Pcr plus and predictor 2013

Sample & Assay Technologies

Assess pathway activity and cellular toxicity with biomarker signature genesRaed N Samara, PhDQIAGEN

Sample & Assay Technologies Outline

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� Introduction

� R&D Development Process

� Pathway Activity Score

� Specificity of Signature Biomarker Genes

� Application Data


3

� Introduction






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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?


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


7

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


RT2 Profiler Plus RT2 Predictor


9

� Introduction





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)


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)


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


19

� Introduction





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


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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



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.


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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


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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


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


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� Introduction






� 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


28

� Introduction





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


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Application Data 1

� What can the gene expression signature be used for and how is it compared to traditional method?


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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


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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


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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?


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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


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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


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


Assess biological function and cellular toxicity using gene expression analysisRaed N Samara, PhDQIAGEN

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Pcr plus and predictor 2013