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