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

2. 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

3. IALT RAND OIZE N=1867 Stage I~III Cisplatin-based adjuvant C/T (n=935)
Lung Cancer V 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

4. 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

5. JBR.10 RAND OIZE N=482 Stage IB, II NSCLC Completed resected
Lung Cancer V 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

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)
NEJM 2005;352:2589

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

8. ERCC-1 staining
NEJM 2007;356:800

9. 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

10. IALT ERCC positive tumor OS HR 1.40 (0.84~1.55) (P=0.40)
NEJM 2006;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)
NEJM 2007;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.
the genomic level (analysis of single-nucleotide polymorphisms),
transcriptional level (reversetranscriptase–polymerase-chain-reaction [RT-PCR] assay)
protein level (immunohistochemicalanalysis)

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