PREVALENCE OF TIDAL INTERACTIONS AMONG LOCAL SEYFERT GALAXIES
BIOMARKER STUDIES IN CLINICAL TRIALS Vicki Seyfert-Margolis, PhD.
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Transcript of BIOMARKER STUDIES IN CLINICAL TRIALS Vicki Seyfert-Margolis, PhD.
BIOMARKER STUDIES IN CLINICAL TRIALS
Vicki Seyfert-Margolis, PhD
CLINICAL DATA (Ontologies)
MECHANISM • Flow Cytometry• Autoantibody• ELISPOT• Cytokine Measures
DISCOVERY• Gene Expression• SNP/Haplotype• Proteomics
ITN Transplant Trial Model
SERIES OF DAYS
WEANING PERIOD
ONE YEAR
Drug Administration• Drug Levels• Drug Effects • Serum Cytokines • Cell Populations • Gene Expressions
Transplant• Graft Assessment • Time 0 Biopsy and Gene Expression• Drug Levels• Drug Effects
IS Withdrawal• Immune Response• Cell Populations - Flow• T Cell Function - IS Effects• Rejection- Gene Expression
Immediate Post Withdrawal• Rejection - Gene Expression• Cell Populations - Flow• T Cell Function
Follow Up: 2-5 years• Tolerance Marker ID• Gene Expression• Regulatory Cells - Flow Cytometry• Th1/Th2 Shift• Serum Profiles• Other Assays
BaselineScreening
End of Study
End of Study
DAY 0
ONE YEAR2-5 YEARS
Start of Study
Start of Study
Integration of domain-specific information
Gene ExpressionCytokine SecretionAntigen Expression
Flow Cytometry
EliSPOT Microarray
High Level Analysis Plan
Original Biopsy Designation
Counts by visit
Classification On left column
AR = Acute RejectionHEP = MildHEP-MOD = ModerateTo Severe
Gene Expression Statistical Framework
DesignComparisons of interestBiological replicates
Pre-processingNormalizationQuality Assurance
InferenceStatistic that incorporates variabilityFold Change (FC) and p-value cutoffFalse Discovery Rate (FDR) estimation to handle multiple testing comparisonsGene class testing, enrichment analysis to facilitate interpretation
Classification
Supervised and supervised approachesSupport Vector Machines (SVM), K-means, Random ForestsIssues with with over fitting dataUsing test set, training set approaches
Validation
Follow-up studyAlternate assay
Mechanism of Action Biomarker
(SI)
(TOL)(CAN) (HC)
Hierarchical Clustering (All Samples, V0, V6)
Hierarchical Clustering(Pearson correlation)All visits
Transcripts filtered for those differentially expressed between V6 and Baseline (V0) at FC >2 and FDR correction
4, 041 transcripts
Blue = baselineYellow = V6Red = FCLB
Baseline = 27FCLB = 21V6 = 12
Hierarchical Clustering (V6 vs. FCLB)
Hierarchical Clustering(Pearson correlation)V6 vs. FCLB
Transcripts filtered for those differentially expressed between FCLB and V6 at FC >1.5 and NO FDR correction
629 transcripts
Blue = V6Red = FCLB
FCLB = 21V6 = 12
Hierarchical Clustering – AR and Non AR FCLB
Hierarchical Clustering(Pearson correlation)FCLB No AR vs.FCLB with AR
Transcripts filtered for those differentially expressed between FCLB NO AR and FCLB with AR at FC >1.5 and NO FDR correction
580 transcripts
Blue = FCLB No ARRed = FCLB with AR
FCLB = 21V6 = 12
ITN Standard Flow Panel
Cell type/function FITC PE PerCP PECy7 APC
DCs CD11c dump* HLA-Dr CD123
DCs/costimulation CD11c CD80 dump* HLA-Dr CD123
CD11c CD86 dump* HLA-Dr CD123
CD11c IFN alpha dump* HLA-Dr CD123
Antigen presentation, activation andcostimulation
CD14 CD4 CD19 CD3 HLA-Dr
monocytes, B cells CD14 CD80 CD19 CD3 CD86
T cells/activation/naïve vs memory CD45RA CD45RO CD8 CD4 CD62L
T cells/activation CD8 CD69 CD4 CD3 CD122
CD8 IL-12R CD4 CD3 HLA-Dr
T regulatory cells CD8 CD25 CD4 CD3 CD62L
Cytokines/chemokines
Th1/Th2 profiles IFNgamma IL-4 CD8 CD3 CD4
Cytotoxic T cells perforin granzyme B CD8 CD3
Th1 cells CD4 CXCR3 CD8 CD3 CCR5
Th2 cells CD4 CCR3 CD8 CD3 CCR4
B cells
Precursors,germinal center, plasma CD38 IgD CD138 CD19 CD10
B cells, immature/mature, naïve CD27 or CD1d IgD CD38 or CD20 CD19 IgM
B cells, mature, naïve CD44 IgD CD38 CD19 CD10
B cells, mature, naïve CD23 IgD CD38 CD19 CD77
B1 cells CD1d CD21 CD5 CD19 CD23
Apoptosis Annexin V CD95 CD20 CD19 CD27
mature, costimulation, Ag. presn. CD27 CD80 HLA-DR CD19 CD86
NK cells CD57 CD56, CD16 CD14 CD3 CD8
NKT cells 6B11 v alpha 24 CD4 CD8
Thistlethwaite – Activated CD3CD4 T Cells (CD62L)
Regulatory T cells
Associations across assays and trials
CD19 IgG1 CD79A CD79B IgJ genes CD19 IgG1 CD79A CD79B IgJ genes
B cells- CD19Naïve B cells- CD27 IgD+ IgMlo
CD20Urine RT - PCR
FlowCytometry
Microarray
Operationally Tolerant Individuals
Data Flow
RawDataRawData
AnalysisPipelineAnalysisPipeline
BiostatisticalRepository
BiostatisticalRepository
Curated‘Results’
(Published)
Curated‘Results’
(Published)
Data Center - Validated Raw Data
TADA - Participant Annotation - Assay review, annotation - Quality Assurance - Normalization
TADA - R or SAS scripting - Analysis Reports - Experimental design, Hypothesis, statistical modeling - Exploratory analyses
Communications & TADA - Camera ready figures - Analysis revised or directed for manuscript, presentation, abstract etc.
National Institute of Allergy & Infectious Diseases
Funded by:
Juvenile Diabetes Research Foundation National Institute of Diabetes & Digestive & Kidney Diseases