the application of single nucleotide polymorphisms (snps) and lung cancer risk zuo-feng zhang, md,...

The Application of Single Nucleotide The Application of Single Nucleotide Polymorphisms (SNPs) Polymorphisms (SNPs) and Lung Cancer Riskand Lung Cancer Risk

Zuo-Feng Zhang, MD, PhD

Epidemiology of Lung CancerEpidemiology of Lung Cancer

Worldwide statistics (2002)– Incidence: ranked #1 (1.35 million cases)– Mortality: ranked #1 (1.18 million deaths)– 5-year survival rate: around 10% in Europe; 8.9% in de

veloping countries

U.S. statistics (2006)– Incidence: ranked #3 (174,470 cases)– Mortality: ranked #1 (162, 460 deaths)– 5-year survival rate: 15%

Risk FactorsRisk Factors for Lung Cancer for Lung Cancer

– Tobacco smoking– ETS– Asbestos– Radon exposure– Occupational exposures– Air pollution– Other radiation– Recurring inflammation– Family history of cancer– Insufficient diet and poor nutritional factors

If DNA damage not repaired

DNA damage repaired

If loose cell cycle control

Defected DNA repair gene

G1

S

G2

M

P53

Cyclin D1

P16

Environmental Carcinogens / Procarcinogens Exposures

Nitrosemins, Xenobiotics, Diet/nutrient

Active carcinogens Detoxified carcinogens

DNA Damage Normal cell

Carcinogenesis Programmed cell death

Tobacco/alcohol infection

Diet and nutritional factors

CYP2E1

GSTP1

mEH mEHNQO1

XRCC3

GSTM1

Theoretical model of gene-environmental interaction pathway for lung cancer Theoretical model of gene-environmental interaction pathway for lung cancer susceptibilitysusceptibility

Ile105Val Ala114Val

Tyr113HisHis139Arg

Tyr113HisHis139Arg

Pro187Ser

Null

Ala146ThrArg72Pro

G870A

G0

Diet/nutrient

Free radicalsOxidative Stress

DNA Repair PathwaysDNA Repair PathwaysDirect Reversal (DR)

Mismatch Repair (MMR)

Base Excision Repair (BER)

Nucleotide Excision Repair (NER)

Double Strand Break Repair (DSBR)

BER PathwayBER Pathway

BER PathwayBER Pathway

Polymorphisms in the BER pathway may alter the repairing capacity of this pathway and modify the risk of lung and head-and-neck cancers.

To examine the individual associations of the – hOGG1 Ser326Cys, – APEX1 Asp148Glu, – PCNA C T (intron 2), – XRCC1 Arg194His and Arg280His, – LIG1 T C (exon 25), A C (exon 6), C T (exon 2), and A G (intr

on 1), – and LIG3 C T (exon 21) polymorphisms – with the risk of lung and head-and-neck cancer, and to investigate whet

her these polymorphisms alter the effects of environmental risk factors, including active and passive cigarette smoking and alcohol drinking, on the development of lung and head-and-neck cancer in the population of Los Angeles County.

Study DesignStudy Design Study design: A population-based case-control study in

Los Angeles County

Subjects Selection Criteria– Patients must be newly diagnosed – Both genders– Ages 18-65 years– Residents of Los Angeles County during the observation period

(1999-2004)– In stable medical condition as determined by their physician

Epidemiology data collection– Interviewed by trained interviewers; 30 ml of buccal cell samples

were collected during the time of interviews

Data sourcesData sources

Data from cancer surveillance program

Questionnaire data Buccal cells were collected

– About 90% of subjects donated buccal cells.

Dietsys USDA food composition data Continuing survey of food intake

for individuals (CSFII)

sociodemographic characteristics

history of tobacco smoking

environmental tobacco smoking

drug use

alcohol use

occupational exposures

environmental exposures

selected clinical factors

food frequency questionnaire

RESPONSE RATES OF THE PARTICIPANTS

Eligible Interviewed (%) Buccal Biospecimen (%)

Control 1540 1040 (67.5) 928 (89.2)

Lung 1577 611 (38.7) 544 (89.0)

Oral 584 303 (51.9) 195 (64.4)

Esophageal 316 108 (34.2) 97 (89.8)

Pharynx 238 100 (42.0) 77 (77.0)

Larynx 226 90 (39.8) 79 (87.8)

Laboratory AssaysLaboratory Assays DNA Extraction and Genotyping

– Genomic DNA was isolated using a modified phenol-chloroform protocol.

– Genotyping of the SNPs was assayed by SNPlexTM techniques.

– PCR-RFLP was used for validation

Confounding VariablesConfounding Variables

Age – <35, 35-36, 37-38, 39-40, 41-42, 43-44, 45-46, 47-48,

49-50, 51-52, 53-54, 55-56, 57-58, and 59-62 Gender Race/ethnicity

– white, Mexican American, African American, Asian American, and other

Education level – years of schooling

Pack-years of smoking Alcohol consumption (for head-and-neck cancer

only) – alcohol drink-years

Logistic Regression Model for Logistic Regression Model for Individual AssociationsIndividual Associations

Logit (R)= α + β1(PCNA) + β2(pack-years) + β3(age) + β4 (gender) + β5(education level) + β6(ethnicity)

Interaction AssessmentInteraction Assessment

To assess a potential gene-environment interaction, a product term is added that allows the effect estimate for one variable to vary within levels of another, and vice versa.

Logit (R)= α + β1(PCNA) + β2(smokingY/N) + β3(age) + β4 (gender) + β5(education level) + β6(ethnicity) + 12 (PCNAsmokingY/N)

False Positive AssessmentFalse Positive Assessment

FPRP = (1-)/[(1-)+(1-)] = 1/{1+[/(1- )][(1- )/]}

: prior probability

: statistical size 1- : probability of rejecting when alternative hypothesis

is true

Wacholder et al., 2004

Haplotype MethodHaplotype Method

SNPs in one gene may have linkage disequilibrium and may be associated with cancer risk as haplotype or linked with unmeasured candidate genes.

Haplotypes were reconstructed using the PHASE version 2.

Statistical AnalysisStatistical Analysis We used SAS 9.1 software to perform data

analyses.

Primarily, unconditional logistic regression was employed to obtain odds ratios and 95% confidence limits.

Stratified analysis was used to assess effect modification.

Demographic characteristics for lung cancer cases and population controlsDemographic characteristics for lung cancer cases and population controlsVariables Lung Cancer Cases (n = 611)

N (%)Controls (n = 1029)

N (%)

With DNA(551)

Without DNA(60)

With DNA(939)

Without DNA(90)

Mean Age (years)(Age range)

52.2 (32 – 59)

52.3(41 – 59)

50.2 (29 – 62)

49.5(30 – 60)

P-value = 0.90 P-value = 0.37

Age (years) < 35 35-44 45-54 55

4 (0.73) 51 (9.3)273 (50)223 (40)

0 (0.0)6 (10)

28 (47)26 (43)

38 (4.1)155 (17)452 (48)294 (31)

3 (3.3)16 (18)47 (52)24 (27)


Race/Ethnicity Caucasian Mexican American African American Asian American Other Missing

320 (58)50 (9.1)90 (16)59 (11)31 (5.6)

1

39 (65)3 (5.0)6 (10)

11 (18)1 (1.7)

0

583 (62)140 (15)79 (8.4)53 (5.7)83 (8.9)

1

45 (50)8 (8.9)23 (25)7 (7.8)7 (7.8)

0

P-value = 0.13 P-value < 0.0001

Gender Male Female

278 (50)273 (50)

25 (42)35 (58)

575 (61)364 (39)

41 (46)49 (54)


Education Level 12 years >12 - 16 years > 16 years Missing

248 (45)248 (45)55 (10)

0

17 (28)27 (45)16 (27)

0

276 (29)422 (45)240 (26)

1

23 (26)49 (54)18 (20)

0

P-value = 0.0003 P-value = 0.22

Association between tobacco smoking and alcohol consumption and lung cancer riskAssociation between tobacco smoking and alcohol consumption and lung cancer risk

Tobacco Smoking

Cases Controls Adjusted OR (95% CL)

Pack-Years*

0 110 484 1.0

>0-20 102 350 1.4 (0.99, 1.9)

>20-40 202 136 8.3 (6.0, 12)

>40 197 58 22 (15, 33)

Trends Ptrend < .0001

Alcohol(drinks/day)**

0 - <2 446 870 1.0

2 164 156 1.1 (0.80, 1.5)

*Adjusted for age, gender, ethnicity, and education.**Adjusted for age, gender, ethnicity, education and pack-years of smoking.

THE ASSOCIATION BETWEEN THE ASSOCIATION BETWEEN PCNAPCNA (C (C T) (rs25406) IN INTRON 2 AND LUNG CANCER RISK T) (rs25406) IN INTRON 2 AND LUNG CANCER RISK

Stratified by Cases N (%)

Controls N (%)

Crude OR(95% CL)

Adjusted OR(95% CL)

PCNA genotypesa

CC 128 (33.3) 259 (36.3) 1.0 1.0

CT 177 (46.1) 357 (50.1) 1.0 (0.76, 1.3) 0.90 (0.65, 1.2)

TT 79 (20.6) 97 (13.6) 1.6 (1.1, 2.4) 1.4 (0.89, 2.1)

Ptrend = 0.0228 Ptrend = 0.3060

CC + CT 305 (79.4) 616 (86.4) 1.0 1.0

TT 79 (20.6) 97 (13.6) 1.6 (1.2, 2.3) 1.5 (0.99, 2.1)

Pack-years of Smokingb

0

CC + CT 55 (85.9) 289 (86.8) 1.0 1.0

TT 9 (14.1) 44 (13.2) 1.1 (0.50, 2.3) 0.89 (0.38, 2.0)

>0 – 20

CC + CT 49 (84.5) 211 (85.4) 1.0 1.0

TT 9 (15.5) 36 (14.6) 1.1 (0.49, 2.4) 0.97 (0.43, 2.2)

>20

CC + CT 201 (76.7) 116 (87.2) 1.0 1.0

TT 61 (23.3) 17 (12.8) 2.1 (1.2, 3.7) 2.4 (1.3, 4.5)a Adjusted for gender, age, education, ethnicity, and tobacco smoking (pack-years).b Adjusted for gender, age, education, and ethnicity.

THE ASSOCIATION BETWEEN THE ASSOCIATION BETWEEN PCNAPCNA (C (C T) (rs25406) IN INTRON 2 T) (rs25406) IN INTRON 2 AND LUNG CANCER RISKAND LUNG CANCER RISK


Controls N (%)

Crude OR(95% CL)

Adjusted OR(95% CL)

Alcohol Drinking (Drinks/Day)a

0 - <2

CC + CT 227 (82.2) 517 (86.6) 1.0 1.0

TT 49 (17.8) 80 (13.4) 1.4 (0.95, 2.1) 1.3 (0.80, 2.0)

2

CC + CT 77 (72.0) 98 (85.2) 1.0 1.0

TT 30 (28.0) 17 (14.8) 2.2 (1.2, 4.4) 2.2 (0.99, 4.9)

a Adjusted for gender, age, education, ethnicity, and tobacco smoking (pack-years).

THE ASSOCIATION BETWEEN THE ASSOCIATION BETWEEN PCNAPCNA (C (C T) (rs25406) IN INTRON 2 T) (rs25406) IN INTRON 2 AND LUNG CANCER RISKAND LUNG CANCER RISK


Controls N (%)

Crude OR(95% CL)

Adjusted OR(95% CL)

Histologya

Large cell

CC + CT 50 (73.5) 616 (86.4) 1.0 1.0

TT 18 (26.5) 97 (13.6) 2.3 (1.3, 4.1) 2.1 (1.1, 4.1)

Small cell

CC + CT 38 (80.8) 616 (86.4) 1.0 1.0

TT 9 (19.2) 97 (13.6) 1.5 (0.71, 3.2) 1.6 (0.67, 3.9)

Squamous cell

CC + CT 45 (83.3) 616 (86.4) 1.0 1.0

TT 9 (16.7) 97 (13.6) 1.3 (0.60, 2.7) 1.2 (0.51, 2.9)

Adenocarcinoma

CC + CT 156 (78.8) 616 (86.4) 1.0 1.0

TT 42 (21.2) 97 (13.6) 1.7 (1.1, 2.6) 1.7 (1.1, 2.6)a Adjusted for gender, age, education, ethnicity, and tobacco smoking (pack-years).

THE ASSOCIATION BETWEEN THE ASSOCIATION BETWEEN PCNAPCNA (C (C T) (rs25406) IN INTRON 2 AND T) (rs25406) IN INTRON 2 AND LUNG CANCER RISKLUNG CANCER RISK


Controls N (%)

Crude OR(95% CL)

Adjusted OR(95% CL)

Ethnicityc

Caucasian

CC + CT 174 (76.0) 386 (86.3) 1.0 1.0

TT 55 (24.0) 61 (13.7) 2.0 (1.3, 3.0) 1.9 (1.1, 3.2)

Mexican/Hispanic

CC + CT 27 (93.1) 101 (91.8) 1.0 1.0

TT 2 (6.9) 9 (8.2) 0.83 (0.17, 4.1) 0.64 (0.11, 3.9)

African American

CC + CT 50 (84.7) 48 (81.4) 1.0 1.0

TT 9 (15.3) 11 (18.6) 0.79 (0.30, 2.1) 0.66 (0.20, 2.2)

Asian

CC + CT 38 (86.4) 28 (80.0) 1.0 1.0

TT 6 (13.6) 7 (20.0) 0.63 (0.19, 2.1) 0.48 (0.12, 1.8)

Other**

CC + CT 16 (72.7) 53 (85.5) 1.0 1.0

TT 6 (27.3) 9 (14.5) 2.2 (0.68, 7.1) 2.7 (0.72, 10)c Adjusted for age, gender, education, and tobacco smoking (pack-years).

Gene SNP

Stratum OR(95%CL)

Power under recessive modela

Reported P value

Prior Probabilityb

.5 .25 .1 .01 .001

PCNA All (TT vs. CC+CT)

1.5 (0.99, 2.1) 0.79 0.018 .022 .065 .172 .695 .958

Heavy smokers 2.4 (1.3, 4.5) 0.25 0.006 .025 .071 .186 .715 .962

Heavy drinkers 2.2 (0.99, 4.9) 0.21 0.054 .203 .434 .697 .962 .996

Large cell 2.1 (1.1, 4.1) 0.26 0.030 .103 .255 .507 .919 .991

Adenocarcinoma 1.7 (1.1, 2.6) 0.49 0.014 .028 .081 .209 .744 .967

Caucasian 1.9 (1.1, 3.2) 0.46 0.016 .033 .093 .236 .773 .972

FALSE POSITIVE REPORT PROBABILITY FOR MAJOR LUNG CANCER FALSE POSITIVE REPORT PROBABILITY FOR MAJOR LUNG CANCER RESULTS RESULTS

a Estimation of the statistical power to detect an OR of 1.5 with level of 0.05.b False-positive report probabilities for the observed odds ratios.

ASSOCIATIONS OF THE ASSOCIATIONS OF THE LIG1 LIG1 HAPLOTYPES OF THE FOUR SNPS HAPLOTYPES OF THE FOUR SNPS AND LUNG CANCERAND LUNG CANCER

TCCA CACA CATA CCCG CCCA CCTA TACA All Others

Overall, n (case/control)

346/695 325/582 112/169 51/65 11/25 18/28 3/8 10/6

Crude OR1.0 1.1

(0.93, 1.4)1.3

(1.0, 1.4)1.6

(1.1, 2.3)0.88

(0.43, 1.8)1.3

(0.70, 2.4)0.75

(0.20, 2.9)3.3

(1.2, 9.3)

Adj ORa

1.0 1.2 (0.95, 1.5)

1.4 (1.0, 1.9)

1.8 (1.1, 2.8)

0.81 (0.35, 1.9)

1.6 (0.78, 3.1)

1.3 (0.31, 5.9)

3.2 (0.99, 11)

* Adjusted for gender, age, education, ethnicity, and tobacco smoking (pack-years).

ASSOCIATIONS OF THE ASSOCIATIONS OF THE LIG1 LIG1 HAPLOTYPES AND LUNG CANCERHAPLOTYPES AND LUNG CANCER

TCCA CACA CATA CCCG CCCA CCTA TACA All Others

Stratified by Smoking status

Never 55/322 68/272 15/71 13/30 1/15 1/15 0/5 1/2

Crude OR 1.01.5

(1.0, 2.2)1.2

(0.66, 2.3)2.5

(1.2, 5.2)0.39

(0.051, 3.0) 0.39

(0.051, 3.0)- 2.9

(0.26, 33)

Adj Orb 1.01.4

(0.94, 2.1)1.2

(0.62, 2.2)2.3

(1.1, 4.8)0.34

(0.043, 2.6)0.40

(0.051, 3.1)- 2.6

(0.22, 30)

Former 209/252 180/208 63/63 27/18 7/9 12/8 2/2 8/4

Crude OR 1.01.0

(0.80, 1.4)1.2

(0.81, 1.8) 1.8

(0.97, 3.4)0.94

(0.34, 2.6)1.8

(0.73, 4.5)1.2

(0.17, 8.6)2.4

(0.72, 8.1)

Adj ORb 1.01.0

(0.79, 1.4)1.2

(0.78, 1.8)1.5

(0.78, 2.8)0.84

(0.30, 2.3)2.1

(0.84, 5.3)1.3

(0.18, 9.1)2.9

(0.84, 9.8)

Current 82/121 77/102 34/35 11/17 3/1 5/5 1/1 1/0

Crude OR 1.01.1

(0.74, 1.7)1.4

(0.83, 2.5)0.96

(0.43, 2.1)4.4

(0.45, 43)1.5

(0.41, 5.3)1.5

(0.091, 24)-

Adj ORb 1.01.1

(0.74, 1.7)1.5

(0.85, 2.6)0.94

(0.41, 2.2)4.4

(0.44, 44)1.3

(0.35, 4.6)1.5

(0.088, 26)-

b Adjusted for gender age, education, and ethnicity.

MULTIGENETIC ASSOCIATION WITH LUNG CANCER MULTIGENETIC ASSOCIATION WITH LUNG CANCER

# of Potential Risk Genotypes

Cases Control Crude OR Adjusted OR*

0 50 (24.5) 157 (34.3) 1.0 1.0

1-2 122 (59.8) 237 (51.7) 1.6 (1.1, 2.4) 1.7 (1.1, 2.7)

3-5 32 (15.7) 64 (14.0) 1.6 (0.92, 2.7) 2.2 (1.1, 4.1)

Ptrend = 0.038 Ptrend = 0.0104

Continuous 1.1 (0.98, 1.3) 1.2 (1.0, 1.5)

* Adjusted for age, gender, education, race/ethnicity and pack-years of smoking.

Discussion on BER ResultsDiscussion on BER Results We observed an overall association of PCNA (C T) SNP with lun

g cancer risk, and the smoking status in pack-years and alcohol drinking status seemed to modify the association of PCNA SNP with lung cancer.

We did not detect any consistent association between individual LIG1 polymorphisms and lung cancer.

When we considered all the SNPs together as haplotypes, we observed the associations between the LIG1 haplotypes and the cancer risks.

The dose-response association of these SNPs in the BER pathway suggested the possible interaction among these SNPs since they are the components involved in this pathway. This also explained why we could not detect the individual SNP association. The SNPs in the same pathway work together to maintain the genome stability.

NER Pathway

Costa RM, 2003

OR (95% CL) XPG Cases N (%)

Controls N (%) Crude Adjusted1

All lung cancer cases

His/His 244 (49.1) 468 (51.9) 1.0 1.0 His/Asp 212 (42.7) 356 (39.5) 1.1 (0.91, 1.4) 1.1 (0.80, 1.4) Asp/Asp 41 (8.2) 78 (8.6) 1.0 (0.67, 1.5) 0.65 (0.39, 1.1) Ptrend=0.51 Ptrend=0.36 His/His+His/Asp 456 (91.8) 824 (91.4) 1.0 1.0 Asp/Asp 41 (8.2) 78 (8.6) 0.95 (0.64, 1.4) 0.62 (0.38, 1.0)

Histology-specific

Adenocarcinoma His/His+His/Asp 221 (91.3) 824 (91.4) 1.0 1.0 Asp/Asp 21 (8.7) 78 (8.6) 1.0 (0.61, 1.7) 0.69 (0.39, 1.2) Squamous cell carcinoma His/His+His/Asp 68 (86.1) 824 (91.4) 1.0 1.0 Asp/Asp 11 (13.9) 78 (8.6) 1.7 (0.87, 3.4) 1.0 (0.45, 2.3) Small-cell lung cancer His/His+His/Asp 60 (93.7) 824 (91.4) 1.0 1.0 Asp/Asp 4 (6.3) 78 (8.6) 0.70 (0.25, 2.0) 0.55 (0.18, 1.7) Large-cell lung cancer His/His+His/Asp 85 (95.5) 824 (91.4) 1.0 1.0 Asp/Asp 4 (4.5) 78 (8.6) 0.50 (0.18, 1.4) 0.31 (0.10, 0.92)

XPG polymorphism and the risk of lung cancer

1Adjusted for age, sex, race-ethnicity, educational level, and tobacco smoking.

Joint effect of tobacco smoking and XPG polymorphism on lung cancer risk

0

5

10

15

20

25

Pack-years: Never 1-20 >20 Never 1-20 >20

XPG: Asp/Asp Asp/Asp Asp/Asp His/His +His/Asp His/His +His/Asp His/His +His/Asp

Departure from additivity:

23-13-1.9+1=9.1, 95% CL=-2.9, 21.7

Joint effect of tobacco, alcohol, and XPG polymorphism on SCCUAT

0

2

4

6

8

10

12

14

16

18

Drinks per day: 1-2 1-2 >=3 >=3 1-2 1-2 >=3 >=3

Packyears: <=20 <=20 <=20 <=20 >20 >20 >20 >20

XPG: Asp/Asp His/His +His/Asp Asp/Asp His/His +His/Asp Asp/Asp His/His +His/Asp Asp/Asp His/His +His/Asp

NER Cases (%) Controls (%) Crude OR Adjusted OR*

All Lung Cancer Cases

ERCC6 Q1413R

Q/Q 251 (62.0%) 451 (61.4) 1.0 1.0

Q/R 125 (30.9%) 254 (34.6%) 0.88 (0.68, 1.15) 1.23 (0.89, 1.70)

R/R 29 (7.2%) 29 (4.0%) 1.80 (1.05, 3.08) 2.10 (1.09, 4.03)

Ptrend=0.02

Q/Q + Q/R 376 (92.8%) 705 (96.1%) 1.0 1.0

R/R 29 (7.2%) 29 (4.0%) 1.88 (1.10, 3.19) 1.94 (1.02, 3.68)

Total 405 (100%) 734 (100%)

ERCC6 R1230P

R/R 420 (85.4%) 756 (86.7%) 1.0 1.0

P/R 67 (13.6%) 107 (12.3%) 1.13 (0.81, 1.56) 1.13 (0.76, 1.69)

P/P 5 (1.0%) 9 (1.0%) 1.00 (0.33, 3.00) 1.37 (0.36, 5.26)

Ptrend=0.46

P/R + P/P 72 (14.6%) 116 (13.3%) 1.0 1.0

R/R 420 (85.4%) 756 (86.7%) 0.90 (0.65, 1.23) 0.87 (0.59, 1.29)

Total 492 (100%) 872 (100%)

TABLE III – ASSOCIATION BETWEEN THE NER POLYMORPHISM AND THE RISK OF LUNG CANCER STRATIFIED BY PATHOLOGICAL DIAGNOSIS

Gene / SNP Cases (%) Controls (%) Crude OR Adjusted OR*

RCC6 Q1413R

Adenocarcinoma

Q/Q + Q/R 184 (92.5%) 705 (96.1%) 1.0 1.0

R/R 15 (7.5%) 29 (4.0%) 1.98 (1.04, 3.77) 2.19 (1.05, 4.59)

Total 199 (100%) 734 (100%)

Squamous

Q/Q + Q/R 59 (95.2%) 705 (96.1%) 1.0 1.0

R/R 3 (4.8%) 29 (4.0%) 1.24 (0.37, 4.18) 1.66 (0.38, 7.19)

Total 62 (100%) 734 (100%)

Small Cell

Q/Q + Q/R 46 (90.2) 705 (96.1%) 1.0 1.0

R/R 5 (9.8%) 29 (4.0%) 2.64 (0.98, 7.15) 2.18 (0.63, 7.47)

Total 51 (100%) 734 (100%)

Large Cell

Q/Q + Q/R 72 (93.5%) 705 (96.1%) 1.0 1.0

R/R 5 (6.5%) 29 (4.0%) 1.69 (0.63, 4.50) 2.19 (0.71, 6.78)

Total 77 (100%) 734 (100%)

SmokerERCC6 Q1413R Cases Controls Adjusted OR*

No Q/Q + Q/R 64 324 1.0

No R/R 2 18 0.59 (0.12, 2.82)

Yes Q/Q + Q/R 312 381 4.41 (3.18, 6.14)

Yes R/R 27 11 19.44 (8.33, 45.35)

OR interaction = 5.82 (1.08, 31.38)

Smoker ERCC6 R1230P Cases Controls Adjusted OR*

No R/R 41 202 1.0

No P/R + P/P 25 140 2.87 (1.28, 6.40)

Yes R/R 210 249 4.29 (3.12, 5.92)

Yes P/R + P/P 129 143 5.63 (3.43, 9.24)

OR interaction = 0.56 (0.25, 1.27)

TABLE III – ASSOCIATION BETWEEN THE NER POLYMORPHISM AND THE RISK OF LUNG CANCER STRATIFIED BY PATHOLOGICAL DIAGNOSIS

Gene / SNP Cases (%) Controls (%) Crude OR Adjusted OR*

RCC6 Q1413R

Adenocarcinoma

Q/Q + Q/R 184 (92.5%) 705 (96.1%) 1.0 1.0

R/R 15 (7.5%) 29 (4.0%) 1.98 (1.04, 3.77) 2.19 (1.05, 4.59)

Total 199 (100%) 734 (100%)

Squamous

Q/Q + Q/R 59 (95.2%) 705 (96.1%) 1.0 1.0

R/R 3 (4.8%) 29 (4.0%) 1.24 (0.37, 4.18) 1.66 (0.38, 7.19)

Total 62 (100%) 734 (100%)

Small Cell

Q/Q + Q/R 46 (90.2) 705 (96.1%) 1.0 1.0

R/R 5 (9.8%) 29 (4.0%) 2.64 (0.98, 7.15) 2.18 (0.63, 7.47)

Total 51 (100%) 734 (100%)

Large Cell

Q/Q + Q/R 72 (93.5%) 705 (96.1%) 1.0 1.0

R/R 5 (6.5%) 29 (4.0%) 1.69 (0.63, 4.50) 2.19 (0.71, 6.78)

Total 77 (100%) 734 (100%)

Double Strand Break RepairDouble Strand Break Repair

NBS1 protein (Nibrin or p95)NBS1 protein (Nibrin or p95)Has 3 known functional regions:N-terminus (1-196 a.a.)Central region (278-343 a.a.)C-terminus (665-693 a.a.)

– Believed to bind site to the MRN complex

Variable Controls (%)HWE p-value

Cases (%) HWE p-value

Crude OR (95% CI)

ORadj (95% CI)*

Ex16+1785 T>C

T/T 369 (45.95) 211 (46.68) 1.0 1.0

C/T 363 (45.21) 0.19 188 (41.59) 0.28 0.91 (0.71, 1.16) 0.85 (0.63, 1.13)

C/C 71 (8.84) 53 (11.73) 1.31 (0.88, 1.94) 1.12 (0.69, 1.80)

Missing 226 159 p-trend= 0.5974

Ex16+572 C>G

C/C 397 (45.74) 223 (45.70) 1.0 1.0

C/G 397 (45.74) 0.83 208 (42.62) 0.41 0.93 (0.74, 1.18) 0.88 (0.67, 1.17)

G/G 74 (8.53) 57 (11.68) 1.37 (0.94, 2.01) 1.35 (0.85, 2.14)

Missing 161 123 p-trend=0.628

Pro672Pro

A/A 356 (44.33) 233 (50.76) 1.0 1.0

A/G 366 (45.58) 0.40 169 (36.82) 0.0043a 0.71 (0.55, 0.90) 0.71 (0.53, 0.95)

G/G 81 (10.09) 57 (12.42) 1.08 (0.74, 1.57) 1.25 (0.80, 1.96)

Missing 226 152 p-trend=0.7126

Table III. Association of Pro672Pro and lung cancer (smoking and Table III. Association of Pro672Pro and lung cancer (smoking and passive smoking exposure)passive smoking exposure)

Pro672Pro Controls (%) Cases (%) Crude OR (95% CI) ORadj (95% CI)*

Never-smokers

wt/wt 170 (45.3) 32 (39.5) 1.0 1.0

wt/var 165 (44) 34 (42.0) 1.10 (0.65, 1.86) 1.27 (0.71, 2.26)

var/var 40 (10.7) 15 (18.5) 1.99 (0.99, 4.03) 1.92 (0.85, 4.36)

Ever-smokers

wt/wt 186 (43.5) 201 (53.2) 1.0 1.0

wt/var 201 (47.0) 135 (35.7) 0.62 (0.46, 0.84) 0.66 (0.48, 0.90)

var/var 41 (9.6) 42 (11.1) 0.95 (0.59, 1.52) 1.22 (0.73, 2.05)

Passive no exposure

wt/wt 40 (46.5) 10 (40.0) 1.0 1.0

wt/var 39 (45.4) 10 (40.0) 1.03 (0.38, 2.74) 1.57 (0.47, 5.23)

var/var 7 (8.1) 5 (20.0) 2.86 (0.75, 10.92) 5.42 (0.88, 33.54)

Yes exposure

wt/wt 130 (45.1) 22 (39.3) 1.0 1.0

wt/var 126 (43.8) 24 (42.9) 1.13 (0.60, 2.11) 1.29 (0.65, 2.55)

var/var 32 (11.1) 10 (17.9) 1.85 (0.80, 4.28) 1.59 (0.61, 4.13)

Table IV. Associations of NBS1 polymorphisms and lung cancer, Table IV. Associations of NBS1 polymorphisms and lung cancer, stratified by smoking and passive smokingstratified by smoking and passive smoking

Variables Ex16+1785 T>C Ex16+572 C>G

Controls Cases ORadj (95% CI)* controls Cases ORadj (95% CI)*

Stratify by smoking

No

wt/wt + wt/var 345 68 1.0 376 75 1.0

var/var 28 10 2.05 (0.86, 4.87) 34 13 1.82 (0.83, 3.99)

Yes

wt/wt + wt/var 387 331 1.0 418 356 1.0

var/var 43 43 1.19 (0.74, 1.92) 40 44 1.35 (0.84, 2.19)

Stratified by passive smoking

No

wt/wt + wt/var 80 24 1.0 90 28 1.0

var/var 6 1 0.61 (0.04, 9.31) 7 1 0.40 (0.03, 5.38)

Yes

wt/wt + wt/var 265 44 1.0 286 47 1.0

var/var 21 9 2.84 (1.11, 7.28) 26 12 2.74 (1.16, 6.47)

DNA Methylation and Risk of DNA Methylation and Risk of Lung CancerLung Cancer

Source: Sharp 2004.

THF cycle

Methionine cycle

DNA Methylation and Lung Cancer DNA Methylation and Lung Cancer RiskRisk

Controls Small Cell Lung Cancer Non-Small Cell Lung Cancer Methylation N (%) N (%) Crude OR

(95% CI) Adjusted OR1

(95% CI) N (%) Crude OR

(95% CI) Adjusted OR1

(95% CI) P16

No 769 (84) 54 (79) 1.00 1.00 379 (82) 1.00 1.00 Yes 146 (16) 14 (21) 1.37 (0.74-2.52) 1.43 (0.69-3.00) 86 (18) 1.20 (0.89-1.60) 1.27 (0.90-1.80)

MGMT

No 721 (82) 50 (81) 1.00 1.00 330 (77) 1.00 1.00 Yes 157 (18) 12 (19) 1.10 (0.57-2.12) 0.99 (0.47-2.10) 100 (23) 1.39 (1.05-1.85) 1.22 (0.87-1.70)

GSTP1

No 675 (88) 44 (85) 1.00 1.00 310 (85) 1.00 1.00 Yes 96 (12) 8 (15) 1.18 (0.52-2.71) 1.35 (0.55-3.31) 53 (15) 1.20 (0.84, 1.73) 1.12 (0.73-1.70)

Number of

0 590 (64) 41 (60) 1.00 1.00 272 (58) 1.00 1.00 1 262 (29) 21 (31) 1.08 (0.60-1.93) 0.94 (0.49-1.78) 150 (32) 1.24 (0.97-1.59) 1.18 (0.88-1.58)

2-3 64 (7) 6 (9) 1.44 (0.59-3.55) 1.81 (0.67-4.91) 43 (9) 1.46 (0.97-2.20) 1.38 (0.85-2.24)

continuous 1.15 (0.81-1.65) 1.16 (0.76-1.76) 1.19 (1.01-1.41) 1.15 (0.95-1.39) Adjusted for age (continuous), sex, race, and pack-year of smoking

P16 hypermethylation and Lung Cancer P16 hypermethylation and Lung Cancer Risk by smoking, drinking, and folate Risk by smoking, drinking, and folate

intakeintakeControls Lung P16

methylation N (%) N (%) Crude OR (95% CI)

Adjusted OR (95% CI)

Smoking (pack-year)

1

Never No 358 (39) 74 (14) 1.00 1.00 Never Yes 72 (8) 21 (4) 1.41 (0.82-2.44) 1.32 (0.75-2.31) >0-40 No 367 (40) 219 (41) 1.00 1.00 >0-40 Yes 66 (7) 45 (8) 1.14 (0.76-1.73) 1.18 (0.77-1.80) >40 No 43 (5) 140 (26) 1.00 1.00 >40 Yes 8 (1) 34 (6) 1.31 (0.56-3.03) 1.36 (0.58-3.18)

Drinking (drinks/day)

2

0-2 No 648 (71) 311 (58) 1.00 1.00 0-2 Yes 122 (13) 73 (14) 1.25 (0.91-1.72) 1.42 (0.98-2.05) 2 No 118 (13) 121 (23) 1.00 1.00 2 Yes 24 (3) 27 (5) 1.10 (0.60-2.01) 0.92 (0.41-2.06)

Folate (g/day)

2

>200 No 276 (36) 156 (31) 1.00 1.00 >200 Yes 53 (7) 45 (9) 1.50 (0.96-2.34) 1.51 (0.89-2.57) 200 No 357 (47) 246 (50) 1.00 1.00 200 Yes 77 (10) 48 (10) 0.90 (0.61-1.34) 0.96 (0.60-1.54) 1. Adjusted for age (continuous), sex, and race. 2. Adjusted for age (continuous), sex, race, and pack-years of smoking

MGMT hypermethylation and Lung cancer MGMT hypermethylation and Lung cancer Risk by smoking, drinking, and folate Risk by smoking, drinking, and folate

intakeintakeControls Lung MGMT methylation

N (%) N (%) Crude OR (95% CI)


Smoking (pack-year)

1



2

0-2 No 607 (69) 276 (56) 1.00 1.00 0-2 Yes 132 (15) 80 (16) 1.33 (0.98-1.82) 1.19 (0.83-1.72) 2 No 112 (13) 103 (21) 1.00 1.00 2 Yes 25 (3) 32 (7) 1.39 (0.77-2.50) 1.18 (0.57-2.46)

Folate (g/day)

2


GSTP1 hypermethylation and Lung GSTP1 hypermethylation and Lung cancer Risk by smoking, drinking, and cancer Risk by smoking, drinking, and

folate intakefolate intakeControls Lung GSTP1 methylation N (%) N (%) Crude OR

(95% CI) Adjusted OR

(95% CI) Smoking

(pack-year) 1



2

0-2 No 562 (73) 251 (61) 1.00 1.00 0-2 Yes 85 (11) 48 (11) 1.26 (0.86-1.86) 1.17 (0.75-1.82) 2 No 111 (14) 102 (25) 1.00 1.00 2 Yes 11 (1) 13 (3) 1.29 (0.55-3.00) 1.19 (0.44-3.22)

Folate (g/day)

2


Number of methylated genes and lung Number of methylated genes and lung cancer Risk by smoking, drinking, and cancer Risk by smoking, drinking, and

folate intakefolate intakeControls Lung Number of

methylated genes N (%) N (%) Crude OR (95% CI)


Smoking (pack-year)

1

Never 0-1 398 (43) 87 (16) 1.00 1.00 Never 2-3 32 (4) 8 (2) 1.14 (0.51-2.57) 0.97 (0.43-2.23) >0-40 0-1 402 (44) 240 (45) 1.00 1.00 >0-40 2-3 31 (3) 24 (5) 1.30 (0.74-2.26) 1.30 (0.74-2.31) >40 0-1 51 (6) 157 (29) 1.00 1.00 >40 2-3 1 (0.1) 17 (3) 5.52 (0.72-42.46) 5.69 (0.73-44.22)


2

0-2 0-1 715 (78) 346 (65) 1.00 1.00 0-2 2-3 55 (6) 38 (7) 1.43 (0.93-2.20) 1.41 (0.86-2.33) 2 0-1 134 (15) 137 (26) 1.00 1.00 2 2-3 9 (1) 11 (2) 1.20 (0.48-2.98) 1.01 (0.31-3.24)

Folate (g/day)

2

>200 0-1 302 (40) 179 (36) 1.00 1.00 >200 2-3 27 (4) 22 (4) 1.37 (0.76-2.49) 1.17 (0.58-2.39) 200 0-1 402 (53) 271 (55) 1.00 1.00 200 2-3 33 (4) 23 (5) 1.03 (0.59-1.80) 1.05 (0.54-2.05) 1. Adjusted for age (continuous), sex, and race. 2. Adjusted for age (continuous), sex, race, and pack-years of smoking

Folate intake, MTHFR C677T Polymorphism Folate intake, MTHFR C677T Polymorphism on Lung Cancer Riskon Lung Cancer Risk

Control SCLC NSCLC

N (%) N (%) Adjusted OR(95% CI)

N (%) Adjusted OR(95% CI)

Folate intake(μg/day)

1 1

>300 102 (12) 4 (6) 1.00 56 (12) 1.00

200-300 265 (31) 17 (25) 1.59 (0.47-5.44) 136 (29) 1.15 (0.72-1.83)

100-200 412 (48) 34 (51) 1.52 (0.45-5.10) 214 (46) 0.83 (0.52-1.33)

≤100 78 (9) 12 (18) 2.14 (0.52-8.85) 63 (13) 0.80 (0.43-1.50)

P for trend 0.3897 0.1640

MTHFR C677T 2 2

CC 391 (42) 32 (49) 1.00 211 (49) 1.00

CT 411 (45) 26 (40) 0.89 (0.48-1.65) 171 (40) 0.78 (0.59-1.04)

TT 118 (13) 7 (11) 1.11 (0.44-2.82) 49 (11) 0.86 (0.56-1.34)

1. Adjusted for age, sex, race, pack-years of smoking, total energy intake and BMI

2. Adjusted for age, sex, race, and pack-years of smoking

Folate on Lung Cancer Risk by Smoking anFolate on Lung Cancer Risk by Smoking and Drinkingd Drinking

Control SCLC NSCLC

N N Adjusted OR(95% CI)

N Adjusted OR(95% CI)

Smoking Folate intake(μg/day)

1 1

Never >200 183 2 1.00 38 1.00

≤200 219 1 0.67 (0.05-5.81) 51 1.07 (0.66-1.74)

Former >200 120 15 1.00 111 1.00

≤200 185 29 1.29 (0.62-2.65) 165 0.92 (0.64-1.33)

Current >200 64 4 1.00 43 1.00

≤200 86 16 3.05 (0.88-10.60) 61 1.05 (0.61-1.81)

Drinking 2 2

Light >200 303 17 1.00 123 1.00

≤200 428 32 0.82 (0.39-1.72) 216 0.76 (0.55-1.06)

Heavy >200 63 4 1.00 69 1.00

≤200 61 14 4.08 (0.85-19.66) 61 0.70 (0.37-1.31)1. Adjusted for age, sex, race, total energy intake, and BMI

2. Adjusted for age, sex, race, pack-years of smoking, total energy intake, and BMI

MTHFR C677T Polymorphism on Lung MTHFR C677T Polymorphism on Lung Cancer Risk by DrinkingCancer Risk by Drinking

Control SCLC NSCLC

Drinking MTHFR

C677T



Light CC 326 25 1.00 153 1.00

CT/TT 450 26 0.95 (0.50-1.81) 152 0.73 (0.54-0.98)

Heavy CC 65 7 1.00 57 1.00

CT/TT 76 7 0.87 (0.23-3.31) 58 1.20 (0.67-2.16)Adjusted for age, sex, race, and pack-years of smoking

Folate and MTHFR C677T Polymorphism on Folate and MTHFR C677T Polymorphism on Lung and Head and Neck Cancer Risks amoLung and Head and Neck Cancer Risks amo

ng Heavy Drinkersng Heavy Drinkers

Control

SCLC NSCLC SCCHN

Heavy Drinker




>200 CC 27 0 28 1.00 27 1.00

>200 CT/TT 31 2 1.00 33 1.12 (0.46-2.71)

29 0.78 (0.34-1.79)

≤200 CC 28 7 25 0.63 (0.24-1.66)

28 1.31 (0.52-3.28)

≤200 CT/TT 26 4 2.67 (0.20-35.60)

32 1.02 (0.40-2.65)

32 2.17 (0.86-5.45)

Adjusted for age, sex, race, pack-years of smoking, total energy intake, and BMI

IL-10IL-10 and and IFNGR1IFNGR1 Polymorphisms and Polymorphisms and Lung CancerLung Cancer

Modulation of Immune ResponseModulation of Immune Response

Achieved through balance of T-helper (Th) cells via cytokine expression – Type 1: pro-inflammatory, cellular immune response

Secrete IFNG

– Type 2: anti-inflammatory, humoral immune response Secrete IL-10

Activation of one Th-cell pathway inactivates the other

Inflammatory Anti-inflammatory

SNPsSNPs

IL-10– -7334 T→C, AKA IL-10-819– -6653 A→C, AKA IL-10-592– TA haplotype associated with low IL-10 production

IFNGR1– Associated with altered immune cell function (mice)

and respiratory disease (humans)– Ex7 +189 T→G

Table 1

Table 2

Table 3

Table 3, cont

IFNGR1 & SmokingCont Case Crude OR variance lower CL upper CL361 77 1.0

7 1 0.67 1.15861 0.08 2.51405 339 3.92 0.02118 2.95 4.95

8 17 9.96 0.19958 4.15 32.38OR-int 3.79 2.84827 0.14 11.67

Never/wNever/vEver/wEver/v

Table 4

ORint = 23.44 – 12.10 – 3.83 + 1 = 8.51

Additive Interaction

AcknowledgmentsAcknowledgmentsDr. Amy Lee

Dr. Yan Cui

Ms. Shu-Chun Chuang

Ms. Lani Park

Mr. Yiren Wang

Mr. Sam Oh

Dr. Barbara Visscher

Dr. Eric Hurwitz

Dr. Donald P. Tashkin

Dr. Jian Yu Rao

Dr. Jenny Papp

Dr. Hal Morgenstern,

Dr. Sander Greenland

Dr. Wei Cao

Dr. Wendy Cozen

Dr. Thomas M. Mack

the application of single nucleotide polymorphisms (snps) and lung cancer risk zuo-feng zhang, md,...

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