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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
Molecular Epidemiology
seeks to identify human cancer risk and carcinogenic
mechanisms to improve cancer prevention strategies
 is multi-disciplinary and translational, going from the
bench to the field and back
 uses biomarkers and state-of-art technologies to gain
mechanistic information from epidemiological studies

1986
Exposure
?
Disease
External Exposure Internal Exposure Biological Effects Disease Progression
Molecular Epidemiology
seeks to identify human cancer risk and carcinogenic
mechanisms to improve cancer prevention strategies
 is multi-disciplinary and translational, going from the
bench to the field and back
 uses biomarkers and state-of-art technologies to gain
mechanistic information from epidemiological studies

2010
Exposure
?
Disease
Genomics Transcriptome Epigenetics Proteomics Metabolome/Metabomics
Smoking and Cancer
Snapshot:
No. (in millions)
US population:
Smokers:
All cancer cases
Lung cancer
Head and neck cancer
300.0
46.5
1.3
0.5
0.04
Genetic susceptibility plays a role !
ACS, 2009
Shared Cancer Deaths
Perinatal factors & growth 5%
Others 7%
Immunity 5%
Tobacco 30%
Genetic factors 5%
Adult diet & Obesity 30%
Alcohol 5%
Radiation 3%
Viral Infection 5%
Harvard School of Public Health, 1998
Sedentary lifestyle 5%
DNA Damage-Response Pathway
DNA
DAMAGE
Altered Expression
BAX, Fas, Bcl2
PIG3
Transcription Dependent Apoptosis
Apoptosis
p53 Protein
Accumulation
Increased Expression
p21WAF1, MDM2, cyclin G,
and GADD45
Cell Cycle
Arrest
Cancer
DNA
Repair
Binding to Transcription
Replication-Repair Factors
• TFIIH (XPB, XPD) and p62
binds to p53
• PCNA (p21WAF1 and GADD45)
Transcription Independent Apoptosis
Modified from Harris, 1994
DNA repair phenotype (DRC)
as a risk factor of lung cancer
Nucleotide
Excision
Repair
NER genes:
XPA
XPC
ERCC1
ERCC2 / XPD
ERCC3 / XPB
XPE / DDB1
ERCC4 / XPF
ERCC5 / XPG
ERCC6 / CSB
ERCC8 / CSA
http://genome.ucsc.edu
Nucleotide Excision Repair of
Tobacco-Induced DNA Damage
Benzo[a]pyrene
BPDE
O
HO
32P-post
MFO
OH
labeling
Bulky DNA Adduct
NER Core
Proteins
Nucleotide Excision
Complex
POL/, ligase
PCNA, RFC
RPA
Ligation
ERCC1
XPA
XPB/ERCC3
XPC
XPD/ERCC2
XPE/DDB1/2
XPE/ERCC4
XPG/ERCC5
Tobacco Smoke
Host-cell reactivation
Normal DNA
Neumann et al., Mol Carcino, 2005
Recombinant Plasmid DNA Expression Vectors
Used in the Host-Cell Reactivation Assay
pCMVcat
pCMVluc
BamH I
EcoR I
Pvu I
Xbal I
Ap
Ap
Bgl II
5000 bp
4863 bp
Enh
Enh
cat
P
BPDE
Hind III
EcoR I
Luc
P
BPDE
Bgl II
Nar I
Xbal I
Transfection Efficiency in
the Host-Cell Reactivation Assay
Cheng et al., BioTechniques, 1997
Risk of Lung Cancer Associated with Low DRC
6
5
Cases = 316
Controls = 316
HCR Assay (plasmid DNA)
Trend test : P < 0.001
4
Odds Ratio
3
2
1
0
Wei et al., JNCI, 2000
6
5
Cases = 221
Controls = 229
In Vitro Adduct Assay (genomic DNA)
Trend test : P < 0.001
4
3
2
1
0
Ist
2nd
3rd
High
4th
Low
DRC (%) by Quartile
Li et al, Cancer Res., 2001
Suboptimal DRC and Cancer Risk
Cancer
# Case/Control
Lung
51/56
316/316
764/677
1522/1672
OR (95%CI)
Reference
5.7 (2.1-15.7)
2.1 (1.5-3.0)
1.5 (1.2-1.9)
1.4 (1.2-1.6)
Wei et al, 1996
Wei et al, 2000
Spitz et al, 2004
unpublished data
2.2 (1.0-4.8)
1.9 (1.5-2.4)
Cheng et al, 1998
Wang et al., 2010
Head & Neck
55/61
744/753
* For the low tertile DRC and others for the low-median DRC; **Recalculated based on published data
Correlation between DNA repair
genotype and phenotype
Known SNPs in the 8 NER Core genes available
in the NIEHS resequencing database
NER
core gene
Nucleotides
/ Protein
Location Function
No. of
SNPs
SNP
No of
No of
Density nsSNPs nsSNPs **
(per kb)
with MAF > 0.05
ERCC1
14kb / 297aa
19q13.2
Endonuclease
73
5.2
1
--
XPA
22kb / 273aa
XPB/ERCC3 37kb / 782aa
XPC
33kb / 940aa
-q13.3
9q22.3
2q21
3p25
Damage detection 140
Helicase
136
Damage detection 145
6.4
3.7
4.4
2
2
12
XPD/ERCC2 19kb / 760aa
19q13.3
Helicase
136
7.2
2
11p12-p11 Damaged DNA
binding
XPF/ERCC4 28kb / 916aa
16p13.3 Endonuclease
- p13.11
XPG/ERCC5 30kb / 1186aa 13q22
Endonuclease
77
3.2
2
--rs2228000( A499V)
rs2228001 (K939Q)
rs1799793 (D312N)
rs13181 (K751Q)
--
214
7.6
7
--
177
5.9
12
rs17655 (D1104H)
Total
1,098
40
5
XPE/DDB2
24kb / 427aa
** XPC codon499 Ala/Val; XPC codon939 Lys/Gln;
XPD codon312 Asp/Asn; XPD codon751 Lys/Gln; XPG codon1104 His/Asp
Genotype Prediction of DRC Phenotype
(UV-damage) in Cancer-free Subjects
Genotype
N
% DRC (SD)
P value
XPD A22541C
AA
AC
CC
22
55
25
9.34
8.18
8.60
(4.65)
(3.13)
(2.47)
Reference
0.208
0.492
XPD Codon 751
Lys/Lys
Lys/Gln
Gln/Gln
46
38
11
8.30
9.51
7.06
(2.22)
(4.22)
(2.49)
Reference
0.096
0.063
XPC Intron 9
SS
SL
LL
36
53
13
8.79
8.81
6.73
(2.42)
(3.86)
(2.47)
Reference
0.978
0.020
Qiao et al., Carcinogenesis, 2002
Correlation between DRC Phenotype (UV-damage)
and Genotype in Cancer-free Subjects
20
LUC Activity (%)
N = 102
P = 0.02
15
10
5
n=9
n=48
n=29
n=10
n=6
W
H
1
2
3
0
None XPC
Hetero
Homozygous
and XPD Genotypes
Three common Variants in XPD and XPC Genes
Qiao et al., Mutat Res, 2002
MDACC Lung Cancer GWAS Study
Genome Wide Association Study of Lung Cancer
– 1200 cases / 1200 controls 317K SNPs
Chris Amos et al., MDACC
Plot for 1806 SNPs in 125 DNA repair genes covered
by the Illumina HumamHap300 (v1.1) BeadChip
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,
DNA repair and clinical outcomes
of lung cancer
Lung Cancer Patient Follow-up
Time Line
275 patients
with NSCLC
Adjusted* Relative Risk of Death
Associated with Efficient DNA Repair
Variable
No.
RR
95% CI
P value
All therapies
No therapy
275
104
1.05
1.01
1.00 - 1.11
0.86 - 1.19
0.054
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
patients had curative surgery without chemotherapy and were dropped from analysis of patients
treated with surgery.
Bosken et al., JNCI, 2002
Effect of Chemotherapy on Survival
in Patients with Stage III/IV NSCLC by DRC
1
0.9
No Chemotherapy (4.9 months)
Fraction Surviving
0.8
Chemotherapy - Efficient DNA Repair
(10.1 months)
0.7
0.6
Chemotherapy - Sub-optimal DNA Repair
(15.8 months)
0.5
0.4
0.3
0.2
0.1
0
0
10
20
30
40
50
60
Survival in Months
Bosken et al., JNCI, 2002
Genetic Variaion in DNA repair genes and
Radiation-induced Pneumonitis (RP) in Cancer
Patients Receiving Radiotherapy
•
Radiation-induced inflammation of the normal lung
tissues
•
RP is the most common dose-limiting complication of
thoracic radiation
•
About 10-20% of the patients experiencing moderate
or severe RP, when the total median radiation dose
was reaching 60-70 Gy
•
Some genetic variants in TGF-β1 predict RP
N=184
158 - 60 to 70 Gy
30 to 58 fractions
147 - chemotherapy
74 - grade 2
36 - grade 3
.
J Clin Oncol. 2009 Jul 10;27(20):3370-8
Some Selected Polymorphisms
of DNA Repair Genes
Genes
# Variants (comm) # AA Changes
Ch
BER
ADPRT / PARP1
APEX1 / APE1
XRCC1
442 (9)
58 (2)
403 (18)
3
1
4
1
14
19
NER
ERCC1
ERCC4 / XPF
149 (6)
330 (2)
0
1
19
19
HR
Rad51
XRCC3
342 ( 0)
198 ( 6)
0
1
1
14
NHEJ
XRCC2
XRCC7 / PRKDC
334 (7)
859 (12)
1
3
7
8
Selected Functional SNPs in DNA
Strand Break Repair Genes
Pathway
Gene
SNPs
Location
BER
XRCC1
APEX1
ADPRT
Q399R
D148E
Val762Ala
Exon
Exon
Exon
NER
ERCC1
ERCC4
8092C>A
R415Q
3’UTR
Exon
DSBR
RAD51
RAD51
XRCC2
XRCC3
XRCC7
-135G>C
-172G>T
Arg188His
T241M
6721G>T
Promoter
Promoter
Exon
Exon
Exon
Association between Selected DNA Repair Gene
SNPs and Risk of Grade ≥ 2 RP in NSCLC
Genes (rs#)
No. (%)
Total #
214 (100)
Hazards Ratio*
P
APEX1 (rs1130409) (BER)
TT
58 (27)
GT
111 (52)
GG
43 (21)
1.00
1.63 (0.89-2.96)
2.27 (1.13-4.59)
0.113
0.022
Rad51 (rs1801320 ) (HR)
GG
173 (81)
AG
35 (17)
AA
5 (2)
1.00
0.48 (0.24-0.96)
1.46 (0.35-6.16)
0.038
0.609
XRCC3 (rs861539 ) (HR)
CC
CT
TT
1.00
0.70 (0.43-1.16)
0.47 (1.24-0.90)
1.169
0.024
63 (29)
92 (17)
59 (2)
*Adjusted for age, sex, race, Karnofsky performance score,
smoking status, tumor histology, and disease stage
Cumulative risk for RP
Cumulative Grade ≥3 RP (hazard)
BER
XRCC3 P = 0.011
CC
DSBR
CT+TT
Time (months)
Unpublished data
CG+CC
Cumulative Survival
Cum survival
RAD51 rs1801320
vs. GG P = 0.009
DSBR
Months
Cum survival
XRCC2 rs3218536 GA+AA
vs. GG P = 0.043
Months
Unpublished data
Conclusions

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

Larger studies are needed to evaluate gene-gene
and gene-environment interactions

High-throughput genotyping/DNA repair phenotyping
should be integrated into risk assessment model
Acknowledgments
 Recruitment of subjects
 Administering questionnaires
 Processing of blood samples
 Extraction of DNA
 Cell culture
 Phenotyping
 Genotyping
 Data entry
 Data analysis
 Manuscripts
• Finding from: NIH-NCI and NIH-NIEHS