tissue intraoperative factors€¦ · sprüssel et al, biotechniques 2004 b a x-fache Änderung...
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Realizing Individualized Cancer Therapy
Tissue Intraoperative FactorsDr. Hartmut Juhl
CEO of Indivumed GmbH and Inostics GmbHAdjunct Professor, Georgetown University and Hamburg University
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Company Structure
Indivumed GmbHHamburg, Germany
Founded: 2002
Indivumed Inc.Kensington, MDFounded: 2005
Inostics GmbHHamburg, Germany
Founded: 2009
B. Vogelstein et al. (JHU)
F.Diehl/H.Juhl
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Therapy 1 Therapy 2 Therapy 3
Indivumed Vision
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Three colon cancer patients:Same disease? Same therapy?
Patient 1 Patient 2 Patient 3
> 1000 different gene damages in various combinations can cause cancer=
Each patient differs with respect to the molecular basis of his/her cancer
The Cancer Problem: Heterogeneity
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Understanding the Molecular Basis of Cancer: Status 1971 (Nixon Declares „War Against Cancer“)
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Understanding the Molecular Basis of Cancer: Status 2010
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PIK3CA
MAPK
Targeting Key Pathways
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FDA-Approved Targeted Cancer Therapeutics(2001-2006)
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Colon cancer: Avastin Breast cancer: Herceptin
Colon cancer: Erbitux Lung cancer: Iressa
Cancer Therapeutics in Clinic
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Targeting Key pathways
?
?
?
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Patient andPresurgical therapy
Medical/SurgicalProcedures
AcquisitionProcessing
StorageTemperatur
Handling, Processingand analytical assays
Specimen is viable and reactive Biomolecules may degrade
Time 0
Challenge for using Tissue as Research and Diagnostic Tool:It is Alive and Reacts to Environmental Changes
Highly defined quality tissues are needed to understand cancer pathways
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Targeting Key pathways
??
?
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Indivumed Research on Critical Variables forScience Guided Biobanking
Postsurgical Processing- Ischemia Time- Location of Biopsy
Intrasurgical Factors- Drugs- Artery Ligation
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Indivumed Research on Critical Variables forScience Guided Biobanking
Postsurgical Processing- Ischemia Time- Location of Biopsy
Intrasurgical Factors- Drugs- Artery Ligation
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Surgical removal
of rectumControl of warm ischemia
Analysis:
Affymetrix
real-time RT-PCR
SELDI-TOF-MS
Impact of cold ischemia: controlled tissue study
Tissue collection following resection:Snap frozen in liquid N2
after 5 min8 min
10 min12 min15 min20 min25 min30 min
Collection of normal and cancer tissue
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Time course of gene expression
70
75
80
85
90
95
100
Ischemia time (min)U
ncha
nged
gen
es (%
)5 8 10 12 15 20 30
[min]
5´
8´
10´15´ 30´
12´20´
Tissue Ischemia and Gene Expression Profiling(Affymetrix cDNA microarray)
Following tumor resection ~ 20-25% of genes are differentially expressedwithin the first 30 minutes !
Sprüssel et al, BioTechniques 2004
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Sprüssel et al, BioTechniques 2004
B
A
x-fa
che
Änd
erun
g (n
orm
alis
iert
e In
tens
ität)
HIF-1α
rel.
Expr
essi
on
time [min]5 8 10 12 15 20 25 30
0,01,02,03,04,0
X-fo
ld c
hang
e(n
orm
alis
ed in
tens
ity) c-fos
time [min]
rel.
expr
essi
on
051015202530
5 8 10 12 15 20 25 30
GAPDHCYCA
HO-1
time [min]
rel.
Expr
essi
on
0,00,51,01,52,02,5
5 8 10 12 15 20 25 30
x-fa
che
Änd
erun
g (n
orm
alis
iert
e In
tens
ität)
Ischemia regulated genes c-fos, HIF-alpha and HO-1
Tissue Ischemia and Gene Expression Profiling(Comparison Affymetrix data and real-time RT-PCR)
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B
A
GAPDHCYCA
CEA
x-fo
ld c
hang
e of
no
rmal
ized
int
ensi
ty
Time [min]
rel.
Expr
essi
on
0,5
1,5
2,5
3,5
0,0
1,0
2,0
3,0
4,0
5 8 10 12 15 20 25 30
x-fa
che
Änd
erun
g (n
orm
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iert
e In
tens
ität)
CK20
Time [min]
rel.
Expr
essi
on1
3
5
7
0
2
4
6
8
5 8 10 12 15 20 25 30
Tumor marker CEA (colorectal cancer biomarker) and cytokeratin CK20
Tissue Ischemia and Gene Expression Profiling(Comparison Affymetrix data and real-time RT-PCR)
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Phosphoprotein Expression:pTyr100 Immunostaining (Ventana)
10 min
20 min
60 min
No clear trend of
pTyr100 expression
within
60 min of cold ischemia
Case BCase A
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Phosphoprotein Expression:pMAPK Immunostaining (Ventana)
10 min
20 min
60 min
Case BCase A
Change of pMAPK expression after 10-20 min cold ischemia
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Phosphoprotein Expression:pmTOR-Immunostaining (Ventana)
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Tumor Tissue Varies in Center and Peripheral Areas
Invasive growth by induction of angiogenesis
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Approx. 40% of proteins are differentially expressed between peripherial and central tumor regions
Localization of Tumor Biopsy Affects Molecular Pattern(Mass-Spectroscopy Analysis)
0 1 0 0 0 0 1 2 5 0 0 1 5 0 0 0
Normal
Center area
Peripheral area
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Patient / tissue
A61 A157 A161 A197 A249
N P C0
1
2
3
4
5
6
rela
tive
RN
A a
mou
nt
N P C N P C N P C N P C
N = NormalP = peripherC = central
Expression of VEGF in Different Tissues: Normal - Periphery - Center
(real-time RT-PCR)
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Indivumed Research on Critical Variables forScience Guided Biobanking
Postsurgical Processing- Ischemia Time- Location of Biopsy
Intrasurgical Factors- Drugs- Artery Ligation
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Drugs Given During Surgery
Number of different commonly usedactive substances during surgery(Indivumed‘s data base):
• Antibiotics: 13• Bronchodilatator: 2• Cardio-drugs: 17• Diuretics & corticosteroids: 5
• GI-tract drugs &antihistaminics: 7
• Infusion &transfusion: 15
• Inhalative narcotics: 5• Local anesthetics: 6• Muscle relaxant: 8• Analgetics &sedatives: 34
Total: 112
Atropin
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T-tests; Grouping: Atropin (normal lig atropin.sta)Group 1: 0Group 2: 1
VariableMean
0Mean
1t-value df p Valid N
0Valid N
1Std.Dev.
0Std.Dev.
1M5939_87M3772_14M6723_51M3555_34
1.15213 1.60276 -2.73669 22 0.012043 17 7 0.15888 0.652400.63586 1.05263 -2.34306 22 0.028574 17 7 0.25252 0.636532.17426 3.41282 -2.31784 22 0.030148 17 7 0.97299 1.633010.71346 0.56601 2.16344 22 0.041640 17 7 0.16216 0.11972
Expression of 4 protein peaks (1.7%) correlates with atropin treatment
Mean Mean±0.95 Conf. Interval Mean±SD
1 0
Atropin
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
M35
55_3
4
Mean Mean±0.95 Conf. Interval Mean±SD
1 0
Atropin
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
M59
39_8
7
Correlation of Colon Tissue Protein Expression with Intrasurgical Application of Atropin
- atropin + atropin - atropin + atropin
- atropin+ atropin
M35
55_3
4
M59
39_8
7
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MesentericMesentericartery inferiorartery inferior XX
Patients receiving left hemicolectomy
Indivumed data base / biobank:Time (min) between artery ligation
and tumor removal
20 25 30 35 40 45 50(min)
Time (min) until freezing10 min
LCM isolation of tumor cells
Gene expression (Affymetrix)
Bioinformatics
Impact of Time between Ligation of Main Artery and Tumor Resectionon Gene Expression in Colon Cancer
(NCI-Indivumed study)
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Warm ischemia (min)20 25 30 35 40 45 50
PCA mapping: grouping of warm ischemia time points
20253035404550
A prospective trial collecting tissue during surgery has been initiated
Impact of Time between Ligation of Main Artery and Tumor Resectionon Gene Expression in Colon Cancer
(NCI-Indivumed study)
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Prospective Trial:Impact of Anesthesia and Surgery on Gene and Protein Expression in
Colon and Liver Tissue
Partner:
OBBR/NCI
Indivumed GmbH
Department of Surgery, Israelitisches Krankenhaus (Dr. Zornig)
Department of Surgery, Diakonieklinikum Alten Eichen (Dr. Dörner)
Department of Hepatobiliary Surgery, University Hospital Hamburg (PI: Dr.
Nashan)
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Impact of Anesthesia and Surgery on Gene and Protein Expression in Colon and Liver Tissue: Study Design
Colon Tissue
Normal Cancer
Start of Surgery
Post-Surgery
10 min
20 min
45 min
Blood
Normal
Before Anesthesia
Before skin incision
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Liver
Normal Met‘s
ArteryClamping
0min
10 min
Post-Surgery
10 min
20 min
45 min
Impact of Anesthesia and Surgery on Gene and Protein Expression in Colon and Liver Tissue: Study Design
Blood
Normal
Before Anesthesia
Before skin incision
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Selected Proteins
Protein Quantification Cellular Distribution
Identification of Surgery Dependent Molecules
pMEK1/2
0
1000
2000
3000
4000
5000
1A85
Sarc
oma
HT29 c
1
A2180
c1
-
B1050
c1
-
MSD
sig
nal [
U]
Gene Expression
Specific Genes
Cancer Normal
Comprehensive Analysis
Indivumed NCI
Impact of Anesthesia and Surgery on Gene and Protein Expression in Colon and Liver Tissue :
Study Design
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Basic Considerations for the Development of Individualized Therapies and Predictive Biomarkers
PredictiveModels
Direct Accessto Patients
Science-based Biobank
ClinicalTrial Center
Technologyfor
Discovery & Development
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All done by Indivumed staff:
• IRB approval• Patient consent• Collection of blood/urine• Documentation of surgery• Documentation of anesthesia• High-speed collection and
processing of biospecimen• Clinical data accrual
• medical history• around surgery• annual follow-up
• treatment• outcome
• Blood/urine during follow-up
• Quality control / SOPs• Molecular analysis• R&D / Service / M&S
Full Integration of Indivumed Staff for Biobanking and Research:Collaboration with 8 Hospitals in Hamburg and 1 in Washington DC
Quality Management SystemISO 9001:2008 Certified
Direct Access to Patients
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Indivumed Standard of Biobanking:> 11,000 cases in the biobank / +2,000/year
Exact documented and very short tissue cold ischemia times of < 12 min (mean 7 min)
Exact tissue localization and standardized fixation
Complete biospecimen sets
Highest tissue quality monitored by visual inspection, H&E staining and microscopic assessment
Native and rapid fluid preparations
Complete specimen data
Complete clinical data
Patients’ confidentiality assured following international standards
10 min
20 min
60 min
Tissue ischemia and protein
phosphorylation
Science-Based Biobank
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Drug Profiling PlatformDrug Response and Predictive Biomarker Development
Piece of tumour tissue after resection(NSCLC, colon, breast)
Cultivation in 24 well plates and drug treatment
Preparation of tissue slices(400 µm)
Drug Response tests:ATP assayCaspase 3/7 assayIHC stainingAntibody based assays (MSD)
Proteomic profiling:• Selected proteins
(e.g., phosphoproteins)• Unselected proteins
Predictive Model
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Identification of Predictive Biomarker within Clinical TrialsCollaboration with the Otto J. Ruesch Center for the Cure of GI-tract Cancer
Screening/Diagnosis
SurgicalTherapy
DrugTherapy (Standard)
DrugTherapy (Targeted)
Biobank/Data and
diagnostics
(research only)
Innovative Diagnostics
Therapy recommendation
Opt
imiz
atio
n of
sta
ndar
d th
erap
yIn
divi
dual
isat
ion
Otto
J. R
uesc
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ente
r Lo
mba
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ance
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ter,
GU
MC
Clinical Trial Center
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Partner for Drug Discovery and Development:A Comprehensive Platform for Discovery and Development
DNA Analysis:•Tissue Analysis• Blood-Based Assays (BEAMing)• Mutation/Methylation/Amplification
Protein Analysis:• Immunohistochemistry• Pathway Analysis• Drug Profiling
Biospecimen Preparations:• Tumor Biobank (> 11,000 Patients)• Primary Cells• Fresh Tissue
Direct Patient Access in 9
Clinical Center
Standardized Biobanking
for R&D
Pharma/Biotech:
Research Service
&
Co-Development (Companion Diagnostics)
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More research is needed to distinguish instable and robust molecules
High-quality biobanks need to have highly standardized processes
and complete documentation of all critical factors
Short ischemia is crucial for analyzing sensitive molecules such as
phosphoproteins
Control of preanalytical factors are a prerequisite to utilize tissue as
diagnostic tool for targeted therapies
Consequences for Research and Patient Care