Integrated Studies Of Breast, Esophageal, And Gastric Cancers Using High Throughput Technologies And Computational Analyses

Maxwell Lee

National Cancer InstituteCenter for Cancer Research

High-dimension Data Analysis Group

March 19, 2014

Collaborators

CCR Howard Yang Huaitian Liu Mitsutaka Kadota

Lalage Wakefield Kent Hunter Beverly Mock Dinah Singer Kevin Gardner Jing Huang Xin Wang Li YangThomas Ried Stephen Hewitt Paul Meltzer Sean Davis

DCEG Phil Taylor Nan Hu Alisa Goldstein Christian AbnetNeal Freedman Sandy Dawsey Gretchen GierachNeil Caporaso Jonine Figueroa

CBIIT Daoud MeerzamanDCP Mark Sherman Barbara Dunn Ronald Lubet Asad UmarDCTD Jay Ji Jason Lih

UMASS Kathleen ArcaroUNC Melissa TroesterPurdue University Sulma MohammedWindber Research Institute Hai HuToyama University Junya Fukuoka

• Breast cancer1. Delineating genetic alterations for tumor progression in the

MCF10A series of cell lines.2. Identifying novel oncogenes and functional studies3. Predicting clinical outcome using DNA methylation and gene

expression signatures

• Esophageal and gastric cancers1. Microarray data analyses2. Whole genome sequencing data analyses

Outline Of The Talk

1. Delineating genetic alterations for tumor progression in the MCF10A series of cell lines.

2. Identifying novel oncogenes and functional studies3. Predicting clinical outcome using DNA methylation and gene

expression signatures

Part 1 Breast Cancer

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.

Breast Cancer Progression Models Of The MCF10A Series Of Cell Lines

Normal Premalignant Low grade Metastatic carcinoma carcinoma

MCF10AM1

MCF10CA1aM4

HRAS G12V

MCF10CA1hM3

MCF10ATM2

Santner SJ, ..., Miller FR. Breast Cancer Res Treat. 2001 Jan;65(2):101-10.

Role of TGF-b

TumorSuppressor

MetastasisPromoter

Tang B, ..., Wakefield LM. J Clin Invest. 2003 Oct;112(7):1116-24.

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.

chro

mos

ome

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

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

CDKN2A/B

PIK3CA Mutation In MCF10CA1h And MCF10CA1a Cells

M1 M2 M3 M4

Chr 3

AG

AG G

G:A = 2:1

DNA Copy Number Alterations In Primary Breast Tumors Detected With SNP Arrays

161 tumors

chro

mos

ome

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

amplificationcommon

high frequency not required but must occur in ≥

1 tumor

Kadota et al Cancer Res. 2009 Sep 15;69(18):7357-65.

Focal Amplification Detected in Primary Breast Tumors

Focal Amplification Of TBL1XR1 In Breast Tumors

Tumor 1

Tumor 2

Frequent Over-expression Of TBL1XR1 In Primary Breast Tumors Using Tissue Microarray (TMA)

Well-diff poorly-diff

negative 16 4

positive 35 15

negative31%

positive69%

N=84

TBL1XR1 expression associates with poorly differentiated tumors withodds ratio = 1.7

In collaboration with Junya Fukuoka at Toyama University

TBL1XR1-shRNA Knockdown in MCF10CA1h (M3) Suppresses In Vivo Tumor Growth

tum

or v

olum

e (m

m3 )

N=10 N=10 N=14implants

Western Blot

In collaboration with Lalage Wakefield

9 of 10 7 of 10 1 of 14tumor incidence

Day 39

p-value = 0.013

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

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.

Polish dataset: K-M survival based on MEI

p = 0.002

Sur

viva

l Pro

babi

lity

Year

Polish dataset: K-M survival using MEI for ER+ and ER- samples

Sur

viva

l Pro

babi

lity

p = 0.009 p = 0.360

Sur

viva

l Pro

babi

lity

ER+ cases ER- cases

Year Year

Validation: K-M survival using MEI for ER+ samples

TCGA ER+ GSE6532 ER+

NKI ER+ METABRIC ER+

Year Year

Year Year

Sur

viva

l Pro

bab

ility

OS

OS

DM

FS

p = 0.004

p = 0.001 p = 0.001

p = 0.00002

1. Microarray data analyses2. Whole genome sequencing data analyses

Part 2 Esophageal And Gastric Cancers

In collaboration with Phil Taylor, Nan Hu, Alisa Goldstein

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 ESCC62 pairs of cardia cancer72 pairs of noncardia cancer

t: tumorn: normal

Anatomy Of Esophageal Cancer And Gastric Cancer

cardia

non-cardia

1,898 ESCC cases 2,100 common control

Illumina 660W Quad chip

P-value derived from logistic regressionmodels adjusted for age and sex

esophageal squamous cell carcinoma (ESCC)

Abnet C, ..., Taylor P. Nat Genet 2010;42:764-767

1,625 gastric cancer1,110 gastric cardia cancer515 gastri non-cardia cancer2,100 common control

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

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

Somatic Structural Variations (SVs) In ESCC And Gastric Cancer

MACROD2 Exon 6 Was Frequently Deleted in Gastric Cancer

O-acetyl-ADP-ribose deacetylase MACROD2 isoform 1

Mutation Substitution Patterns

Mutation Substitution Patterns

Collaborators

CCR Howard Yang Huaitian Liu Mitsutaka Kadota

Lalage Wakefield Kent Hunter Beverly Mock Dinah Singer Kevin Gardner Jing Huang Xin Wang Li YangThomas Ried Stephen Hewitt Paul Meltzer Sean Davis

DCEG Phil Taylor Nan Hu Alisa Goldstein Christian AbnetNeal Freedman Sandy Dawsey Gretchen GierachNeil Caporaso Jonine Figueroa

CBIIT Daoud MeerzamanDCP Mark Sherman Barbara Dunn Ronald Lubet Asad UmarDCTD Jay Ji Jason Lih

UMASS Kathleen ArcaroUNC Melissa TroesterPurdue University Sulma MohammedWindber Research Institute Hai HuToyama University Junya Fukuoka

Dr. Robert Wiltrout

Dr. Glenn Merlino