Cancer Genetic Markers of Susceptibility

Stephen J Chanock, M.D. November 29, 2006

http://cgems.cancer.gov

Mission of CGEMS

Conduct genome-wide SNP scans in Prostate cancer (1 in 8 men) Breast cancer (1 in 9 women) Analyze and publish findings

Rapid sequential replication studies Aggressive timeline Initial scan in nested case-control studies from

Prostate, Lung, Colon, Ovary (PLCO) Project Nurses’ Health Study

Replication Strategy for Prostate Cancer

Initial Study 1150 cases/1150 controls >500,000 Tag SNPs

Replication Study #1

3000 cases/ 3000 controls

Replication Study #2

2400 cases/ 2400 controls

Replication Study #3

2500 cases/ 2500 controls

~24,000 SNPs

~1,500 SNPs

200+ New ht-SNPs

25-50 Loci

Fin e

ly m

appe

d ha

plot

ypes

Power of the first two phases of CGEMS Point wise significance 10-7 ; "genome wide" significance 0.05

1

Power

0.8

0.6

0.4

AdditiveGRR : 1.4

0.10 0.2 0.3 0.4 0.5

Minor Allele Frequency

Recessive GRR : 2

Dominant GRR : 1.5

Multiplicative GRR : 1.3

0.2

0

GRR AA Aa aa

Recessive 2.0 1.0 1.0 2.0

Dominant 1.5 1.0 1.5 1.5 Continuous line : power for direct detection (r2 = 1) Additive 1.4 1.0 1.4 1.8 Dashed line : power for r2 = 0.8

Multiplicative 1.3 1.0 1.3 1.69 Skol et al. Nat Genet (2006)

CGEMS Scans

Prostate Cancer Breast Cancer T

Two Scans One Scan Illumina Illumina

317k 240k 550k (available) (Feb 2007)

(March 2007)

Recruitment Incidence Density SamplingNb. of selections

1st medic. end 1st end 2nd end 3rd end 4th end 5th

Rec

ruitm

ent f

rom

1 c

ente

r

random selection of 5 controls amongthese

random selection of 1 controls amongthese

random selection of 2 controls amongthese

random selection of 3 controls amongthese

random selection of 2 controls amongthese

5 pairs of 1 pair of 2 pairs of 3 pairs of 2 pairs of 3 pairs of prevalent incident incident incident incident incident

cases/controls case/control case/control case/control case/control case/control

1

1 1

1 2

2

2 1

2 1 1

1

as asexamination period period period period period control case

1111111111111111

16 pairs of case/control

25 DNAs to type

5 periods 6 strata

Aggressive Prostate Cancer

• High priority to examine early vs aggressive• Cohort based studies (screening)

– Bias towards early cases • Enrich primary scan with >55%

aggressive:45% early – Aggressive defined as:

• Gleason>7 +/or Stage C/D – Follow-up studies in cohorts

• Comparable distributions for early/advanced

Inclusion in CGEMS from PLCOof prostate cancer patients

1994

Oct 2001

Oct 2003

28 521 eligible participants

Aggressive Cancer0 0

737 624Non-aggressive Cancer

Matching with controls was performed for 737 aggressive cases and 493 randomly selected non-aggressive cases.

Non aggressive : stage <=2 (non invasive) and Gleason score <=6 Aggressive : stage >=3 (invasive) and Gleason score >=7

Distribution of genotyped individuals usedfor the search of association

Prostate cancer status Number of times selected as controls at start of CGEMS project 0 1 2 3 Total

durin

g fo

llow

-up

Always negative 0 1 082 22 1 1 105 "controls"

Diagnosed with 461 26 1 0 488non-aggressive C. 1 177 casesDiagnosed with 673 16 0 0 689aggressive C.

Total 1 134 1 124 23 1 2 282

dropped : 1 XX DNA 4 unexpected dup 1 173

"controls" dropped : 1 XX DNA dropped :

2 unexpected dup 1 XX DNA 3 failed genotype 4 failed genotype

Buccal Cell DNA and InfiniumTM II:ACS:CGEMS Pilot

23 matched blood and buccalArchived Buccal samples (2001/2002 in CPS-II)

Swish with ScopeTM and store after centrifugation

Extracted simultaneously with Autopure (Gentra)

Target 50ng/uL by QDNA (picogreen)4 outliers (0.5ng/uL- 35ng/uL)

HumanHap300 InfiniumTM II protocol Completion 99.02% Concordance 99.96%

PLCO WGS QC Removal of Inconsistent Genotypes

Low Completion Rate (<95%) Duplicates: HapMap & PLCO qc samples

Fitness for HW Proportion in controls Exclusion Cut-off: <0.001

Re-Map SNP Positions Examine adjacent bps of SNPs Heterogeneity in Cases/Controls

Cryptic stratification STRUCTURE (Pritchard) Principal Component Analysis (Price Nat Gen 2006) Study Center (9 for PLCO)

Discordance rate

Mean discordance

rate 2 10-4

Mean discordance

rate 2 10-4

28 individuals(with 24 duplicates)

Mean discordance

rate 1.4 10-3

PLCO CEPH-CGEMS CEPH-49 duplicate pairs 74 duplicate pairs HapMap

log 1

0(p-

valu

e)

log-log quantile plot ofp-value for Hardy-Weinberg proportion

-2

-3

-4

-5

-6

-7 -2-3-4-5-6

20 simulations

Observed values

expected : 244 observed : 586

expected : 2600 observed : 3340

Exact test , 299 779 SNPs log10(quantile)

QQ plot for ~300k SNPsQuantile

0

0.2

0.4

0.6

0.8

1.0

0 0.2 0.4 0.6 0.8 1.0p value

Log1

0(pv

alue

)Log-Log quantile plot for p-value for the 4

statistical tests used307,256 SNPs

-3

-4

-5

Log10(quantile) -3-4-5

Sing. Sampl. No cov

Sing. Sampl. with cov

Incid. Den. Sampl. No cov

Incid. Den. Sampl with cov

-6

Log(p-value)

Log(quantile)

Log/log quantile plot of p value (observed)

0

-2

-4

-6

-6 -4 -2 Log(quantile) 0

-1.9

-2.0

-2.1 -1.9-2.0-2.1

Log(p-value) or Log(quantile)

Log(quantile)

Log(p-value) Genomic control parameter = 0.99

PLCO Recruitment SitesOpportunity to look at

geographic differences

Admixture coefficient in PLCO samples

Asia Method : run merged PLCO data + HapMap data on STRUCTURE with 6000 SNPs having no pairwise r2 and high FST values. The population of origin of the HapMap samples is specified Result : Reliable identification of 3 outliers. They are all three control DNAs. and have to be removed from subsequent analysis

control

case

Africa Europe

Log-Log quantile plot for p-values of 101 SNPs that differentiate the populations of South and North of Europe

0

-1

-2

Log10(quantile) 0-1-2

Sing. Sampl. No cov

Sing. Sampl. with cov

Incid. Den. Sampl. No cov

Incid. Den. Sampl with cov

Seldin et al. PLOS Genetics 2:1339-1351 (2006)

Log1

0(p-

valu

e)

Lactase region Log10(p-value for association) LCT 0

rs2117511

rs6739713

rs1438307

rs6430585

rs9287442

rs1469996 rs4954633

rs2322659

rs2874874

rs3754690

rs3754689

rs309126

rs12478902

rs309160

136300k 136350k 136400k 136450k 136500k

-1

log10(0.05) rs4988235 rs182549

-2

-3

Bersaglieri et al. AJHG 74:1111-1120 (2004) position

-2

-3Log 1

0Pva

lue

Log10Pvalue of the 4 d.f. χ2 test plotted against the position of the 8q24 SNP (rs#1447295)* in build 35

0

-1

-4

-5 126000000 127000000 128000000 129000000 130000000 131000000

mapinfoPosition in build35 *Amundadottir Nat Genet 2006

*Freedman PNAS 2006

Characteristics of the SNPs demonstrating the strongest signal of association in 8q24

+-----------------------------------------------------------+| position Pval HW completion || rsnumber (b.35)

MAF controls rate

|-------------------------------------------------------|298. | rs4242382 128586755 .14 .7604 1 | 299. | rs7017300 128594450 .18 .1629 1 | 300. | rs7837688 128608542 .14 .8663 .999 | 301. | rs1447295 128554220 .14 .6012

1

| +------------------------------------------------------------+

Linkage disequilibrium (r2) with rs1447295 of the SNPs demonstrating the strongest signal of association

r2 with

| rs# position rs1447295 passoc | (b. 35)

| rs4242382 128586755 .94 .00007 || rs7017300 128594450 .71 .00009 || rs7837688 128608542 .84 .00003 || rs1447295 128554220 - .0003 |

Prostate Scan8q24 Region

Genotype RR for Indolent Genotype RR for aggressive

rs number susceptibility

allele allele

frequency Heterozyg. Homozig. Heterozyg. Homozig.

rs1447295 A 0.1 1.08 1.45 1.24 1.46 rs4242382 A 0.1 1.13 1.39 1.27 1.39 rs7017300 C 0.13 1.14 1.63 1.17 1.37 rs7837688 T 0.1 1.14 1.36 1.26 1.54

Key Findings: 1. Comparable risk as original reports in Nat Genet and PNAS

2. Comparable risk for BPC3 (~6500 cases/controls)

3. Discovery of 1 and perhaps 2 additional loci

Value-Added Analysis CGEMS Opportunity to investigate

• Gene:environment • Covariates: BMI, smoking, serum levels

• Multi-SNP Analysis • Gene:gene interactions

• Explore pathways • Follow-up in cohort studies in CGEMS

http://cgems.cancer.gov

CGEMS: caBIG PostingPre-Computed Analysis

Pre-computed Analysis No Restrictions

Raw Genotype Case/control Age (in 5 yrs) Family Hx (+/-)

Registration

Association Finding

Association Finding Report

Population Frequency Report

Acknowledgements

NCI Gilles Thomas Robert Hoover Joseph Fraumeni Daniela Gerhard Kevin Jacobs Zhaoming Wang Meredith Yeager Robert Welch Richard Hayes Sholom Wacholder Nilanjan Chatterjee Kai Yu Margaret Tucker Marianne Rivera-Silva

HSPH David Hunter Peter Kraft

ACS Heather Feigelson Carmen Rodriguez Eugene Calle Michael Thun