tranSMART v1.2 Case Study

for PredicTox

April 2015

Agenda What is PredicTox? Brief tranSMART overview Answering scientific questions with

tranSMART’s help: A case study maximizing data value

Questions?

Agenda

A public private partnership, led by the Reagan-Udall Foundation for the FDA, with the goals of: Applying systems-based approaches to better

understand Adverse Events (AEs) Developing predictive models

Pilot project --- one drug class &AE Use TranSMART as platform for Integration of

clinical, preclinical and molecular data

PredictTox

Agenda What is PredicTox? Brief tranSMART overview Answering scientific questions with

tranSMART’s help: A case study maximizing data value

Questions?

Agenda

tranSMART Data Warehouse Structure

Szalma S.; Koka, VC.; Khasanova, T.; Perakslis, E. :Effective knowledge management in translational medicineJournal of Translational Medicine 2010, 8:68

Analytical and visualization

tools

SecurityAccess

(enterprise vs. project level)

Patient Privacy

Diverse Data

Warehouse structure

PredictTox

tranSMART Data for PredicTox (so far) 18 gene expression data sets from GEO

Human white blood cells having to do with left ventricular dysfunction, and the drugs Imantinib, Sunitinib, and Trastuzumab.

Preclinical studies with gene expression data from heart tissue of rats dosed with imatinib.

These datasets may provide confirmatory gene expression profiles as it differentiates left ventricular dysfunction from other cardiac disease.

Information gleaned from these data may provide mechanistic insight into the cardiotoxicity of tyrosine kinase inhibitors.

Agenda What is PredicTox? Brief tranSMART overview Answering scientific questions with

tranSMART’s help: A case study maximizing data value

Questions?

Agenda

Case Study GSE21125 Blood Signature of Pre-heart Failure: A

Microarray Study (Smih et al) Human white blood cells from healthy, heart failure risk patients,

asymptomatic left ventricular dysfunction patients, chronic heart failure, acute heart failure patients

Platform - RNG-MRC_HU25k_NICE PLoS ONE 6(6): e20414. doi:10.1371/journal.pone.0020414

GSE2535 In chronic myeloid leukemia white cells from cytogenetic responders and non-responders to imatinib have very similar gene expression signatures (Crossman et al) Analysis of peripheral blood and bone marrow of chronic myelogenous

leukemia (CML) patients prior to imatinib (Gleevec) treatment. This study attempts to determine transcriptional signature of imatinib non-responders.

Platform - Affymetrix Human Genome U95 Version 2 Array Haematologica 2005; 90:459-464

Case Study Data

Analytical Rationale Imatinib is the first targeted therapy used to treat

Philadelphia chromosome positive CML Targets and inhibits the catalytic activity of constitutively

active tyrosine kinase Bcr-Alb. Also associated with reduced left ventricular ejection

volume indicative of left ventricular dysfunction With data loaded into tranSMART we investigated the

correlation between gene expression signatures of patients with ALVD and those associated with imatinib response Do these profiles have overlapping genes?

What are the functions of the overlapping genes? Do these profiles show effects on similar pathways? Can apply the signature of one data set to cluster gene

expression profiles from the other?

Case Study

Analysis WorkflowMarker

Selection Analyses

Gene Lists

Investigate the Role of

Shared Genes

Biomarkers?

Pathway Enrichment Analysis

Mechanistic Similarities?

Clustering Analysis

Hierarchical clustering using swapped gene list

Case Study

GSE2535 Marker Selection

Calculates the most differentiating genes between two datasets

Case Study

Gene List Creation Gene lists gathered using Marker Selection

workflow in tranSMART were edited to remove control genes, repeats, unrecognized loci and ORFs GSE21125 Marker Selection list yielded 54

recognizable gene symbols when loaded into tranSMART when comparing healthy controls with ALVD patients

GSE2535 Marker Selection list yielded 92 recognizable gene symbols when loaded into tranSMART when comparing responders vs. non-responders

Case Study

Analysis WorkflowMarker

Selection Analyses

Gene Lists

Investigate the Role of

Shared Genes

Biomarkers?

Pathway Enrichment Analysis

Mechanistic

Similarities?

Clustering Analysis

Hierarchical clustering using swapped gene list

Case Study

Venn Diagram GSE21125 and GSE2535

Compared gene lists from GSE21125 and GSE2535 Shared gene CACNA2D2

CACNA2D2 – voltage-dependent calcium channel Homozygous mutation is associated with epileptic encephalopathy Null mutants in mice display seizures, cardiac abnormalities and premature

death. Known to promote tumorigenesis and over expression is associated with

increased cell proliferation. Oncogene. 2015 Jan 26. doi: 10.1038/onc.2014.467

Search in PubMed for CACNA2D2 and left ventricular dysfunction yielded no results

Case Study

CACNA2D2 Expression

CACNA2D2 down-regulated in patients with ALVD p=6.41 x 10-6

GSE21125 GSE2535

• CACNA2D2 slightly down-regulated in patients unresponsive to imatinib treatment p= 0.011

• Gene expression data for CACNA2D2 show a statistically significant difference in these comparisons

Case Study

CACNA2D2 in GSE21125 CACNA2D2 seems to

highly differentiate the investigated pathologies. Not part of the blood

gene expression signature in Smih et al

Pairwise t-Test

Case Study

Analysis WorkflowMarker

Selection Analyses

Gene Lists

Investigate the Role of

Shared Genes

Biomarkers?

Pathway Enrichment Analysis

Mechanistic

Similarities?

Clustering Analysis

Hierarchical clustering using swapped gene list

Case Study

Pathway Enrichment AnalysisGSE21125 Smih et al

GSE2535 Crossman et al -log 0.05≈ 1.3

Case Study

Analysis WorkflowMarker

Selection Analyses

Gene Lists

Investigate the Role of

Shared Genes

Biomarkers?

Pathway Enrichment Analysis

Mechanistic Similarities

?

Clustering Analysis

Hierarchical clustering using swapped gene list

Case Study

GSE21125 Hierarchical Clustering with GSE2535 Marker Selection List

Clustering based on the GSE2535 gene list shows separation of Control profiles but does not effectively differentiate ALVD patients.

Case Study

GSE2535 Hierarchical Clustering with GSE21125 Marker Selection List

Applying the GSE21125 Marker Selection List does not distinguish imatinib responders vs non-responders

Case Study

Further inspection of GSE2535

GSM48360 GSM48357 GSM48368 GSM4835441806_at38013_at39583_at

1968_g_at36397_at35923_at34582_at38767_at

1892_s_at37866_at

39015_f_at526_s_at41476_at1114_at

32331_at680_s_at1197_at

41542_at40340_at32918_at593_s_at36475_at31520_at

37745_s_at36094_at37346_at41559_at40842_at38371_at37739_at41060_at40242_at39096_at1894_f_at33325_at

1011_s_atAFFX-HUMGAPDH/M33197_5_at

256_s_at

Column13Column15

Cluster1(n=6877)

GSM48360 GSM48355 GSM48357 GSM48373 GSM48368 GSM48369 GSM483541194_g_at

41032_at39326_at34042_at36139_at34029_at37880_at40006_at40280_at

40986_s_at37251_s_at

405_at32258_r_at

32733_at34120_r_at

846_s_at38591_at41256_at1929_at

31862_at36566_at40236_at32622_at

35825_s_at37162_at1284_at

41409_at38802_at201_s_at33326_at1512_at717_at

38824_at41202_s_at

32119_at37603_at40853_at195_s_at

Column13Column15

Cluster2(n=5748)

-4.00 4.00

Column13

Non-responder

Responder

Column15

Leipzig

Mannheim

Batch effect

Case Study

Summary Marker Selection analysis GEO data sets GSE21125 and

GSE2535 in tranSMART yielded gene lists of 54 and 92 gene respectively

These lists had one gene in common CACNA2D2 voltage-dependent calcium channel This gene is down regulated in GSE21125 in patients with

ALVD and distinguishes ALVD from the other pathologies studied

Down regulated in non-responders to imatinib Null mutants in mice display seizures, cardiac abnormalities

and premature death

Case Study

Summary Pathway enrichment analysis

GSE21125 – pathways involved in cell adhesion and cytoskeleton remodeling

GSE2535 – pathways involved in VEGF signaling and ESR1 activation The datasets share one pathway – “Cytoskeleton remodeling Role of PKA

in cytoskeleton reorganization” the significance of which remains to be investigated

Hierarchical clustering analysis shows poor performance with swapped gene lists

Case Study

Agenda What is PredicTox? Brief tranSMART overview Answering scientific questions with

tranSMART’s help: A case study maximizing data value

Questions?

Agenda

How to Get Involved Looking for partners: data, expertise,

funding, and other resources Steering Committee and work groups

forming soon--- stay tuned for updates No membership fee --- funding is raised

from a variety of public and private sources For more info: contact

nbeck@reaganudall.org or see her after the talk

Extra Slides

Lessons learned The more attributes for the samples the

better The more data the better!

Need same tissue, same species, similar treatments, and similar measurements!

Case Study

Current Project Activities

Securing data sharing agreements with pharma companies

Gathering publically available data Building the ontology of Cardiac Adverse

Events Establishing the project governance structure Building out tranSMART instance

Rancho developed use case using GEO data sets to demonstrate utility…

Pilot project – develop centralized knowledge base that includes publically available clinical and molecular data having to do with tyrosine kinase inhibitors (TKIs) and mAbs and cardiac AEs; specifically left ventricular dysfunction.

Data goes into tranSMART infrastructure Integrated knowledgebase Mine information on biomarkers, non-clinical

and clinical screens Assist in hypothesis generation and mechanistic

level understanding

PredictTox

Concept of tranSMART on data level

tranSMART

Examples Of Data Stored In tranSMART Data from clinical trials

Demographics, medical history Treatment information Clinical outcomes, including AEs OMICs type data (gene expression, proteomics, RBM, SNPs)

Pre-clinical Studies PK/PD data OMICs type data for animal models and cell lines Toxicology data

Warehouse structure

Concept of tranSMART

Discovery Group

Preclinical Group

Clinical Development

tranSMART

Cytoskeleton remodeling Role of PKA in cytoskeleton reorganization

Case Study

Conclusions We went over tranSMART and explored main

functionality of the platform We used platform to answer a scientific

question Great sets from GEO were curated and

loaded into this tranSMART instance – they serve as a starting point and will provide useful comparisons for new, exciting data that is yet to come

We need more data!

Case Study

Marker Selection vs. Signature from Smih et al.

Tested the performance of the blood signature from supporting reference and the Marker Selection gene list

Performed hierarchical clustering analysis using “Marker selection list” generated by comparing

gene expression from healthy controls and ALVD patients

7 gene list from the paper

Case Study

GSE21125 Clustering with Marker Selection List

• The marker selection list was able to differentiate patient samples based on pathology

Case Study

Clustering with Smih et al Gene List

The list from Smih et al. performs well at clustering samples from patients with ALVD

Case Study