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Association of ERCC1 gene expression
with outcome in stage II-III esophageal
adenocarcinoma patients treated with
preoperative platinum-based
chemoradiation in a phase II cooperative
group study (SWOG S0356).
P. Bohanes1, B. H. Goldman2, J. K. Benedetti2, C. Blanke3, L. P.
Leichman4, S. Iqbal1, C. R. Thomas5, C. L. Corless5, K. G.
Billingsley5, K. D. Danenberg6, P. J. Gold7, and H.J. Lenz1
1University
of Southern California, Los Angeles, CA; 2SWOG Statistical Center, Seattle WA;
3University of British Columbia Vancouver, BC, Canada; 4Desert Regional Medical Center,
Palm Springs, CA; 5Oregon Health and Science University, Portland, OR; 6Response
Genetics, Inc, Los Angeles, CA; 7Swedish Cancer Institute, Seattle, WA
Author’s Disclosures
P. Bohanes: None
 B.H. Goldman: None
 L. Leichman: None
 C. Blanke: Sanofi-Aventis
 S. Iqbal: Sanofi-Aventis
 C.R. Thomas: None
 C.L. Corless: None
 J.K. Benedetti: None
 K.G. Billingsley: None
 K.D. Danenberg: Response Genetics
 P. Gold: None
 H.J. Lenz: Sanofi-Aventis, Response Genetics

Background

There is no worldwide accepted standard treatment for
locally advanced esophageal adenocarcinoma.
◦ Neoadjuvant chemo-radiation prior to surgery is one of the
accepted treatment strategies
◦ Platinum agents are often used as the chemotherapy backbone
for patients treated with trimodality therapy

In contrast to the growing number of predictive
biomarkers for anti-cancer agents, there are no
established biomarkers to select patients who will
benefit most from chemo-radiation.

Utilization of predictive biomarkers to select therapy
should lead to higher cure rates.
Background – ERCC1

ERCC1 has been shown to be a critical gene in DNA repair
◦ NER pathway - recognizes and removes platinum-induced DNA adducts
◦ DSBR pathway- repairs radiation-induced damage
(Bohanes et al. Clin Colorectal Cancer. In Press; Ahmad et al. Moll Cell Biol 2008)

A prospective clinical trial demonstrated in advanced NSCLC
that customized therapy based on ERCC1 mRNA levels
increased the response rate to cisplatin-based chemotherapy
administered to patients with low ERCC1 mRNA levels (Cobo et al.
J Clin Oncol 2007)
ERCC1 down-regulation sensitizes cells
to all platinum compounds
90
80
Cell viability (%)
70
60
50
control
40
siRNA
30
20
10
0
Cisplatin (4 uM)
Oxaliplatin (2 uM)
Carboplatin (20 uM)
Youn et al. Cancer Res 2004
Tumor ERCC1 mRNA Levels
Type of Cancer
NSCLC
Colorectal
Esophageal
Median Value
1.65
1.15
1.92
Range
0.14-13.4
0.34-4.66
0.33-5.29
% with low
expression
57%
78%
42%
References
Cobo et al.
JCO.2007
Lenz et al.
ASCO. 2008
Current study
ERCC1 in Gastrointestinal Malignancies
Author
Tumor
Setting
Patients
Cutoff
Results
Shirota et al.
J Clin Oncol 2001
Colorectal
FOLFOX
50
> 1.7 x 10-3
 OS
Lenz et al.
ASCO 2008
Colorectal
FOLFOX
191
> 1.7 x 10-3
 RR,  OS
Uchida et al.
BMC Cancer 2006
Colorectal
Oxaliplatin +
capecitabine
91
> 3.4 x 10-3
 TTR
Metzger et al.
J Clin Oncol 1998
Gastric
Cisplatin + 5-FU
38
> 1.46 x 10-3
 OS
Wei et al.
Br J Cancer 2008
Gastric
FOLFOX
76
> 2.2 x 10-3
OS
Langer et al.
Clin Cancer Res 2008
Esophageal
Cisplatin + 5-FU
± Paclitaxel
38
--
NS for RR
Warnecke-Eberz et al.
Clin Cancer Res 2004
Esophageal
Oxaliplatin +
5FU + EBRT
36
> 1.1 x 10-3
 RR
Joshi et al.
Clin Cancer Res 2005
Esophageal
Cisplatin + 5-FU
+ EBRT
99
> 3.0 x 10-3
OS
ERCC1 in Gastrointestinal Malignancies
Author
Tumor
Setting
Patients
Cutoff
Results
Shirota et al.
J Clin Oncol 2001
Colorectal
FOLFOX
50
> 1.7 x 10-3
 OS
Lenz et al.
ASCO 2008
Colorectal
FOLFOX
191
> 1.7 x 10-3
 RR,  OS
Uchida et al.
BMC Cancer 2006
Colorectal
Oxaliplatin +
capecitabine
91
> 3.4 x 10-3
 TTR
Metzger et al.
J Clin Oncol 1998
Gastric
Cisplatin + 5-FU
38
> 1.46 x 10-3
 OS
Wei et al.
Br J Cancer 2008
Gastric
FOLFOX
76
> 2.2 x 10-3
OS
Langer et al.
Clin Cancer Res 2008
Esophageal
Cisplatin + 5-FU
± Paclitaxel
38
--
NS for RR
Warnecke-Eberz et al.
Clin Cancer Res 2004
Esophageal
Oxaliplatin +
5FU + EBRT
36
> 1.1 x 10-3
 RR
Joshi et al.
Clin Cancer Res 2005
Esophageal
Cisplatin + 5-FU
+ EBRT
99
> 3.0 x 10-3
OS
ERCC1 in Gastrointestinal Malignancies
Author
Tumor
Setting
Patients
Cutoff
Results
Shirota et al.
J Clin Oncol 2001
Colorectal
FOLFOX
50
> 1.7 x 10-3
 OS
Lenz et al.
ASCO 2008
Colorectal
FOLFOX
191
> 1.7 x 10-3
 RR,  OS
Uchida et al.
BMC Cancer 2006
Colorectal
Oxaliplatin +
capecitabine
91
> 3.4 x 10-3
 TTR
Metzger et al.
J Clin Oncol 1998
Gastric
Cisplatin + 5-FU
38
> 1.46 x 10-3
 OS
Wei et al.
Br J Cancer 2008
Gastric
FOLFOX
76
> 2.2 x 10-3
OS
Langer et al.
Clin Cancer Res 2008
Esophageal
Cisplatin + 5-FU
± Paclitaxel
38
--
NS for RR
Warnecke-Eberz et al.
Clin Cancer Res 2004
Esophageal
Oxaliplatin +
5FU + EBRT
36
> 1.1 x 10-3
 RR
Joshi et al.
Clin Cancer Res 2005
Esophageal
Cisplatin + 5-FU
+ EBRT
99
> 3.0 x 10-3
OS
ERCC1 in Gastrointestinal Malignancies
Author
Tumor
Setting
Patients
Cutoff
Results
Shirota et al.
J Clin Oncol 2001
Colorectal
FOLFOX
50
> 1.7 x 10-3
 OS
Lenz et al.
ASCO 2008
Colorectal
FOLFOX
191
> 1.7 x 10-3
 RR,  OS
Uchida et al.
BMC Cancer 2006
Colorectal
Oxaliplatin +
capecitabine
91
> 3.4 x 10-3
 TTR
Metzger et al.
J Clin Oncol 1998
Gastric
Cisplatin + 5-FU
38
> 1.46 x 10-3
 OS
Wei et al.
Br J Cancer 2008
Gastric
FOLFOX
76
> 2.2 x 10-3
OS
Langer et al.
Clin Cancer Res 2008
Esophageal
Cisplatin + 5-FU
± Paclitaxel
38
--
NS for RR
Warnecke-Eberz et al.
Clin Cancer Res 2004
Esophageal
Oxaliplatin +
5FU + EBRT
36
> 1.1 x 10-3
 RR
Joshi et al.
Clin Cancer Res 2005
Esophageal
Cisplatin + 5-FU
+ EBRT
99
> 3.0 x 10-3
OS
ERCC1 in Gastrointestinal Malignancies
Author
Tumor
Setting
Patients
Cutoff
Results
Shirota et al.
J Clin Oncol 2001
Colorectal
FOLFOX
50
> 1.7 x 10-3
 OS
Lenz et al.
ASCO 2008
Colorectal
FOLFOX
191
> 1.7 x 10-3
 RR,  OS
Uchida et al.
BMC Cancer 2006
Colorectal
Oxaliplatin +
capecitabine
91
> 3.4 x 10-3
 TTR
Metzger et al.
J Clin Oncol 1998
Gastric
Cisplatin + 5-FU
38
> 1.46 x 10-3
 OS
Wei et al.
Br J Cancer 2008
Gastric
FOLFOX
76
> 2.2 x 10-3
OS
Langer et al.
Clin Cancer Res 2008
Esophageal
Cisplatin + 5-FU
± Paclitaxel
38
--
NS for RR
Warnecke-Eberz et al.
Clin Cancer Res 2004
Esophageal
Oxaliplatin +
5FU + EBRT
36
> 1.1 x 10-3
 RR
Joshi et al.
Clin Cancer Res 2005
Esophageal
Cisplatin + 5-FU
+ EBRT
99
> 3.0 x 10-3
OS
Main Objective

To validate prospectively ERCC1 gene
expression (predefined cutoff of 1.7) as a
biomarker predicting outcome in patients treated
with oxaliplatin-based chemotherapy in
combination with radiation in the SWOG S0356
trial.
Secondary Objectives

To explore the association between outcome
and :
◦ Other baseline mRNA levels of genes involved in
DNA repair (XPD, RRM1) and 5-FU metabolism (TS,
TP, DPD and GSTP1)
◦ Functional polymorphisms in genes involved in DNA
repair (ERCC1, XPD, RAD51, XRCC1, XRCC3) and
5-FU metabolism (TS, MTHFR, GSTP1)
SWOG S0356 Treatment Design
Tumor biopsy
D1
D8
D15
D22
D29
D36
D43
D22
D29
D36
D43
CTX + EBRT
Surgery
CTX
D1
D8
OHP 85 mg/m2
D15
PI 5FU 180 mg/m2/day
Inclusion Criteria

Esophageal or gastroesophageal junction
adenocarcinoma
◦ Patients > 18 years
◦ Clinical stage II or III; Zubrod PS ≤ 2
◦ Endoscopic ultrasound only for tumors that do not
form a clear mass on CT scan
◦ Pre-tx PET scans mandatory
◦ Thoracic esophagus or gastroesophageal junction
◦ Tumors < 2 cm into the gastric cardia (Siewert I-II)
◦ Standard hematologic/non-hematologic parameters
Patient Characteristics
N=92
February 2005 to August 2008
98 patients registered
6 ineligible patients*
92 eligible for clinical outcome
evaluation and this study
Median Age (range)
62.0 (41.6-83.1)
Gender
Male
Female
86 (93%)
6 (7%)
Race
White
Other
Missing
85 (96%)
4 (4%)
3
Performance Status
0/1
Missing
54/37 (59%/41%)
1
Primary Site
Esophagus
GE Junction
Missing
*2 squamous tumors, 2 met disease, 2 with biopsy/scans performed >28 days from protocol entry
54 (60%)
36 (40%)
2
Pathologic Response


26 (28.2%) patients = pCR (centrally confirmed)
10 patients had either Tin situ N0M0 or T1N0M0
Leichman et al. Annual Meeting of the ASTRO, San Diego, 2010
Progression-Free Survival (PFS)
100%
80%
N
60%
92
Events
55
Median in Months
20.8
2-year PFS
40%
45.6%
20%
0%
0
2
4
Years After Registration
6
Overall Survival (OS)
100%
N
92
80%
60%
Events
47
Median in Months
33.7
2-year OS
40%
55.4%
20%
0%
0
2
4
Years After Registration
6
Median follow-up
of 36.8 months
Leichman et al. Annual Meeting of the ASTRO, San Diego, 2010
Gene Expression
RNA Extracted
RNA
Laser Capture Micro-dissection
Reverse Transcription
cDNA
PCR with TaqMan®
Data Analysis
Statistical Considerations

Cox regression used for univariate analyses of
biomarker association with outcome
◦ Adjustment for baseline factors did not affect any
results

Recursive partitioning used to look for optimal
cut points for continuous markers
◦ Also used for building “regression trees”
◦ P values adjusted for the multiple comparisons
implied by this technique
Genes Analyzed for mRNA
Levels
92 patients -> 90 pre-treatment samples -> 55 with sufficient tumor tissue
Genes
Number of patients
Median expression level (Range)
GSTP1
55
1.61 (0.52-8.73)
ERCC1
53
1.92 (0.33-5.29)
TP
52
8.93 (1.36-38.45)
TS
50
3.32 (0.92-8.62)
DPD
39
0.57 (0-2.22)
RRM1
26
1.75 (0.35-5.81)
XPD
19
1.88 (0.76-3.60)
Overall
(N=92)
Gene Expression
Dataset (N=55)
62.0 (41.6-83.1)
63.9 (43.6-83.1)
Gender
Male
Female
86 (93%)
6 (7%)
51 (93%)
4 (7%)
Race
White
Other
Missing
85 (96%)
4 (4%)
3
51 (94%)
3 (6%)
1
54/37 (59%/41%)
1
34/21 (62%/38%)
0
Primary Site
Esophagus
GE Junction
Missing
54 (60%)
36 (40%)
2
34 (62%)
21 (38%)
0
pCR
26 (28%)
14 (25%)
Median age (range)
Performance status
0/1
Missing
PFS by ERCC1 mRNA Levels
100%
≤1.7
>1.7
80%
N
22
31
Median 2-year PFS HR (95% CI)
NR
67%
1.0 (ref)
14.8 mos
17%
2.97 (1.37-6.45)
p*
0.0058
60%
40%
Median follow-up
of 36.8 months
20%
0%
0
1
2
3
Years After Registration
4
5
OS by ERCC1 mRNA Levels
100%
≤ 1.7
> 1.7
N
Median
22
31
NR
22.4 mos
2-year OS
72%
37%
p*
HR (95% CI)
1.0 (ref)
2.32 (1.01-5.31) 0.047
80%
60%
40%
Median follow-up
of 36.8 months
20%
0%
0
1
2
3
Years After Registration
4
5
Other Results

An analysis of PFS using a recursive partitioning
model found the optimal split for ERCC1 gene
expression to be 1.66 (adjusted p=0.04).

ERCC1 mRNA levels were not associated with pCR

None of the other accessed mRNA levels were
associated with outcome (either univariate or in
recursive partitioning)
◦ DNA-repair: XPD, RRM1
◦ Platinum detoxification: GSTP1
◦ 5-FU metabolism: TS, TP, DPD
Genotyping

DNA was extracted from blood (Qiagen, CA, USA).

Genotyping was performed by using PCR-RFLP technique.

Performed in 91 patients (out of 92 eligible)

ERCC1 118C>T

MTHFR 677C>T

ERCC1 8092C>A

MTHFR 1298A>C

GSTP1 Ile105Val

TS 3’UTR 6bp+/6bp-

RAD51 135G>C

TS 5’UTR VNTR

XPD 156A>C

TS 5’UTR G/C SNP

XPD Lys751Gln

XRCC1 Arg391Gln

XRCC3 Thr241Met
None are significant
after adjusting for
multiple comparisons
Summary

In this trial, using oxaliplatin and protracted-infusion 5FU
with radiation, low intratumoral ERCC1 from the primary
esophageal cancer predicted for PFS and OS.

Pre-established ERCC1 mRNA cutoff of 1.7 was
confirmed in this trial.

This pre-specified cutoff was further validated by using
recursive partitioning (optimal cutoff of 1.66).

Genomic polymorphisms analyzed were not associated
with outcome in this study.
Study Limitations

Small sample size.

Lack of sufficient tumor tissue collection.

No differentiation between clinical stage II and
clinical stage III patients
Conclusions

ERCC1 mRNA level is a very promising pre-treatment
biomarker in patients with localized esophageal and
gastroesophageal adenocarcinoma treated with
trimodality treatment.

Biomarker studies are feasible within cooperative
groups.

Based on these and published data, the SWOG is
planning a prospective biomarker-driven clinical trial.
Acknowledgments
The patients and their families and Investigators
who participated in SWOG S0356
Response Genetics: Kathleen D. Danenberg.
SWOG Statistical Center: Bryan Goldman.
Funded by SWOG award 5-U10-CA058882-18
Pierre Bohanes was partially funded by Cancer & Solidarité Fondation