Transcript ERCC 1 isoform expression and DNA repair in NSCLC NEJM 2013

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
Predictive
EGFR
Benefit from EGFR TKI
KRAS
Lack benefit from
platinum/vinorelbine or EGFR TKI
ALK fusion gene
Benefit from ALK inhibitor
High ERCC1
expression
Lack benefit from platinum –based
chemotherapy
Prognostic
Poor survival
Better survival
IALT
N=1867
Stage I~III
NSCLC
Completed resected
Optional RT
R
A
N
DR
O
I
Z
E
Cisplatin-based adjuvant C/T (n=935)
Primary:
OS
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
N=482
Stage IB, II
NSCLC
Completed resected
Age ≥18
PS 0-1
R
A
N
DR
O
I
Z
E
CDDP+Vinorelbine adjuvant C/T (n=242)
Primary:
OS
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)
ERCC negative tumor
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.
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 cisplatinbased 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 ERCCpositive 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 ERCC1deficient 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 cisplatininduced DNA damage.
• The unique functional isoform ERCC1-202
might a more accurate predictor marker
Thanks for the listening
Discussion and Comments