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 EGFR Benefit from EGFR TKI KRAS Lack benefit from platinum/vinorelbine or EGFR TKI Poor survival ALK fusion gene Benefit from ALK inhibitor High ERCC1 expression Lack benefit from platinum –based chemotherapy Better survival

IALT RAND OIZE N=1867 Stage I~III Cisplatin-based adjuvant C/T (n=935) Lung Cancer V5- 08-11-06.ppt 二○一七年四月二十二日 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

IALT 5yr OS 44.5% vs 40.4% (P<0.03) 5yr RFS 39.4% vs 34.3% (P<0.003) NEJM 2004;350:351

JBR.10 RAND OIZE N=482 Stage IB, II NSCLC Completed resected Lung Cancer V5- 08-11-06.ppt 二○一七年四月二十二日 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) NEJM 2005;352:2589 JCO 2010;28:29

JBR.10 5-yr RFS rate 39.4% vs 34.3% (P<0.001) 5-yr OS rate 69% vs 54% (P=0.009) NEJM 2005;352:2589

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

ERCC-1 staining NEJM 2007;356:800

IALT ERCC negative tumor OS HR 0.65 (0.50~0.86) (P=0.002) DFS HR 0.65 (0.50~0.85) (P=0.001) NEJM 2006;305:983

IALT ERCC positive tumor OS HR 1.40 (0.84~1.55) (P=0.40) NEJM 2006;305:983

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) NEJM 2007;356:800

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. the genomic level (analysis of single-nucleotide polymorphisms), transcriptional level (reversetranscriptase–polymerase-chain-reaction [RT-PCR] assay) protein level (immunohistochemicalanalysis)

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

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

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

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

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.

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)

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

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

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

Mapping ERCC Abs across different isoforms Highly immunogenic region

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)

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

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)

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.

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

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

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

Thanks for the listening Discussion and Comments