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

1. 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

2. ? Exposure Disease 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 External Exposure Internal Exposure Biological Effects Disease Progression

3. 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 TranscriptomeEpigenetics Proteomics Metabolome/Metabomics

4. Smoking and Cancer Snapshot: No. (in millions) US population: 300.0 Smokers: 46.5 All cancer cases 1.3 Lung cancer 0.5 Head and neck cancer 0.04 Genetic susceptibility plays a role ! ACS, 2009

5. 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% Sedentary lifestyle 5% Harvard School of Public Health, 1998

6. Altered Expression BAX, Fas, Bcl2 PIG3 Increased Expression p21WAF1, MDM2, cyclin G, and GADD45 • Binding to Transcription Replication-Repair Factors • TFIIH (XPB, XPD) and p62 • binds to p53 • PCNA (p21WAF1 and GADD45) DNA Damage-Response Pathway Transcription Dependent Apoptosis DNA DAMAGE Apoptosis Cell Cycle Arrest Cancer p53 Protein Accumulation DNA Repair Transcription Independent Apoptosis Modified from Harris, 1994

7. DNA repair phenotype (DRC) as a risk factor of lung cancer

8. 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

9. Benzo[a]pyrene O MFO BPDE HO OH Bulky DNA Adduct Tobacco Smoke Nucleotide Excision Complex POL/, ligase PCNA, RFC RPA Normal DNA Ligation Nucleotide Excision Repair of Tobacco-Induced DNA Damage 32P-post labeling ERCC1 XPA XPB/ERCC3 XPC XPD/ERCC2 XPE/DDB1/2 XPE/ERCC4 XPG/ERCC5 NER Core Proteins Host-cell reactivation Neumann et al., Mol Carcino, 2005

10. pCMVcat pCMVluc BamH I EcoR I Pvu I Ap Ap Xbal I Bgl II 5000 bp 4863 bp Enh Enh Xbal I cat Luc EcoR I P P Hind III Nar I Bgl II Recombinant Plasmid DNA Expression Vectors Used in the Host-Cell Reactivation Assay BPDE BPDE

11. Transfection Efficiency in the Host-Cell Reactivation Assay Cheng et al., BioTechniques, 1997

12. Risk of Lung Cancer Associated with Low DRC HCR Assay (plasmid DNA) Cases = 316 Controls = 316 Trend test : P < 0.001 Odds Ratio Wei et al., JNCI, 2000 Cases = 221 Controls = 229 In Vitro Adduct Assay (genomic DNA) Trend test : P < 0.001 High Low DRC (%) by Quartile Li et al, Cancer Res., 2001

13. Suboptimal DRC and Cancer Risk Cancer # Case/Control OR (95%CI) Reference Lung 51/56 5.7 (2.1-15.7) Wei et al, 1996 316/316 2.1 (1.5-3.0) Wei et al, 2000 764/677 1.5 (1.2-1.9) Spitz et al, 2004 1522/1672 1.4 (1.2-1.6) unpublished data Head & Neck 55/61 2.2 (1.0-4.8) Cheng et al, 1998 744/753 1.9 (1.5-2.4) Wang et al., 2010 * For the low tertile DRC and others for the low-median DRC; **Recalculated based on published data

14. Correlation between DNA repair genotype and phenotype

15. Known SNPs in the 8 NER Core genes available in the NIEHS resequencing database NER Nucleotides Location Function No. of SNP No of No of core gene / Protein SNPs Density nsSNPsnsSNPs ** (per kb) with MAF > 0.05 ERCC1 14kb / 297aa 19q13.2 Endonuclease 73 5.2 1 -- -q13.3 XPA 22kb / 273aa 9q22.3 Damage detection 140 6.4 2 -- XPB/ERCC3 37kb / 782aa 2q21 Helicase 136 3.7 2 -- XPC 33kb / 940aa 3p25 Damage detection 145 4.4 12 rs2228000( A499V) rs2228001 (K939Q) XPD/ERCC2 19kb / 760aa 19q13.3 Helicase 136 7.2 2 rs1799793 (D312N) rs13181 (K751Q) XPE/DDB2 24kb / 427aa 11p12-p11 Damaged DNA 77 3.2 2 -- binding XPF/ERCC4 28kb / 916aa 16p13.3 Endonuclease 214 7.6 7 -- - p13.11 XPG/ERCC5 30kb / 1186aa 13q22 Endonuclease 177 5.9 12 rs17655 (D1104H) Total 1,098 40 5 ** XPC codon499 Ala/Val; XPC codon939 Lys/Gln; XPD codon312 Asp/Asn; XPD codon751 Lys/Gln; XPG codon1104 His/Asp

16. Genotype Prediction of DRC Phenotype (UV-damage) in Cancer-free Subjects Genotype N % DRC (SD) P value 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 Qiao et al., Carcinogenesis, 2002

17. Correlation between DRC Phenotype (UV-damage) and Genotype in Cancer-free Subjects N = 102 P = 0.02 None Hetero Homozygous Three common Variants in XPD and XPC Genes Qiao et al., Mutat Res, 2002

18. MDACC Lung Cancer GWAS Study Genome Wide Association Study of Lung Cancer – 1200 cases / 1200 controls 317K SNPs Chris Amos et al., MDACC

19. 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,

20. DNA repair and clinical outcomes of lung cancer

21. Lung Cancer Patient Follow-up Time Line Death or Loss to Follow-up Last Clinic Visit Enrollment in Study Treatment Diagnosis 275 patients with NSCLC

22. Adjusted* Relative Risk of Death Associated 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 patients had curative surgery without chemotherapy and were dropped from analysis of patients treated with surgery. Bosken et al., JNCI, 2002

23. 1 0.9 No Chemotherapy (4.9 months) 0.8 Chemotherapy - Efficient DNA Repair (10.1 months) 0.7 0.6 Chemotherapy - Sub-optimal DNA Repair (15.8 months) Fraction Surviving 0.5 0.4 0.3 0.2 0.1 0 0 10 20 30 40 50 60 Effect of Chemotherapy on Survival in Patients with Stage III/IV NSCLC by DRC Survival in Months Bosken et al., JNCI, 2002

24. 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

25. 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

26. Some Selected Polymorphisms of DNA Repair Genes Genes # Variants (comm) # AA Changes Ch BER ADPRT / PARP1 442 (9) 3 1 APEX1 / APE1 58 (2) 1 14 XRCC1 403 (18) 4 19 NER ERCC1 149 (6) 0 19 ERCC4/ XPF 330 (2) 1 19 HR Rad51 342 ( 0) 0 1 XRCC3 198 ( 6) 1 14 NHEJ XRCC2 334 (7) 1 7 XRCC7 / PRKDC 859 (12) 3 8

27. Selected Functional SNPs in DNA Strand Break Repair Genes Pathway Gene SNPs Location BER XRCC1 Q399R Exon APEX1 D148E Exon ADPRT Val762Ala Exon NER ERCC1 8092C>A 3’UTR ERCC4 R415Q Exon DSBR RAD51 -135G>C Promoter RAD51 -172G>T Promoter XRCC2 Arg188His Exon XRCC3 T241M Exon XRCC7 6721G>T Exon

28. 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) 1.00 GT 111 (52) 1.63 (0.89-2.96) 0.113 GG 43 (21) 2.27 (1.13-4.59) 0.022 Rad51 (rs1801320 ) (HR) GG 173 (81) 1.00 AG 35 (17) 0.48 (0.24-0.96) 0.038 AA 5 (2) 1.46 (0.35-6.16) 0.609 XRCC3 (rs861539 ) (HR) CC 63 (29) 1.00 CT 92 (17) 0.70 (0.43-1.16) 1.169 TT 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

29. Cumulative risk for RP BER XRCC3P = 0.011 CC DSBR Cumulative Grade ≥3 RP (hazard) CT+TT Time (months) Unpublished data

30. RAD51 rs1801320 CG+CC vs. GG P = 0.009 Cum survival Months XRCC2 rs3218536GA+AA vs. GG P = 0.043 Cum survival Months Cumulative Survival DSBR Unpublished data

31. 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

32. 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

33. Thank You!