pcr plus and predictor 2013
TRANSCRIPT
Sample & Assay Technologies
Assess pathway activity and cellular toxicity with biomarker signature genesRaed N Samara, PhDQIAGEN
Sample & Assay Technologies Outline
2
� Introduction
� R&D Development Process
� Pathway Activity Score
� Specificity of Signature Biomarker Genes
� Application Data
Sample & Assay Technologies Outline
3
� Introduction
� R&D Development Process
� Pathway Activity Score
� Specificity of Signature Biomarker Genes
� Application Data
Sample & Assay Technologies
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Gene expression for biological functional readout
� Use real-time PCR technology to develop a numerical score for biological functional readout.
protein activity pathway activity toxicityphysiological
functionpathological
condition
gene expression change
Pathway and Toxicity Gene Profiling
Sample & Assay Technologies Pathway and Toxicity Gene Profiling
Title, Location, Date 5
Gene profiling assesses whether genes in a certain pathway are upregulated or downregulated. But did pathway activity change?
Sample & Assay Technologies Where to measure pathway activity?
Title, Location, Date 6
LRP
FzdWNT
Dsh
GSK3ββββ
APC Axin
ββββ-catenin
TCF/LEF1
ββββ-catenin
P
Myc
CCND1
physiological
LRP
WNT
Dsh
GSK3ββββ
Axin
TCF/LEF1
Pββββ-catenin
ββββ-catenin
APC
MycCCND1
Fzd
pathological
PathwayActivity?
Downstream effect!
� Traditional indicators provide insufficient information of the regulatory status of the whole pathway signaling
� Multi-gene expression based methods measure pathway alteration as a function of the downstream effect of pathway regulation on multiple gene expression changes
Sample & Assay Technologies
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Gene expression for biological functional readout
� Use real-time PCR technology to develop a numeric score for biological functional readout.
protein activity pathway activity toxicityphysiological
functionpathological
condition
gene expression change
gene panel gene panel gene panel gene panel gene panel
Specific gene panel with companion score to monitor individual phenotype
measure by real-time PCR
Signature gene
Sample & Assay Technologies Pathway and Toxicity Gene Profiling & Activity
Title, Location, Date 8
RT2 Profiler Plus RT2 Predictor
Sample & Assay Technologies Outline
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� Introduction
� R&D Development Process
� Pathway Activity Score
� Specificity of Signature Biomarker Genes
� Application Data
Sample & Assay Technologies R&D Development Process
Signaling pathway Toxicity pathway
Sample & Assay Technologies Signature Genes and Mathematical Model Derivation
� Bootstrap resampling method was used to randomly split training samples set into two parts to evaluate classifier model performance without selection bias.
� Classification performance was estimated by using the out-of-bootstrap-sample activity scores from the 250 iterations with their corresponding top 16 genes and classifier models.
� The median rank of each gene was calculated from 250 iterations and the top 16 genes were selected as the final signature biomarker gene set for assessing specific pathway activity.
Sample & Assay Technologies Current available product
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RT2 Profiler Plus PCR Array
RT2 Predictor PCR ArrayHuman WNT Signaling Pathway Activity
Human IL6/STAT3 Signaling Pathway Activity
Human TGFb Signaling Pathway Activity
Human Notch Signaling Pathway Activity
Human NFkB Signaling Pathway Activity
Human Oxidative Stress Toxicity
Human Hypoxia Signaling Pathway Activity
Human Unfolded Protein Response Toxicity
Human Mitochondrial Energy Metabolism Toxicity
Human WNT Signaling Pathway Plus
Human IL6/STAT3 Signaling Pathway Plus
Human TGFb/BMP Signaling Pathway Plus
Human Notch Signaling Pathway Plus
Human NFkB Signaling Pathway Plus
Human Oxidative Stress Plus
Human Hypoxia Signaling Pathway Plus
Human Unfolded Protein Response Plus
Human Mitochondrial Energy Metabolism Plus
Sample & Assay Technologies Product Formats – RT2 Profiler Plus PCR Array (96)
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Profiling+ SCORE
Profiling� Same workflow as current RT2 Profile PCR array
� Content (per sample)�Signature genes (16)�Pathway relevant genes (68)�HK gene (5)�GDC (1)�RTC (3)�PPC (3)
� Data analysis �Profiling�Pathway activity readout
Sample & Assay Technologies Product Formats – RT2 Profiler Plus PCR Array (384)
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Profiling+ SCORE
Profiling
� Same workflow as current RT2 Profile PCR array
� Content (per sample)�Signature genes (16)�Pathway relevant genes (68)�HK gene (5)�GDC (1)�RTC (3)�PPC (3)
� Data analysis �Profiling�Pathway activity readout
Sample & Assay Technologies Product Formats – RT2 Predictor PCR Array (96 well)
Title, Location, Date 15
SCORE Only� Same workflow as current RT2 PCR array
� Content (per sample)�Signature gene (16)�HK gene (5)�GDC (1)�RTC (1)�PPC (1)
� Data analysis �Pathway activity readout
Sample & Assay Technologies Product Formats – RT2 Predictor PCR Array (384 well)
Title, Location, Date 16
SCORE Only� Same workflow as current RT2 PCR array
� Content (per sample)�Signature gene (16)�HK gene (5)�GDC (1)�RTC (1)�PPC (1)
� Data analysis �Pathway activity readout
Sample & Assay Technologies Workflow – RT2 Profiler Plus PCR Array
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Purify RNA
Prepare cDNA from purified RNA using RT2 First Strand Kit
Add cDNA to RT2 SYBR Green Mastermix
Aliquote mix into RT2 Profile Plus PCR Arrays
Cycle in real-time cycler
Analyze results
Sample & Assay Technologies Workflow – RT2 Predictor PCR Array
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Purify RNA (FFPE samples NOT recommended)
Prepare cDNA from purified RNA using RT2 First Strand Kit
Add cDNA to RT2 SYBR Green Mastermix
Aliquote mix into RT2 Profile Plus PCR Arrays
Cycle in real-time cycler
Analyze results
Sample & Assay Technologies Outline
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� Introduction
� R&D Development Process
� Pathway Activity Score
� Specificity of Signature Biomarker Genes
� Application Data
Sample & Assay Technologies Pathway Activity Data Analysis Principle
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� Calculate ∆Ct of each signature gene vs average of HK genes of each sample
� Calculate ∆∆Ct of each signature gene between experimental and control samples
� Compare ∆∆Ct of signature genes in experiment sample to ∆∆Ct of signature genes in training data set
� A probability of pathway activity regulation is determined based on similarity between experimental sample and training data
� For Toxicity pathways:� 0 to 0.3: – no toxicity� 0.3 to 1: Toxicity
� For Signaling pathways:� -1 to -0.3: Pathway activity repressed� -0.3 to 0.3: No change in activity� 0.3 to 1: Pathway activity stimulated
Sample & Assay Technologies
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Data analysis Set Up – RT2 Profiler Plus & Predictor
Data analysis(Plus Array)
Pathway activity readout
Upload data
Select PlusArray catalog
Upload Ct in excel format
Sample & Assay Technologies
Title, Location, Date 22
Prediction of Pathway Activity in Experimental Sample
� A new set of 50 bootstrapped samples were drawn and used to train the final model ensemble with selected 16 biomarker signature genes.
� The resulting 50 models are used to predict the pathway activity score of test samples.
� All 50 predictions for a test sample are shown as a box-and-whisker plot.
Sample & Assay Technologies
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Data analysis Output – RT2 Profiler Plus & Predictor
Positive score indicates stimulation of pathway activity while negative score indicates repression. *Samples with P value <0.05 as well as probability score >0.3 or <-0.3 indicate significant pathway activity change in experimental sample as compared to control sample.
Training data used to derive biomarker signature genes and classifier algorithm
Summary of Pathway Activity Scores
0.82C
0.010.904624B
-0.925142A
P valuePathway activity score Sample
0.01
C
0.010.904624B
A
P value*Sample
0.146092
Sample & Assay Technologies
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Data analysis Output – RT2 Profiler Plus & Predictor
*Samples with P value <0.05 and probability score >0.3 indicate significant pathway activity change in experimental sample as compared to control sample.
Training data used to derive biomarker signature genes and classifier algorithm
Summary of Pathway Activity Scores
0.0010.474624C
0.0010.745142B
0.146092A
P valuePathway activity score Sample
C
B
0.146092A
P value*Sample
0.56
Sample & Assay Technologies Interpretation of score
Title, Location, Date 25
Array Score P value Prediction
Signaling
-1 to -0.3P<0.05 Pathway repressed
P>0.05 Unchanged
-0.3 to 0.3P<0.05 Unchanged
P>0.05 Unchanged
0.3 to 1P<0.05 Pathway activated
P>0.05 Unchanged
Toxicity
0 to 0.3P<0.05 No toxicity induced
P>0.05 Unchanged
0.3 to 1P<0.05 Toxicity induced
P>0.05 Unchanged
Sample & Assay Technologies Outline
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� Introduction
� R&D Development Process
� Pathway Activity Score
� Specificity of Signature Biomarker Genes
� Application Data
Sample & Assay Technologies
� Possible cross talk between Notch and NFκB, Wnt and TGFβ� Overall signatures seem to be specific to individual signaling pathway� It is expected to have some degree of cross talks given the nature of interactions between
different signaling pathways
+ - + - + - + - + -Notch Wnt IL6 NFκB TGFβ
-1.2
-0.9
-0.6
-0.3
0
0.3
0.6
0.9
1.2
-1.2
-0.9
-0.6
-0.3
0
0.3
0.6
0.9
1.2
Pat
hway
act
ivity
sco
re
WntArray
NotchArray
Specificity of Signature Biomarker Genes
Sample & Assay Technologies Outline
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� Introduction
� R&D Development Process
� Pathway Activity Score
� Specificity of Signature Biomarker Genes
� Application Data
Sample & Assay Technologies Application of Gene Expression Signature
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� Application Data 1: Pharmacodynamic marker� Efficacy measurement after drug treatment� Target validation after drug treatment
� Application Data 2: Pathway activity changes during neural stem cell differentiation
� Application Data 3 & 4: Dissection of mechanisms that regulate pathway activity� Crosstalk between pathways� Study cancer stem cells
Sample & Assay Technologies
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Application Data 1
� What can the gene expression signature be used for and how is it compared to traditional method?
Sample & Assay Technologies
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Application Data 1� HCT116 or 293H cells were treated with CCT036477 to inhibit WNT pathway activity or CHIR99021 to stimulate WNT
pathway activity, respectively� Human WNT Signaling Pathway Predictor Arrays and Western blots to detect active ββββ-catenin were used to assess
pathway activity changes
� Gene expression signature successfully indicated dose-response activation or repression of pathway activity after CHIR99021 or CCT036477 treatment, respectively
� Detection of decreased Wnt/ββββ-catenin signaling activity by gene expression signature preceded the detection of down-regulation of active ββββ-catenin protein levels by Western blot after CCT036477 treatment
Sample & Assay Technologies
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Application Data 2
� Neural stem cells were spontaneously differentiated for 10 days by withdrawing FGF and EGF from culture medium
� RT2 Predictor array indicted activation of Notch and TGFββββ pathway activity while repression of Wnt signaling during this differentiation process
NSC (undifferentiated) NSC (differentiated 10d)
Notch
Wnt
IL6/STAT3
NFkB
TGFβ
Pathway activity changes during neural stem cell differentiation
Sample & Assay Technologies
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Application Data 3
� What is the benefit of profiling changes in the expression of pathway-relevant genes coupled with pathway activity scores?
Sample & Assay Technologies
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Application Data 3� Notch signaling has been shown to be able to interact with WNT signaling� U105MG cells were treated with Notch NICD adenovirus and WNT Signaling Pathway Plus Array was used to
exam WNT pathway activity and associated mechanism
� Expression profiling indicated the upregulation of WNT target genes, Myc and LEF1, suggesting the activation of WNT/ββββ-catenin pathway activity.
Was the pathway activated?� Pathway activity score indicated that WNT/ββββ-catenin pathway activity was marginally activated in U105MG cells
after NICD treatmentWhat was the mechanism involved?� Gene expression profiling indicated upregulation of FZD4, a receptor for WNT signaling� However, the upregulation of WNT11, a non canonical WNT ligand, suggesting further study is needed to examine
the exact mechanism
Sample & Assay Technologies
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Application Data 4
CD133+
CD
133-
Wnt pathway activity confers chemoresistance to cancer stem-like cells in a neuroblastoma cell line
Tumour Biol. 2012 Aug 11. [Epub ahead of print]
Wnt Signaling RT2 PCR Array Plus uncovered:� Differential Wnt pathway gene expression profiles
associated with CD133+ (stem like) and CD133-neuroblastoma cells
� Activation of Wnt pathway signaling in CD133+ cells (sample 1) while repression of Wnt signaling in CD133- cells (sample 3) upon Dox treatment.
Sample & Assay Technologies Benefits of Profiler Plus & Predictor
Title, Location, Date 36
Profiler Plus� Ability to profile and determine pathway activity score� Pathway activity score is experimentally verified� Determine pathway activity score in a medium-throughput
manner� Simple workflow
Predictor� Ability to determine pathway activity score� Pathway activity score is experimentally verified� Determine pathway activity score in a high-throughput
manner� Simple workflow
Sample & Assay Technologies
Assess biological function and cellular toxicity using gene expression analysisRaed N Samara, PhDQIAGEN
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