r
m o re
G ra d e 1/ 2
S a m p le s
P a t ie n t
To ta l
P a t ie n t s w/
o ne o r
m o re
G ra d e 3
S a m p le s
P a t ie n t s
w/ o n e o r
m o re
Lym p h
N o de
D is s e c t e d
S a m p le s
P a t ie n t s w/
o ne o r
m o re
Lym p h
N o de
P o s it iv e
S a m p le s
P a t ie n t s w/
o ne o r
m o re
E s t ro g e n
Re c e p
P o s it iv e
S a m p le s
P a t ie n t s w/
o ne o r
m o re
H e r2 N e u
P o s it iv e
S a m p le s
P a t ie n t s w/ P a t ie n t s w/
o ne o r
o ne o r
m o re S t a g e m o re S t a g e
I/ II
III/ IV
S a m p le s
S a m p le s
2
1
0
2
0
1
0
2
0
19
9
10
19
11
5
5
13
6
85223 Lobular
2
0
1
2
2
1
1
0
2
89803 Carcinosarcoma NOS
1
0
1
1
0
0
0
1
0
Carcinoma Ductular
85213 Infiltrating
Carcinoma Duct Infiltrating &
©2002 VCU
Patient w/
Patient w/
Patient w/
Patient w/
one or more one or more one or more
Description
ICD9_Code
one or more
Patient w/ one
Path Stage
or more Path
Patient
Grade 1/2
Grade 3
Path Stage
III/IV
Invasion
Total
Samples
Samples
I/II Samples
Samples
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 w/one or Patient w/one or
Patient more Diagnostic more Remission
ICD9_Code
Description
Total
Samples
Samples
Patient w/one or
Patient w/one
Patient w/one
more Remiss
or more
or more
Post BMT
Relapseon
Unknown
Samples
Samples
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
©2002 VCU
Clinical Data Model (VCU) - Primary: Data Collection
CERNER
TISSBK & 1o
CLINICAL & Consent
Study ID
Study ID
Tissue ID
SSN
Sample ID
Sub-sample ID
REGISTRY
PathShadw
MRN
SSN
Path Accsn
MRN
Histopath
Risk Factors
SSN
Path Dx
SEQ
ACCSN
Clin Lab
CLAIMS
Reg Shadw
Clinical Risk
Factors
MRN
SSN
PAN
Tables:
Extract Info
StorageInfo
Usage Info
etc
Tables:
Histopath
parameters
Path Dxs
SNOMED
Text Repts
Tables:
Demogrphs
Risk Factrs
Nutirtion
Comorbidty
etc
SPOTTED
Study ID
Lab ID
Treatments
Tissue ID
Outcomes
Run ID
Secondary: Queries, Data Reduction,
Clinical Data Repository
Anonymization
Table:
Consent
Info
AFFY
Tables:
Tumor info
Treatment
Follow-up
etc
Tables: Surg
Tx Medical
Tx Radiatin
Tx other dxs
GeneX
CEL file data
Spot data
Experimental
(Metadata)
Tertiary: Analysis & Hypothesis Testing
Gene
Expression
Non-genetic
predictors
Treatments
Outcomes
Expanded
GeneX
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.
©2002 VCU
CTRF CA GENOMICS TISSUE
UTILIZATION - PLAN
©2002 VCU
Choice of the RNA Extraction Procedure
for Best Microarray Results (I)
Starting material: 10 mm OCT sections of snapfrozen tissue (in liquid N2)
•TRIZOL (Invitrogen)
Or
•TRIZOL (Invitrogen) + RNeasy cleanup (QIAGEN)
©2002 VCU
RNA extraction
ds cDNA synthesis
TRIZOL + RNeasy cleanup
28S/18S ratio: 1.8
TRIZOL
©2002 VCU
Fluorescence
28S/18S ratio: 1.9
~ 50 bp
1,500 bp
Migration Time
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
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)
©2002 VCU
RNA extraction
ds cDNA synthesis
TRIZOL + RNeasy cleanup
28S/18S ratio: 1.9
28S/18S ratio: 0.2
Fluorescence
1,500 bp
500 bp
RNeasy Isolation
©2002 VCU
Migration Time
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 to….
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
©2002 VCU
Devitalization of Tissue
Dr. Nasim and Dr. Grant
©2002 VCU
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
©2002 VCU
GMU - Quality Control Protocol for Custom
Spotted Arrays (Process for Single Run)
2 X 5 Labeling Reactions
Cy3
cDNA
Cy5
–
5000 Probes
- Probe Excess
Pool
- Includes Control Genes
and Lambda
1
2
Slides A thru E
3
4
5
A
B
C
D
E
Quality Control Protocol for Custom Spotted
Arrays (Process for Single Run)
Slide A
Chamber
1
Slide B
Slide C
Slide D
Slide E
Chamber Chamber Chamber Chamber
2
3
4
Hybrid Chambers
5
Hybridization Oven
Quality Control Protocol for Custom Spotted
Arrays (Process for Single Run)
A
B
C
Measures 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
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 between
different days and different chambers
Same day, different chambers
Same chamber, different days
©2002 VCU
Filtering of the data based on the value
of negative controls
Ratio between Cy5 and Cy3
Normalization with the median
©2002 VCU
Frequency distribution of Cy5/Cy3 ratio
day 1
n.genes
750
Day 1
Day 2
Day 3
Day 4
250
0
0.0
0.5
1.0
1.5
2.0
2.5
ratio
day 3
day 2
750
n.genes
n.genes
500
500
250
250
0
0.0
day 4
750
750
n.genes
0.5 < ratio < 2
96.3 %
97.8 %
99.2 %
99.6 %
500
0.5
1.0
1.5
ratio
©2002 VCU
2.0
2.5
0
0.0
500
250
0.5
1.0
1.5
ratio
2.0
2.5
0
0.0
0.5
1.0
1.5
ratio
2.0
2.5
Frequency distribution of the
% error of Cy5/Cy3 ratio
Day 1 (n=5)
Day 2 (n=5)
Day 3 (n=5)
Day 4 (n=5)
All slides (n=20)
n.genes
1500
1000
500
0
0
10
20
% error
©2002 VCU
30
40
% error
14.2
10.1
5.5
7.4
6.8
Percent of genes detected in Ch1, Ch2, and both channels.
Total genes: 5297
Day1 Slide 1
Slide 2
Slide 3
Slide 4
Slide 5
All slides
Day3 Slide 1
Slide 2
Slide 3
Slide 4
Slide 5
All slides
©2002 VCU
Ch1
Ch2
Both
45.3
37.1
46.0
42.8
28.6
49.7
53.4
24.5
52.7
44.7
42.3
36.0
24.0
40.2
28.0
34.0
Ch1
Ch2
Both
40.9
46.1
17.9
45.5
56.8
59.4
54.6
40.8
58.2
58.6
40.9
45.6
17.7
45.3
53.4
46.0
Day2 Slide 1
Slide 2
Slide 3
Slide 4
Slide 5
All slides
Day4 Slide 1
Slide 2
Slide 3
Slide 4
Slide 5
All slides
Ch1
Ch2
Both
32.1
36.6
39.9
31.4
40.3
39.7
29.2
35.7
36.2
37.4
31.6
26.5
33.2
29.5
35.2
31.0
Ch1
Ch2
Both
65.1
54.3
45.2
52.6
61.4
79.6
52.6
46.7
64.3
62.2
64.7
50.1
42.9
52.2
57.8
54.0
CTRF – Promoting Focus Group
Activity
 Establish Standing Weekly or
Biweekly Meeting Dates and
Times
Document Discussions
and Progress Using
Listservs
Complete the Milestone
Updates
©2002 VCU
Communication Amongst
Members and Focus Groups








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
©2002 VCU
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

Old
Business
New Business
 Annual
Report due December 31,
2002 – Infrastructure created
Samples collected
Samples extracted & arrayed
Samples analyzed
Publications
Grants submitted/awarded
©2002 VCU
Updates for
Annual Report
Needed NOW!!!
©2002 VCU
Next Leadership Meeting
Monday
January 13, 2002
9:30am
©2002 VCU