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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