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
Dia
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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