The 6th Chinese Conference on Oncology &the 9th Cross-Strait Academic Conference on Oncology

May 21-23, Shanghai, China

Genetic Variation in DNA Repair and Clinical Outcomes of Lung Cancer

Qingyi Wei, MD, PhD

Department of Epidemiology

External Exposure External Exposure Internal Exposure Internal Exposure Biological Effects Biological Effects DiseaseDisease Progression Progression

Molecular EpidemiologyMolecular Epidemiology seeks to identify human cancer risk and carcinogenicseeks to identify human cancer risk and carcinogenic mechanisms to improve cancer prevention strategies mechanisms to improve cancer prevention strategies

is multi-disciplinary and translational, going from the is multi-disciplinary and translational, going from the

bench to the field and back bench to the field and back

uses biomarkers and state-of-art technologies to gainuses biomarkers and state-of-art technologies to gain

mechanistic information from epidemiological studiesmechanistic information from epidemiological studies

ExposureExposure DiseaseDisease??

19861986

Genomics Genomics TranscriptomeTranscriptome Epigenetics Epigenetics Proteomics Proteomics Metabolome/Metabomics Metabolome/Metabomics

Molecular EpidemiologyMolecular Epidemiology seeks to identify human cancer risk and carcinogenicseeks to identify human cancer risk and carcinogenic mechanisms to improve cancer prevention strategies mechanisms to improve cancer prevention strategies

is multi-disciplinary and translational, going from the is multi-disciplinary and translational, going from the

bench to the field and back bench to the field and back

uses biomarkers and state-of-art technologies to gainuses biomarkers and state-of-art technologies to gain

mechanistic information from epidemiological studiesmechanistic information from epidemiological studies

ExposureExposure DiseaseDisease??

20102010

Snapshot:Snapshot: No. (in millions)No. (in millions)

US population: US population: 300.0300.0 Smokers:Smokers: 46.5 46.5 All cancer casesAll cancer cases 1.3 1.3 Lung cancer Lung cancer 0.5 0.5 Head and neck cancerHead and neck cancer 0.04 0.04

Smoking and CancerSmoking and Cancer

ACS, 2009ACS, 2009

Genetic susceptibility plays a role !Genetic susceptibility plays a role !

Shared Cancer DeathsShared Cancer Deaths

Radiation 3%Radiation 3%

Tobacco 30%Tobacco 30%

Alcohol 5%Alcohol 5%Adult diet & Obesity 30% Adult diet & Obesity 30%

Others 7%Others 7%

Immunity 5%Immunity 5%

Sedentary lifestyle 5%Sedentary lifestyle 5%

Genetic factors 5%Genetic factors 5%

Viral Infection 5% Viral Infection 5%

Perinatal factors & growth 5%Perinatal factors & growth 5%

Harvard School of Public Health, 1998Harvard School of Public Health, 1998

Cell CycleCell CycleArrestArrest

DNA DNA RepairRepair

CancerCancer

ApoptosisApoptosis

Transcription Dependent ApoptosisTranscription Dependent Apoptosis

Transcription Independent ApoptosisTranscription Independent Apoptosis

DNA Damage-Response PathwayDNA Damage-Response PathwayDNA Damage-Response PathwayDNA Damage-Response Pathway

p53 Proteinp53 Protein

Accumulation Accumulation

DNADNA

DAMAGEDAMAGE

Altered ExpressionAltered ExpressionBAX, Fas, Bcl2BAX, Fas, Bcl2PIG3PIG3

Binding to Transcription Binding to Transcription

Replication-Repair FactorsReplication-Repair Factors

• TFIIH (XPB, XPD) and p62 TFIIH (XPB, XPD) and p62

binds to p53binds to p53

• PCNA (p21PCNA (p21WAF1WAF1 and GADD45) and GADD45)

Increased ExpressionIncreased Expression p21p21WAF1WAF1, MDM2, cyclin G, , MDM2, cyclin G, and GADD45and GADD45

Modified from Harris, 1994Modified from Harris, 1994

DNA repair phenotype (DRC) DNA repair phenotype (DRC)

as a risk factor of lung canceras a risk factor of lung cancer

DNA repair phenotype (DRC) DNA repair phenotype (DRC)

as a risk factor of lung canceras a risk factor of lung cancer

Nucleotide Nucleotide Excision Excision RepairRepair

http://genome.ucsc.eduhttp://genome.ucsc.edu

NER genes:

XPAXPCERCC1ERCC2 / XPDERCC3 / XPBXPE / DDB1ERCC4 / XPFERCC5 / XPGERCC6 / CSBERCC8 / CSA

Nucleotide Excision Repair of Nucleotide Excision Repair of Tobacco-Induced DNA Damage Tobacco-Induced DNA Damage Nucleotide Excision Repair of Nucleotide Excision Repair of

Tobacco-Induced DNA Damage Tobacco-Induced DNA Damage

OHOH

BPDEBPDE OO

HOHO

MFOMFO

Bulky DNA AdductBulky DNA Adduct

Normal DNANormal DNA

Benzo[a]pyreneBenzo[a]pyrene

Tobacco Smoke Tobacco Smoke

Nucleotide Excision Nucleotide Excision ComplexComplex

LigationLigation

POLPOL//, ligase, ligasePCNA, RFCPCNA, RFC RPARPA

NER Core Proteins

ERCC1XPAXPB/ERCC3XPCXPD/ERCC2XPE/DDB1/2XPE/ERCC4XPG/ERCC5

Neumann et al., Mol Carcino, 2005Neumann et al., Mol Carcino, 2005

3232P-post labelingP-post labeling

Host-cell reactivationHost-cell reactivation

Recombinant Plasmid DNA Expression Vectors

Used in the Host-Cell Reactivation Assay

4863 bp

Ap

Enh

PLuc

Bgl II

Xbal I

Pvu I

BamH I

Nar I

Bgl II

pCMVlucpCMVcat

5000 bp

Ap

Enh

P

cat

Hind III

Xbal I

EcoR I

EcoR I BPDE BPDE

Transfection Efficiency in Transfection Efficiency in the Host-Cell Reactivation Assaythe Host-Cell Reactivation Assay

Cheng et al., BioTechniques, 1997Cheng et al., BioTechniques, 1997

Od

ds

Ra

tio

Od

ds

Ra

tio

DRC (%) by QuartileDRC (%) by Quartile

Risk of Lung Cancer Associated with Low DRCRisk of Lung Cancer Associated with Low DRC

High LowHigh Low

0

1

2

3

4

5

6

Ist 2nd 3rd 4th

0

1

2

3

4

5

6Cases = 316Cases = 316Controls = 316Controls = 316

Cases = 221Cases = 221Controls = 229Controls = 229

HCR Assay (plasmid DNA)HCR Assay (plasmid DNA)

In Vitro Adduct Assay (genomic DNA)In Vitro Adduct Assay (genomic DNA)

Trend test : Trend test : PP < 0.001 < 0.001

Trend test : Trend test : PP < 0.001 < 0.001

Li et al, Cancer Res., 2001Li et al, Cancer Res., 2001

Wei et al., JNCI, 2000Wei et al., JNCI, 2000

* * For the low tertile DRC and others for the low-median DRC; **Recalculated based on published dataFor the low tertile DRC and others for the low-median DRC; **Recalculated based on published data

Suboptimal DRC and Cancer RiskSuboptimal DRC and Cancer Risk

LungLung 51/56 51/56 5.7 (2.1-15.7) 5.7 (2.1-15.7) Wei et al, 1996Wei et al, 1996316/316316/316 2.1 (1.5-3.0) 2.1 (1.5-3.0) Wei et al, 2000Wei et al, 2000764/677764/677 1.5 (1.2-1.9) 1.5 (1.2-1.9) Spitz et al, 2004Spitz et al, 2004

1522/16721522/1672 1.4 (1.2-1.6) 1.4 (1.2-1.6) unpublished dataunpublished data

Head & NeckHead & Neck 55/6155/61 2.2 (1.0-4.8) 2.2 (1.0-4.8) Cheng et al, 1998Cheng et al, 1998744/753744/753 1.9 (1.5-2.4) 1.9 (1.5-2.4) Wang et al., 2010Wang et al., 2010

Cancer # Case/Control OR (95%CI)Cancer # Case/Control OR (95%CI) ReferenceReference

Correlation between DNA repair Correlation between DNA repair

genotype and phenotypegenotype and phenotype

Correlation between DNA repair Correlation between DNA repair

genotype and phenotypegenotype and phenotype

Known SNPs in the 8 NER Core genes available Known SNPs in the 8 NER Core genes available in the NIEHS resequencing database in the NIEHS resequencing database

NER NER Nucleotides Nucleotides Location Location Function Function No. of No. of SNP SNP No of No of No of No of

core genecore gene / Protein/ Protein SNPsSNPs Density Density nsSNPs nsSNPs **nsSNPs nsSNPs **

(per kb)(per kb) with MAF > 0.05 with MAF > 0.05

ERCC1ERCC1 14kb / 297aa14kb / 297aa 19q13.2 19q13.2 EndonucleaseEndonuclease 7373 5.25.2 11 ----

-q13.3-q13.3

XPAXPA 22kb / 273aa22kb / 273aa 9q22.39q22.3 Damage detectionDamage detection 140140 6.46.4 22 ----

XPB/ERCC3XPB/ERCC3 37kb / 782aa37kb / 782aa 2q212q21 HelicaseHelicase 136136 3.73.7 22 ----

XPCXPC 33kb / 940aa33kb / 940aa 3p253p25 Damage detectionDamage detection 145145 4.44.4 1212 rs2228000( A499V)rs2228000( A499V)

rs2228001 (K939Q)rs2228001 (K939Q)

XPD/ERCC2XPD/ERCC2 19kb / 760aa19kb / 760aa 19q13.319q13.3 HelicaseHelicase 136136 7.27.2 22 rs1799793 (D312N)rs1799793 (D312N)

rs13181 (K751Q)rs13181 (K751Q)

XPE/DDB2XPE/DDB2 24kb / 427aa24kb / 427aa 11p12-p1111p12-p11 Damaged DNA Damaged DNA 7777 3.23.2 22 ----

binding binding

XPF/ERCC4XPF/ERCC4 28kb / 916aa28kb / 916aa 16p13.3 16p13.3 EndonucleaseEndonuclease 214214 7.67.6 77 ----

- p13.11- p13.11

XPG/ERCC5XPG/ERCC5 30kb / 1186aa30kb / 1186aa 13q2213q22 EndonucleaseEndonuclease 177177 5.95.9 1212 rs17655 (D1104H)rs17655 (D1104H)

TotalTotal 1,0981,098 4040 55

** ** XPC codon499 Ala/Val; XPC codon939 Lys/Gln; XPC codon499 Ala/Val; XPC codon939 Lys/Gln; XPD codon312 Asp/Asn; XPD codon751 Lys/Gln; XPG codon1104XPD codon312 Asp/Asn; XPD codon751 Lys/Gln; XPG codon1104 His/Asp His/Asp

Genotype Prediction of DRC Phenotype Genotype Prediction of DRC Phenotype (UV-damage) in Cancer-free Subjects(UV-damage) in Cancer-free Subjects

XPD A22541C A A 22 9.34 (4.65) Reference A C 55 8.18 (3.13) 0.208 C C 25 8.60 (2.47) 0.492

XPD Codon 751

Lys/Lys 46 8.30 (2.22) Reference Lys/Gln 38 9.51 (4.22) 0.096 Gln/Gln 11 7.06 (2.49) 0.063

XPC Intron 9

SS 36 8.79 (2.42) Reference SL 53 8.81 (3.86) 0.978 LL 13 6.73 (2.47) 0.020

XPD A22541C A A 22 9.34 (4.65) Reference A C 55 8.18 (3.13) 0.208 C C 25 8.60 (2.47) 0.492

XPD Codon 751

Lys/Lys 46 8.30 (2.22) Reference Lys/Gln 38 9.51 (4.22) 0.096 Gln/Gln 11 7.06 (2.49) 0.063

XPC Intron 9

SS 36 8.79 (2.42) Reference SL 53 8.81 (3.86) 0.978 LL 13 6.73 (2.47) 0.020

Genotype N % DRC (SD) P valueGenotype N % DRC (SD) P value

Qiao et al., Carcinogenesis, 2002

W H 1 2 30

5

10

15

20

n=9 n=6n=10n=48 n=29

XPC and XPD Genotypes

LU

C A

cti

vit

y (

%)

Correlation between DRC Phenotype (UV-damage) Correlation between DRC Phenotype (UV-damage) and Genotype in Cancer-free Subjectsand Genotype in Cancer-free Subjects

None Hetero Homozygous None Hetero Homozygous Three common Variants in Three common Variants in XPDXPD and and XPCXPC Genes Genes

N = 102N = 102PP = 0.02 = 0.02

Qiao et al., Mutat Res, 2002Qiao et al., Mutat Res, 2002

MDACC Lung Cancer GWAS StudyMDACC Lung Cancer GWAS StudyMDACC Lung Cancer GWAS StudyMDACC Lung Cancer GWAS Study

Genome Wide Association Study of Lung Cancer

– 1200 cases / 1200 controls 317K SNPs

Chris Amos et al., MDACC

Genes that have at least one significant SNP:GTF2H5, MSH3, RPA3, RBBP8, DCLRE1C, WRN, MGMT, PARP1, SMUG1, SHFM1, BRCA2, XRCC4, XRCC5, RAD54B, LIG4, PER1, FANCL, FANCL,

Plot for 1806 SNPs in 125 DNA repair genes covered by the Illumina HumamHap300 (v1.1) BeadChip

DNA repair and clinical outcomes DNA repair and clinical outcomes

of lung cancerof lung cancer

DNA repair and clinical outcomes DNA repair and clinical outcomes

of lung cancerof lung cancer

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Lung Cancer Patient Follow-up Lung Cancer Patient Follow-up Time LineTime Line

275 patients 275 patients with NSCLC with NSCLC 275 patients 275 patients with NSCLC with NSCLC

Adjusted* Relative Risk of Death Adjusted* Relative Risk of Death Associated with Efficient DNA RepairAssociated with Efficient DNA Repair

Variable No. RR 95% CI P value

All therapies 275 1.05 1.00 - 1.11 0.054

No therapy 104 1.01 0.86 - 1.19 0.860

Chemotherapy only 86 1.12 1.04 - 1.22 0.005

Surgery only 36 0.88 0.77 - 1.09 0.250

**Adjusted for age, sex, pack-years, entry into study date, and clinical stage. Only 2 stage IV Adjusted for age, sex, pack-years, entry into study date, and clinical stage. Only 2 stage IV patients had curative surgery without chemotherapy and were dropped from analysis of patients patients had curative surgery without chemotherapy and were dropped from analysis of patients treated with surgery. treated with surgery.

Bosken et al., JNCI, 2002Bosken et al., JNCI, 2002

Survival in MonthsSurvival in Months

00

0.10.1

0.20.2

0.30.3

0.40.4

0.50.5

0.60.6

0.70.7

0.80.8

0.90.9

11

00 1010 2020 3030 4040 5050 6060

Fra

cti

on

Su

rviv

ing

Fra

cti

on

Su

rviv

ing

No Chemotherapy (4.9 months)

Chemotherapy - Efficient DNA Repair (10.1 months))

Chemotherapy - Sub-optimal DNA Repair (15.8 months)

Effect of Chemotherapy on Survival Effect of Chemotherapy on Survival in Patients with Stage III/IV NSCLC by DRC in Patients with Stage III/IV NSCLC by DRC

Bosken et al., JNCI, 2002Bosken et al., JNCI, 2002

• Radiation-induced inflammation of the normal lung Radiation-induced inflammation of the normal lung tissuestissues

• RP is the most common dose-limiting complication of RP is the most common dose-limiting complication of thoracic radiationthoracic radiation

• About 10-20% of the patients experiencing moderate About 10-20% of the patients experiencing moderate or severe RP, when the total median radiation dose or severe RP, when the total median radiation dose was reaching 60-70 Gy was reaching 60-70 Gy

• Some genetic variants in TGF-Some genetic variants in TGF-ββ1 predict RP1 predict RP

Genetic Variaion in DNA repair genes and Genetic Variaion in DNA repair genes and Radiation-induced Pneumonitis (RP) in Cancer Radiation-induced Pneumonitis (RP) in Cancer

Patients Receiving Radiotherapy Patients Receiving Radiotherapy

N=184

158 - 60 to 70 Gy30 to 58 fractions 147 - chemotherapy74 - grade 236 - grade 3.

J Clin Oncol. 2009 Jul 10;27(20):3370-8

Some Selected Polymorphismsof DNA Repair Genes

Genes # Variants (comm) # AA Changes Ch

BERADPRT / PARP1 442 (9) 3 1APEX1 / APE1 58 (2) 1 14XRCC1 403 (18) 4 19

NERERCC1 149 (6) 0 19ERCC4 / XPF 330 (2) 1 19

HRRad51 342 ( 0) 0 1XRCC3 198 ( 6) 1 14

NHEJXRCC2 334 (7) 1 7XRCC7 / PRKDC 859 (12) 3 8

PathwayPathway GeneGene SNPsSNPs Location Location

BERBER XRCC1 XRCC1 Q399RQ399R ExonExonAPEX1APEX1 D148ED148E ExonExonADPRTADPRT Val762AlaVal762Ala ExonExon

NERNER ERCC1ERCC1 8092C>A8092C>A 3’UTR3’UTRERCC4ERCC4 R415Q R415Q ExonExon

DSBRDSBR RAD51RAD51 -135G>C-135G>C PromoterPromoterRAD51RAD51 -172G>T -172G>T PromoterPromoterXRCC2 XRCC2 Arg188HisArg188His ExonExonXRCC3XRCC3 T241MT241M ExonExonXRCC7XRCC7 6721G>T6721G>T ExonExon

Selected Functional SNPs in DNA Selected Functional SNPs in DNA Strand Break Repair GenesStrand Break Repair Genes

Association between Selected DNA Repair Gene SNPs and Risk of Grade ≥ 2 RP in NSCLC

Genes (rs#) No. (%) Hazards Ratio* P

Total # 214 (100)

APEX1 (rs1130409) (BER)TT 58 (27) 1.00GT 111 (52) 1.63 (0.89-2.96) 0.113GG 43 (21) 2.27 (1.13-4.59) 0.022

Rad51 (rs1801320 ) (HR)GG 173 (81) 1.00AG 35 (17) 0.48 (0.24-0.96) 0.038AA 5 (2) 1.46 (0.35-6.16) 0.609

XRCC3 (rs861539 ) (HR)CC 63 (29) 1.00CT 92 (17) 0.70 (0.43-1.16) 1.169TT 59 (2) 0.47 (1.24-0.90) 0.024

*Adjusted for age, sex, race, Karnofsky performance score, smoking status, tumor histology, and disease stage

Time (months)Time (months)

Cum

ulat

ive

Gra

de

Cum

ulat

ive

Gra

de ≥≥

3 R

P (h

azar

d)3

RP

(haz

ard) XRCC3 P = 0.011

CT+TTCT+TT

CCCC

Unpublished dataUnpublished data

BER

DSBR

Cumulative risk for RPCumulative risk for RP

Unpublished dataUnpublished data

DSBR

Cumulative SurvivalCumulative SurvivalRAD51 rs1801320 CG+CC vs. GG P = 0.009

Months

Cu

m s

urv

iva

l

XRCC2 rs3218536 GA+AA vs. GG P = 0.043

Months

Cu

m s

urv

iva

l

ConclusionsConclusionsConclusionsConclusions

High-throughput genotyping/DNA repair phenotyping should be integrated into risk assessment model

High-throughput genotyping/DNA repair phenotyping should be integrated into risk assessment model

Suboptimal DRC is a risk factor for developing lung cancer but lead to better response in the treatment

Suboptimal DRC is a risk factor for developing lung cancer but lead to better response in the treatment

Some genotypes of DNA repair genes may be correlated with the DRC phenotype, which warrants further investigations

Some genotypes of DNA repair genes may be correlated with the DRC phenotype, which warrants further investigations

Larger studies are needed to evaluate gene-gene and gene-environment interactions

Larger studies are needed to evaluate gene-gene and gene-environment interactions

AcknowledgmentsAcknowledgmentsAcknowledgmentsAcknowledgments Recruitment of subjectsRecruitment of subjects Administering questionnairesAdministering questionnaires Processing of blood samplesProcessing of blood samples Extraction of DNAExtraction of DNA Cell cultureCell culture PhenotypingPhenotyping GenotypingGenotyping Data entryData entry Data analysisData analysis ManuscriptsManuscripts

• Finding from: NIH-NCI and NIH-NIEHSFinding from: NIH-NCI and NIH-NIEHS