1. Integrated Studies Of Breast, Esophageal, And Gastric Cancers Using High Throughput Technologies And Computational Analyses Maxwell Lee National Cancer Institute Center for Cancer Research High-dimension Data Analysis Group March 19, 2014

2. Collaborators CCRHoward YangHuaitian Liu Mitsutaka Kadota Lalage WakefieldKent HunterBeverly MockDinah Singer Kevin GardnerJing HuangXin WangLi Yang Thomas RiedStephen HewittPaul MeltzerSean Davis DCEGPhil TaylorNan HuAlisa GoldsteinChristian Abnet Neal FreedmanSandy DawseyGretchen GierachNeil Caporaso Jonine Figueroa CBIITDaoud Meerzaman DCPMark ShermanBarbara DunnRonald LubetAsad Umar DCTDJay JiJason Lih UMASSKathleen Arcaro UNCMelissa Troester Purdue UniversitySulma Mohammed Windber Research InstituteHai Hu Toyama UniversityJunya Fukuoka

3. Breast cancer 1.Delineating genetic alterations for tumor progression in the MCF10A series of cell lines. 2.Identifying novel oncogenes and functional studies 3.Predicting clinical outcome using DNA methylation and gene expression signatures Esophageal and gastric cancers 1.Microarray data analyses 2.Whole genome sequencing data analyses Outline Of The Talk

4. 1.Delineating genetic alterations for tumor progression in the MCF10A series of cell lines. 2.Identifying novel oncogenes and functional studies 3.Predicting clinical outcome using DNA methylation and gene expression signatures Part 1 Breast Cancer

5. Modified Wellings-Jensen Model of Breast Cancer Evolution Lee el al. Breast Cancer Res. 2006;8(1):R6.Wellings et al. J Natl Cancer Inst. 1975 Aug;55(2):231-73.

6. Breast Cancer Progression Models Of The MCF10A Series Of Cell Lines NormalPremalignantLow gradeMetastaticcarcinoma MCF10A M1 MCF10CA1a M4 HRAS G12V MCF10CA1h M3 MCF10AT M2 Santner SJ,..., Miller FR. Breast Cancer Res Treat. 2001 Jan;65(2):101-10. Role of TGF-  Tumor Suppressor Metastasis Promoter Tang B,..., Wakefield LM. J Clin Invest. 2003 Oct;112(7):1116-24.

7. DNA Copy Number Alterations In MCF10A Series Of Cells Detected Using The Affymetrix 500K SNP Arrays Kadota et al PLoS One. 2010 Feb 15;5(2):e9201. chromosome

8. MYC Amplification In MCF10A Series Of Cells Detected By DNA Copy Number Analysis Using SNP Arrays Kadota et al PLoS One. 2010 Feb 15;5(2):e9201. MYC

9. CDKN2A/CDKN2B Deletions In MCF10A Series Of Cells Detected By DNA Copy Number Analysis Using SNP Arrays CDKN2A/B

10. PIK3CA Mutation In MCF10CA1h And MCF10CA1a Cells M1M2M3M4 Chr 3 A G A GG G:A = 2:1

11. DNA Copy Number Alterations In Primary Breast Tumors Detected With SNP Arrays 161 tumors chromosome 161 breast tumors putative novel oncogenes Affymetrix SNP5 array 1q 8q traditional approach my approach size of focal amplification multiple genes 1 gene frequency of tumors with amplification common high frequency not required but must occur in ≥ 1 tumor Kadota et al Cancer Res. 2009 Sep 15;69(18):7357-65.

12. Focal Amplification Detected in Primary Breast Tumors

13. Focal Amplification Of TBL1XR1 In Breast Tumors Tumor 1 Tumor 2

14. Frequent Over-expression Of TBL1XR1 In Primary Breast Tumors Using Tissue Microarray (TMA) Well-diffpoorly-diff negative164 positive3515 negative 31% positive 69% N=84 TBL1XR1 expression associates with poorly differentiated tumors with odds ratio = 1.7 In collaboration with Junya Fukuoka at Toyama University

15. TBL1XR1-shRNA Knockdown in MCF10CA1h (M3) Suppresses In Vivo Tumor Growth tumor volume (mm 3 ) N=10 N=14implants Western Blot In collaboration with Lalage Wakefield 9 of 107 of 101 of 14tumor incidence Day 39 p-value = 0.013

16. The Polish Breast Cancer Study (PBCS) The PBCS included 2386 cases and 2502 controls They resided in Warsaw, Poland from 2000-2003 Median follow-up: 8 years 208 PBCS tumors with Illumina HumanRef-8 v2 Expression data 226 PBCS tumors with Illumina Human Methylation27 data In collaboration with Mark Sherman, Jonine Figueroa, Paul Meltzer, Sean Davis, and Melissa Troester

17. An Algorithm For Methylation And Expression Index (MEI) Illumina Infinium HumanMethylation27 BeadChip Illumina HumanRef-8 v2 Expression BeadChip Differential methylation based on IHC (positive vs. negative for ER, PR, Her2, EGFR, or CK5) 2227 methylation markers in 1162 genes Top 3% most variable gene expression 541 genes 128 methylation markers in 65 genes MEI: the weighted sum of the gene expression where the weights are the negative numbers of the Spearman correlations.

18. Polish dataset: K-M survival based on MEI p = 0.002 Survival Probability Year

19. Polish dataset: K-M survival using MEI for ER+ and ER- samples Survival Probability p = 0.009p = 0.360 Survival Probability ER+ cases ER- cases Year

20. Validation: K-M survival using MEI for ER+ samples TCGA ER+GSE6532 ER+ NKI ER+ METABRIC ER+ Year Year Survival ProbabilityOS OSDMFS p = 0.004 p = 0.001 p = 0.00002

21. 1.Microarray data analyses 2.Whole genome sequencing data analyses Part 2 Esophageal And Gastric Cancers In collaboration with Phil Taylor, Nan Hu, Alisa Goldstein

22. Clustering Of ESCC And Gastric Cancers Using The Affymetrix U133 Gene Expression Data Wang et al. PLoS One. 2013 May 22;8(5):e63826. 54 pairs of ESCC 62 pairs of cardia cancer 72 pairs of noncardia cancer t: tumor n: normal

23. Anatomy Of Esophageal Cancer And Gastric Cancer cardia non-cardia

24. 1,898 ESCC cases 2,100 common control Illumina 660W Quad chip P-value derived from logistic regression models adjusted for age and sex esophageal squamous cell carcinoma (ESCC) Abnet C,..., Taylor P. Nat Genet 2010;42:764-767

25. 1,625gastric cancer 1,110gastric cardia cancer 515gastri non-cardia cancer 2,100common control

26. Analyses of Whole Genome Sequencing (WGS) Data Generated By The Complete Genomics Inc. 4 pairs of ESCC and blood 4 pairs of gastric non-cardia cancer and blood 7 pairs of gastric cardia cancer and blood

27. Circos Plot Of WGS data from A Gastric Cardia Cancer: Somatic SVs, CNAs, And SNVs

28. Somatic Structural Variations (SVs) In ESCC And Gastric Cancer

29. MACROD2 Exon 6 Was Frequently Deleted in Gastric Cancer O-acetyl-ADP-ribose deacetylase MACROD2 isoform 1

30. Mutation Substitution Patterns

32. Collaborators CCRHoward YangHuaitian Liu Mitsutaka Kadota Lalage WakefieldKent HunterBeverly MockDinah Singer Kevin GardnerJing HuangXin WangLi Yang Thomas RiedStephen HewittPaul MeltzerSean Davis DCEGPhil TaylorNan HuAlisa GoldsteinChristian Abnet Neal FreedmanSandy DawseyGretchen GierachNeil Caporaso Jonine Figueroa CBIITDaoud Meerzaman DCPMark ShermanBarbara DunnRonald LubetAsad Umar DCTDJay JiJason Lih UMASSKathleen Arcaro UNCMelissa Troester Purdue UniversitySulma Mohammed Windber Research InstituteHai Hu Toyama UniversityJunya Fukuoka

33. Dr. Robert Wiltrout Dr. Glenn Merlino