into False Positive Responses in the Local Lymph Node Assay (LLNA) - - PowerPoint PPT Presentation

Toxicogenomic Investigation into False Positive Responses in the Local Lymph Node Assay (LLNA)

Darrell Boverhof, Ph.D. Toxicology & Environmental Research and Consulting (TERC) The Dow Chemical Company Midland, MI Rboverhof@dow.com

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Acknowledgements

 DOW  David Adenuga  Michael Woolhiser  Bhaskar Gollapudi  Lindsay Sosinski  Rachel Golden  University of Manchester, UK  Ian Kimber  Rebecca Dearman  The Hamner Institutes for Health Sciences  Russell Thomas  Michael Black

Study supported by: CEFIC-LRI

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Proliferation/memory T-cells

LLNA GPMT

(Guinea pig maximization test)

Background-

Allergic Contact Dermatitis

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http://sensitive-learning.eu/mod/resource/view.php?id=77

Background-

Local Lymph Node Assay

Assay which detects the contact sensitization potential of chemicals (sensitization phase)

Treat Days 1, 2, 3 Inject 3H-thymidine Day 6 Rest Days 4, 5

3H-thymidine Incorporated

into DNA of dividing cells Remove Lymph Nodes Day 6 5 hours Sensitization / Lymph Node Proliferation Prepare Single, Cell Suspension Measure 3H-thymidine Incorporation Auricular Nodes Treat Days 1, 2, 3 Inject 3H-thymidine Day 6 Rest Days 4, 5

3H-thymidine Incorporated

into DNA of dividing cells Remove Lymph Nodes Day 6 5 hours Sensitization / Lymph Node Proliferation Prepare Single, Cell Suspension Measure 3H-thymidine Incorporation Auricular Nodes

 Reported as a stimulation index (SI)  Ratio Treatment/Vehicle  Cutoff for positive response SI >= 3

Dorsal surface of ears

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The Problem of False Positive Chemistries in the LLNA

The Problem of False Positive Chemistries in the LLNA

Can differential gene expression responses (toxicogenomics) in the lymph node be applied to distinguish true sensitizers from false positives in the LLNA? Functional insights into different mechanisms Development of molecular classifiers to distinguish between these two classes

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Study Approach

Critical Elements:

Chemical Selection

Selection of false positive chemistries # of Chemistries

Dose

All chemicals tested at equipotent doses in LLNA

Time

Examined multiple time points

Comprehensive

Anchor gene expression responses to traditional LLNA endpoint 2 Phases- Development and Confirmation

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Study Approach- Comprehensive

Phase 1

 9 Sensitizers  7 False positives  Functional evaluation of differential gene expression

Whole genome array

 Molecular classifier development and assessment

Phase 2

 6 Sensitizers  6 False Positives  Confirmation of functional gene expression responses

Focused QRTPCR arrays

 Evaluation of classifier performance

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Phase 1 Test Materials

 Equipotent doses (SI 6-9) calculated based on results from screening LLNA

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Sensitizer Class Test Material Vehicle Dose Dinitrochlorobenzene (DNCB) Acetone 0.10% Hexylcinnamic aldehyde (HCA) Acetone 25% Isoeugenol Acetone 10% para-phenylene diamine(PPD) Acetone 1% Hydroquinone (HQ) Acetone 0.25% Methyldibromo glutaronitrile (MDBGN) Acetone 20% Toluene diisocyanate (TDI) Acetone 0.04% Trimellitic anhydride (TMA) Acetone 0.65% Ammonium Hexachloroplatinate (AHCP) DMSO 0.70% Oleic acid DMSO 50% Maleic acid DMSO 11.50% Sodium lauryl sulphate (SLS) DMSO 25% Tetraethylene glycol Monotetradecyl ether (TGME) Acetone 20% Polyaminofunctional siloxane Acetone 45% N-decylphenol polyethyleneglycol ether (DPP) Acetone 35% Hexadecan-1-ol Ethoxylated (EO2) C16 (HDE) Acetone 30% Presumptive False Positives Sensitizers

Phase 1- Stimulation Index response

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Phase 1- Toxicogenomic evaluation

Isolate RNA Reverse Transcription Cy3 Isolate Auricular nodes Hybridize on array Data Extraction and Analysis Isolate RNA Reverse Transcription Cy3 Isolate RNA Reverse Transcription Cy3 Cy3 Isolate Auricular nodes Hybridize on array Data Extraction and Analysis

 Test materials grouped into a 3 class coding scheme –

 Vehicles controls  Sensitizers  False positives

 Data filtered on expression (± 1.5 FC) and 2 way ANOVA linear contrasts (FDR < 0.05)

 Commonly regulated genes  Responses unique to Sens and FP

 Functional evaluation- Gene Ontology

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Toxicogenomic evaluation

Commonly Expressed Genes

Genes similarly regulated by sensitizers and false positives relative to controls

 Functional Categories involved in Cell Proliferation

Initiation of mitosis Cell cycle regulation The metaphase checkpoint Chromosome condensation in prometaphase Spindle assembly and chromosome separation

• Indicates both Sensitizers and False positives

induce a LN proliferative response

• Offer no ability to discriminate between sensitizers

and false positives

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Genes differentially regulated by Sens relative to both FPs and Controls

Toxicogenomic evaluation Sensitizer-Specific Genes

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Key functional categories –

• 1. Positive regulation of immune system process
• 2. Leukocyte activation and migration

Normalized Mean Intensity (Log2)

Untreated Acetone DMSO AHCP TDI TMA DNCB HCA HQ Isoeugenol PPD MDBGN DPP HDE Siloxane TGME Maleic Oleic SLS

• 4
• 2

2 4

Fxyd4 Thbs4 Cphx Lgals7 Il21

Vehicles Sensitizers False Positives

Genes differentially regulated by FPs relative to both Sens and Controls

Toxicogenomic evaluation False Positive-Specific Genes

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Key functional categories –

• 1. Acute inflammatory response
• 2. Innate defense response (Neut/Mac markers – Mpo, Lcn2)

Phase 2 Test Materials

 Equipotent doses (SI 6-9) calculated based on results from screening LLNA

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Sensitizer Class Test Material Conc. DNCB 0.05% Benzoquinone (BZQ) 0.10% 2-hydroxyethyl acrylate (2-HEA) 20% Phenyl acetaldehyde (PA) 20% Citral 30% Propyl Gallate (PG) 3% DPP 35% Benzalkonium Chloride (BZC) 1% Squalene 50% Methyl Oleate (MO) 50% Hexaethylene glycol monododecyl ether (HGME) 25% Linolenic Acid (LA) 50% Sensitizers Presumptive False Positives

Stimulation Index

Acetone DNCB BZQ 2-HEA PA Citral PG DPP BZC Squalene MO HGME LA 5 10 15

Sensitizers False Positives

SI=3

Phase 2- Stimulation Index response

Phase 2 Sensitizer-Specific Genes

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Key functional categories –

• 1. Positive regulation of immune system process
• 2. Leukocyte activation and migration

Fold change to Veh

DNCB BQ 2-HEA PA Citral Propyl galate DPP Benzalk Cl Squalene Me oleate HGME Linoleic acid

3 6 9 12 15

Cphx Il21 Lgals7 Thbs4

Sensitizers False Positives

Phase 2 False Positive-Specific Genes

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Key functional categories –

• 1. Acute inflammatory response
• 2. Innate defense response (Neut/Mac markers – Mpo, Lcn2)

Fold change to Veh

DNCB BQ 2-HEA PA Citral Propyl galate DPP Benzalk Cl Squalene Me oleate HGME Linoleic acid

3 6 9 12 15 18

Cd5l Dao Defa-rs2 Il12b Syn3

Sensitizers False Positives

Development of Statistical Classifiers- Phase 1

Partition Data into 5 Sets Gene Expression Microarrays

Accuracy Sensitivity Specificity AUC

Set 1 Set Aside Select Features Build Model Predict Hold- Out Set

Repeat 10X – Tested in a total of 84

• ptimized models

Averaged parameter values provide broad- based evaluation

• f predictive

performance Total of 50 iterations

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Development and Evaluation of Statistical Classifiers

30 genes

PHASE 1 PHASE 2

Summary/Conclusions

Sensitizer- and False Positive-specific gene expression responses were identified

Sens- antigen-mediated T-cell response. FPs- consistent with a pro-inflammatory response

Class-specific gene expression responses were reproducible in an independent experiment Molecular classifiers were developed that had very good performance Genes that made up the classifier were consistent with those identified through the functional analysis Approach could be used as part of a WoE analysis for suspected false positives

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Acknowledgements

 DOW  David Adenuga  Michael Woolhiser  Bhaskar Gollapudi  Lindsay Sosinski  Rachel Golden  University of Manchester, UK  Ian Kimber  Rebecca Dearman  The Hamner Institutes for Health Sciences  Russell Thomas  Michael Black

Study supported by: CEFIC-LRI

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