1. Affymetrix Gene Expression Microarrays Application to Pulmonary Arterial Hypertension Bob Stearman 02/24/2014

2. Pulmonary Arterial Hypertension (PAH) Rare Disease: USA ~1000 new cases/yr More Common in Females (2:1) with Earlier Presentation (30s-40s vs 40s-50s) BMPR2 Mutations Found in HPAH with Incomplete Penetrance Combination Drug Therapy Common PDE5 and Endothelin Inhibitors plus Prostacyclin Slows Progression But No Cure (5-yr 66%) Elevations of Pulmonary Hemodynamics

3. Pulmonary Arterial Hypertension (PAH) Hyperproliferation of PAECs and SMCs Structural Changes in Lung Vasculature Stacher et al., AJRCCM 186, 261 (2012) Vessel ThickeningMuscularization (SMCs)Plexiform Lesion (ECs)

4. Pulmonary Arterial Hypertension (PAH) Elevation in Pulmonary Hemodynamics Echocardiogram and Right Heart Catherization Studies Increased Inflammation Stacher et al., AJRCCM 186, 261 (2012) Vessel Thickening vs mPAP (p-value 0.03; r=0.27) Perivascular Inflammation

5. Multi-Center Lung Tissue Procurement 9 Geographically Spread Procurement Centers “Excessively” Complex Tissue Dissection 3+ Hours To Carryout 1 Central Tissue Repository Genomics Core & Secondary Tissue Repository (Frozen and RNALater) Rare Disease = Slow Recruitment Pulmonary Hypertension Breakthrough Initiative

6. PHBI Clinical Information Stacher et al., AJRCCM 186, 261 (2012)

7. PHBI Pathological Information Vessel Thickness (measured and fractional) Total, Intima, Media, Internal Diameter Plexiform Lesions Number and Density Inflammation Thrombi Pulmonary Vein Remodeling Stacher et al., AJRCCM 186, 261 (2012)

8. RUL = right upper lobe RML = right middle lobe RLL = right lower lobe H= Hilum. PHBI Lung Tissue Harvest Stacher et al., AJRCCM 186, 261 (2012)

9. PAH Patients: End-stage disease getting transplant 4 General Types of PAH: HPAH, IPAH, APAH, Other Failed Donors (FDs; controls): No match for lungs but available for research Slow Recruitment: MAs run in 3 “annual” batches PAH: Female Bias (2:1 F:M) and older FDs: Male Bias (1:2 F:M) and younger compared to PAH Pulmonary Hypertension Breakthrough Initiative: Sample Summary

10. Workflow of Gene Expression Reddy Gali, Ph.D. Microarray Analysis Using R/Bioconductor Biological question Experimental design Tissue / sample preparation Extraction of Total RNA Microarray hybridization & processing Image analysis Probe amplification & labeling Data analysis Expression measures - Normalization - Statistical Filtering - Clustering - Pathway analysis Biological Verification QC

11. PHBI: Experimental Design PAH Patients vs. FD Disease-specific gene expression patterns? 4 General Types of PAH: HPAH, IPAH, APAH, Other Will Gene Expression Patterns Detect Clinical Subclasses? PAH Patient Gender Bias PAH vs FD Gender and Age (?) Bias Microarrays run in 3 “annual” batches Different lots of arrays and reagents Changes in labeling methodology

12. PHBI Lung RNA Samples (in RNALater) Are Variable

13. PHBI: Normalization Uncorrected PHBI Data

14. PHBI: Normalization Corrected PHBI Data By Batch and Gender

15. PHBI: Normalization Corrected PHBI Data By Batch and Gender Low Variance Genes Removed (33K->6K)

16. PHBI: Clustering

17. PHBI: Expression Correlation With Clinical Parameters

19. Sources of Reproducibility (Experimental Design- Randomization) C4 Hybridized Sample B & A1 (Day 1); A6 & A15 (Day 2) [while same day samples had very similar #DE genes as C1, C2, and C3] C5 Hybridized Samples B & A1 (Day 1); A6 (Day 2); A15 (Day 3) C2 and C3 Stored Male Arrays ON, 4 o C while Female Arrays were Wash/Stain (Yang et al 2008)

20. Sources of Reproducibility (Experimental Design- Randomization) (Yang et al 2008) Each dataset is self-consistent Training on Center 3 generally groups M and F by sample type Training on Center 4 produces poor correlation between M and F by sample type

21. Stability of Results Critical Review of Published Microarray Studies for Cancer Outcome and Guidelines on Statistical Analysis and Reporting Alain Dupuy, Richard M. Simon (JNCI 2007) “Simon Says Top 40”

22. Nature Biotechnology 28, 827 (2010)

23. Affymetrix Gene Expression Microarrays Report Expectations Experimental Design and Questions Summary Signal Intensities Before and After Normalization PCA plots Significant Genes Listing including FC, p-value, FDR analysis Cluster Diagram (+/- Signal Intensities) Over-represented Gene Ontology Categories RDAVID?? Experimental Conclusions and Discussion

24. Affymetrix Gene Expression Microarrays Report Expectations