Pcr plus and predictor 2013
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Pcr plus and predictor 2013 Pcr plus and predictor 2013 Presentation Transcript

  • Sample & Assay Technologies Assess pathway activity and cellular toxicity with biomarker signature genes Raed N Samara, PhD QIAGEN
  • Sample & Assay Technologies Outline Introduction R&D Development Process Pathway Activity Score Specificity of Signature Biomarker Genes Application Data 2
  • Sample & Assay Technologies Outline Introduction R&D Development Process Pathway Activity Score Specificity of Signature Biomarker Genes Application Data 3
  • Sample & Assay Technologies Gene expression for biological functional readout Use real-time PCR technology to develop a numerical score for biological functional readout. protein activity pathway activity toxicity physiological function pathological condition gene expression change Pathway and Toxicity Gene Profiling 4
  • Sample & Assay Technologies Pathway and Toxicity Gene Profiling Gene profiling assesses whether genes in a certain pathway are upregulated or downregulated. But did pathway activity change? Title, Location, Date 5
  • Sample & Assay Technologies Where to measure pathway activity? physiological pathological WNT WNT Fzd Fzd L R P Dsh Pathway Activity? APC Axin GSK3β β P TCF/ LEF1 Dsh APC Axin GSK3β β P β -catenin β -catenin L R P β -catenin β -catenin Myc TCF/ LEF1 CCND1 Myc CCND1 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 Title, Location, Date 6
  • Sample & Assay Technologies Gene expression for biological functional readout Use real-time PCR technology to develop a numeric score for biological functional readout. protein activity pathway activity toxicity physiological function pathological condition gene expression change Signature gene gene panel gene panel gene panel gene panel gene panel measure by real-time PCR Specific gene panel with companion score to monitor individual phenotype 7
  • Sample & Assay Technologies Pathway and Toxicity Gene Profiling & Activity RT2 Profiler Plus Title, Location, Date RT2 Predictor 8
  • Sample & Assay Technologies Outline Introduction R&D Development Process Pathway Activity Score Specificity of Signature Biomarker Genes Application Data 9
  • Sample & Assay Technologies Signaling pathway R&D Development Process 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 RT2 Profiler Plus PCR Array 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 RT2 Predictor PCR Array Human 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 12
  • Sample & Assay Technologies Product Formats – RT2 Profiler Plus PCR Array (96) 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 Profiling + SCORE 13
  • Sample & Assay Technologies Product Formats – RT2 Profiler Plus PCR Array (384) 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 Profiling + SCORE 14
  • 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 Title, Location, Date 15
  • 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 Title, Location, Date 16
  • Sample & Assay Technologies Workflow – RT2 Profiler Plus PCR Array 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 17
  • Sample & Assay Technologies Workflow – RT2 Predictor PCR Array 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 18
  • Sample & Assay Technologies Outline Introduction R&D Development Process Pathway Activity Score Specificity of Signature Biomarker Genes Application Data 19
  • Sample & Assay Technologies Pathway Activity Data Analysis Principle 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 20
  • Sample & Assay Technologies Data analysis Set Up – RT2 Profiler Plus & Predictor Upload data Select Plus Array catalog Upload Ct in excel format Data analysis (Plus Array) Pathway activity readout 21
  • Sample & Assay Technologies 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-andwhisker plot. Title, Location, Date 22
  • Sample & Assay Technologies Data analysis Output – RT2 Profiler Plus & Predictor Summary of Pathway Activity Scores Sample Pathway activity score P value value* A -0.925142 0.01 B 0.904624 0.01 C 0.146092 0.82 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 23
  • Sample & Assay Technologies Data analysis Output – RT2 Profiler Plus & Predictor Summary of Pathway Activity Scores Sample Pathway activity score P value value* A 0.146092 0.56 B 0.745142 0.001 C 0.474624 0.001 *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 24
  • Sample & Assay Technologies Array Interpretation of score Score -1 to -0.3 Signaling -0.3 to 0.3 0.3 to 1 0 to 0.3 Toxicity 0.3 to 1 Title, Location, Date P value Prediction P<0.05 Pathway repressed P>0.05 Unchanged P<0.05 Unchanged P>0.05 Unchanged P<0.05 Pathway activated P>0.05 Unchanged P<0.05 No toxicity induced P>0.05 Unchanged P<0.05 Toxicity induced P>0.05 Unchanged 25
  • Sample & Assay Technologies Outline Introduction R&D Development Process Pathway Activity Score Specificity of Signature Biomarker Genes Application Data 26
  • Sample & Assay Technologies Notch + - Specificity of Signature Biomarker Genes + Wnt - + IL6 - NFκB + - + TGFβ 1.2 0.9 Wnt Array 0.6 Pathway activity score 0.3 0 -0.3 -0.6 -0.9 -1.2 1.2 0.9 0.6 0.3 0 -0.3 Notch Array -0.6 -0.9 -1.2 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 -
  • Sample & Assay Technologies Outline Introduction R&D Development Process Pathway Activity Score Specificity of Signature Biomarker Genes Application Data 28
  • Sample & Assay Technologies Application of Gene Expression Signature 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 29
  • Sample & Assay Technologies Application Data 1 What can the gene expression signature be used for and how is it compared to traditional method? 30
  • Sample & Assay Technologies 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 β -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/β-catenin signaling activity by gene expression signature preceded the detection of downβ regulation of active β -catenin protein levels by Western blot after CCT036477 treatment 31
  • Sample & Assay Technologies Pathway activity changes during neural stem cell differentiation Application Data 2 NSC (undifferentiated) NSC (differentiated 10d) Notch Wnt IL6/STAT3 NFkB TGFβ 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 TGFβ pathway activity while repression of Wnt β signaling during this differentiation process 32
  • Sample & Assay Technologies Application Data 3 What is the benefit of profiling changes in the expression of pathway-relevant genes coupled with pathway activity scores? 33
  • Sample & Assay Technologies 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/β-catenin pathway activity. β Was the pathway activated? Pathway activity score indicated that WNT/β-catenin pathway activity was marginally activated in U105MG cells β after NICD treatment What 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 34
  • Sample & Assay Technologies Application Data 4 Wnt pathway activity confers chemoresistance to cancer stem-like cells in a neuroblastoma cell line CD133- 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 CD133neuroblastoma cells CD133+ Activation of Wnt pathway signaling in CD133+ cells (sample 1) while repression of Wnt signaling in CD133- cells (sample 3) upon Dox treatment. 35
  • 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 Title, Location, Date 36
  • Sample & Assay Technologies Questions Assess biological function and cellular toxicity using gene expression analysis Raed N Samara, PhD QIAGEN