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ERCC 1 isoform expression and DNA repair in NSCLC

ERCC 1 isoform expression and DNA repair in NSCLC. NEJM 2013;368:1101 Reporter: 胡名宏 Supervisor: 邱宗傑 2013.06.03. Predictive/Prognostic factors in NSCLC. IALT. RAND OIZE. N=1867 Stage I~III NSCLC Completed resected Optional RT. Primary: OS. Cisplatin-based adjuvant C/T (n=935).

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ERCC 1 isoform expression and DNA repair in NSCLC

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  1. ERCC 1 isoform expression and DNA repair in NSCLC NEJM 2013;368:1101 Reporter: 胡名宏 Supervisor: 邱宗傑 2013.06.03

  2. Predictive/Prognostic factors in NSCLC

  3. IALT RAND OIZE N=1867 Stage I~III NSCLC Completed resected Optional RT Primary: OS Cisplatin-based adjuvant C/T (n=935) R Observation (n=932) NEJM 2004;350:351 JCO 2010;28:35

  4. IALT 5yr OS44.5% vs 40.4%(P<0.03) 5yr RFS39.4% vs 34.3%(P<0.003) NEJM 2004;350:351

  5. JBR.10 RAND OIZE N=482 Stage IB, II NSCLC Completed resected Age ≥18 PS 0-1 Primary: OS CDDP+Vinorelbine adjuvant C/T (n=242) R Observation (n=240) NEJM2005;352:2589 JCO2010;28:29

  6. JBR.10 5-yr RFS rate 39.4% vs 34.3% (P<0.001) 5-yr OS rate 69% vs 54% (P=0.009) NEJM2005;352:2589

  7. Excision repair cross-complementation group 1 (ERCC1) Ribonucleotide reductase M1 (RRM1)

  8. ERCC-1 staining NEJM2007;356:800

  9. IALT ERCC negative tumor OS HR 0.65 (0.50~0.86) (P=0.002) ERCC negative tumor DFS HR 0.65 (0.50~0.85) (P=0.001) NEJM2006;305:983

  10. IALT ERCC positive tumor OS HR 1.40 (0.84~1.55) (P=0.40) NEJM2006;305:983

  11. DFS and OS for high RRM1/ERCC1 NSCLC DFS > 120m in highRRM1/high ERCC1 (P=0.01) OS > 120m in highRRM1/high ERCC1 (P=0.02) NEJM2007;356:800

  12. Background • DNA repair capacity is a major determinant of cisplatin resistance • ERCC1 protein plays an essential role in nucleotide excision repair. • ERCC1 as a biomarker of patient survival, treatment efficacy, or both has been studied at the genomic level, transcriptional level and protein level in both retrospective and prospective studies.

  13. Background • The ERCC1 gene generates four isoforms (designated 201, 202, 203, and 204) by alternative splicing • ERCC1-201 and ERCC1-203 isoform have appeared to be nonfunctional in nucleotide excision repair capacity • Previous study revealed level of expression of ERCC1 in NSCLC tumors was prognostic or predictive, or both, of a benefit from cisplatin-based adjuvant chemotherapy

  14. Method • Tumor samples from the IALT, Cancer and Leukemia Group B (CALGB) 9633, and National Cancer Institute of Canada Clinical Trials Group JBR.10 trials are included in the LACE Biology biomarker project. • GALGB 9633 (180 P’t) and JBR.10 (314 P’t): validation set • 589 P’t from IALT could be stained again

  15. Method • Mouse monoclonal antibody against ERCC1 (clone 8F1) were used • Both set were evaluated by experienced pathologist in a blinded fashion • Stroma, epitheliuim and endothelium cells were taken into account • Staining intensity scale 0~3 (percentage of positive tumor nuclei 0% for 0, 0~9% for 0.1, 10~49% for 0.5, >50% for 1.0) • ERCC H score>1 was ERCC1-positive

  16. A549 cell line • ERCC1-deficient cells after knocked out ERCC1 gene • High sensitivity to cisplatin and a low rate of repair of cisplatin–DNA adducts

  17. Results • ERCC1 was scored as positive (H score >1) in 78% of samples (494 patients in the validation set) • Among patients with ERCC1-negative or ERCC-positive tumors, overall survival did not differ significantly between the chemotherapy and control groups.

  18. OS in ERCC-neg and ERCC-pos with chemotherapy HR 1.16; 95% [CI]0.64 to 2.10 (P = 0.62) HR 0.78; 95% [CI] 0.58 to 1.05 ( P = 0.09)

  19. Results • Discrepancy of ERCC1 tumor staining between 2006 and 2011 was noted: • ERCC1 (+) only 44% of the IALT Biology cohort in 2006, but , 77% were scored as positive using current 8F1 antibody batch

  20. Discrepancy of ERCC1 tumor staining • Discordant sample 36% • Possible change in 8F1 antibody batch might increase sensitivity

  21. Results • In addition to 8F1 and FL-297, 14 other commercial Ab used for detecting ERCC • None of the 16 Abs was specific for only one ERCC1 isoforms • Using RT-PCR and Western blot, 4 isoforms of ERCC1 could not recognized specifically

  22. Mapping ERCC Abs across different isoforms Highly immunogenic region

  23. Quantification of removal of cisplatin-DNA adducts • A459: wild type • ERCC1 –deficient clone 216 and 375 (control vector) • Cells expressing single isoforms • 2-hr cisplatin treatment (25umol/L)

  24. Tumor volumes after treating ERCC1-deficient cell • 105 ERCC1-deficient cell with single isoforms expression (201, 202,203,204) • Nude mice • Twice weekly IP cisplatin infection

  25. IC 50 of cisplatin • All cell line treated for 48 hrs with increasing dose of cisplatin • Significant differen -ces from wild type cell (P<0.05)

  26. Discussion • A number of clinical studies suggest ERCC1 was a prognostic factors or a predictive biomarkers. • In this study, ERCC1 failed to correlate with overall survival.

  27. Possible explanations • The current tools used to evaluate ERCC1 expression are inadequate • differences between two 8F1 batches could be related to distinct Ab titration, affinity, purity or even epitope recognition

  28. Possible explanations • The level of biologic complexity has been underestimated: four ERCC1 protein isoforms have not been correctly assessed • strong homology among the four protein isoforms • nonfunctional isoforms lead to a false classification as ERCC1-positive

  29. ERCC1-202 • Only the reintroduction of the ERCC1-202 isoform rescued nucleotide excision repair activity and the capacity to repair cisplatin-induced DNA damage. • The unique functional isoform ERCC1-202 might a more accurate predictor marker

  30. Thanks for the listening Discussion and Comments

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