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MDR1 and ERCC1 Expression Predict Outcome of Patients with Locally Advanced Bladder Cancer Receiving Adjuvant Chemotherapy

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  • KMG Klinik Silbermühle GmbH

Abstract and Figures

PURPOSE: The role of adjuvant chemotherapy in patients with locally advanced bladder cancer still remains to be defined. We hypothesized that assessing the gene expression of the chemotherapy response modifiers multidrug resistance gene 1 (MDR1) and excision repair crosscomplementing 1 (ERCC1) may help identify the group of patients benefiting from cisplatin-based adjuvant chemotherapy. EXPERIMENTAL DESIGN: Formalin-fixed paraffin-embedded tumor samples from 108 patients with locally advanced bladder cancer, who had been enrolled in AUO-AB05/95, a phase 3trial randomizing a maximum of three courses of adjuvant cisplatin and methotrexate (CM) versus methotrexate, vinblastine, epirubicin, and cisplatin (M VEC), were included in the study. Tumor cells were retrieved by laser-captured microdissection and analyzed for MDR1 and ERCC1 expression using a quantitative real-time reverse transcription-polymerase chain reaction assay. Gene expression levels were correlated with clinical outcomes by multivariate Cox proportional hazards regression analysis. RESULTS: Expressions of MDR1 and ERCC1 were independently associated with overall progression-free survival (P = .001, relative risk = 2.9 and P = .01, relative risk = 2.24, respectively). The correlation of high MDR1 expression with inferior outcome was stronger in patients receiving M-VEC, whereas ERCC1 analysis performed equally in the CM and M-VEC groups. CONCLUSIONS: High MDR1 and ERCC1 gene expressions are associated with inferior outcome after cisplatin-based adjuvant chemotherapy for locally advanced bladder cancer. Prospective studies are warranted to define a role for MDR1 and ERCC1 analysis in individualizing multimodality treatment in locally advanced bladder cancer.
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MDR1 and ERCC1 Expression
Predict Outcome of Patients
with Locally Advanced
Bladder Cancer Receiving
Adjuvant Chemotherapy
1,2,3
Andreas-Claudius Hoffmann*
,,
, Peter Wild
§
,
Christina Leicht
, Simone Bertz
#
,
Kathleen D. Danenberg
, Peter V. Danenberg
,
Robert Stöhr
#
, Michael Stöckle**, Jan Lehmann
††
,
Martin Schuler*and Arndt Hartmann
#
*Department of Medicine (Cancer Research), West German
Cancer Center, University Duisburg-Essen, Essen, Germany;
Department of Biochemistry and Molecular Biology, and
Norris Comprehensive Cancer Center, University of Southern
California, Los Angeles, CA, USA;
Response Genetics, Inc,
Los Angeles, CA, USA;
§
ETH Zurich, Zurich, Switzerland;
Department of Pathology, University Hospital Regensburg,
Regensburg, Germany;
#
Department of Pathology,
University Hospital Erlangen, Erlangen, Germany;
**Department of Urology, Saarland University, Homburg,
Germany;
††
Urology Practice Prüner Gang, Kiel, Germany
Abstract
PURPOSE: The role of adjuvant chemotherapy in patients with locally advanced bladder cancer still remains to be defined. We hy-
pothesized that assessing the gene expression of the chemotherapy response modifiers multidrug resistance gene 1 (MDR1)and
excision repair cross-complementing 1 (ERCC1) may help identify the group of patients benefiting from cisplatin-based adjuvant
chemotherapy. EXPERIMENTAL DESIGN: Formalin-fixed paraffin-embedded tumor samples from 108 patients with locally ad-
vanced bladder cancer, who had been enrolled in AUO-AB05/95, a phase 3 trial randomizing a maximum of three courses of adjuvant
cisplatin and methotrexate (CM) versus methotrexate, vinblastine, epirubicin, and cisplatin (M-VEC), were included in the study.
Tumor cells were retrieved by laser-captured microdissection and analyzed for MDR1 and ERCC1 expression using a quantitative
real-time reverse transcriptionpolymerase chain reaction assay. Gene expression levels were correlated with clinical outcomes by
multivariate Cox proportional hazards regression analysis. RESULTS: Expressions of MDR1 and ERCC1 were independently asso-
ciated with overall progression-free survival (P= .001, relative risk = 2.9 and P= .01, relative risk = 2.24, respectively). The cor-
relation of high MDR1 expression with inferior outcome was stronger in patients receiving M-VEC, whereas ERCC1 analysis
performed equally in the CM and M-VEC groups. CONCLUSIONS: High MDR1 and ERCC1 gene expressions are associated with
inferior outcome after cisplatin-based adjuvant chemotherapy for locally advanced bladder cancer. Prospective studies are war-
ranted to define a role for MDR1 and ERCC1 analysis in individualizing multimodality treatment in locally advanced bladder cancer.
Neoplasia (2010) 12, 628636
Abbreviations: MDR1,multidrug resistance gene 1;ERCC1,excision repair cross-complementing 1; FFPE, formalin-fixed paraffin-embedded; M-VEC, methotrexate, vinblastine,
epirubicin, and cisplatin; CM, methotrexate and cisplatin; Pgp, P-glycoprotein
Address all correspondence to: Andreas-Claudius Hoffmann, MD, Department of Medicine (Cancer Research), Molecular Oncology Risk Profile Evaluation, West German Cancer
Center, University Hospital Essen, Hufelandstrasse 55, 45147 Essen, Germany. E-mail: ach@o117.com
1
Authorsdisclosures of potential conflicts of interest: Kathleen D. Danenberg, Response Genetics, Inc, leadership role (CEO), stock ownership. Peter V. Danenberg, Response
Genetics, Inc, advisory role, stock ownership. Andreas-Claudius Hoffmann, Response Genetics, Inc, consultant, research funding. Peter Wild, Christina Leicht, Simone Bertz,
Robert Stöhr, Michael Stöckle, Jan Lehmann, Martin Schuler, and Arndt Hartmannnone. Response Genetics, Inc, funded the gene expression analysis. A.-C.H. received
research funding from Kampf dem KrebseV of the German Cancer Society (Deutsche Krebsgesellschaft).
2
The results of this study were presented at the American Society of Clinical Oncology Annual Meeting 2009.
3
This is the first description of the suitability of MDR1 and ERCC1 expression to predict overall survival and progression-free survival in patients enrolled in a prospective
randomized phase 3 trial of adjuvant chemotherapy for locally advanced bladder cancer. These findings represent an important step toward the development of biomarkers for
individualizing adjuvant treatment decisions in bladder cancer patients.
Received 11 March 2010; Revised 22 May 2010; Accepted 27 May 2010
Copyright © 2010 Neoplasia Press, Inc. All rights reserved 1522-8002/10/$25.00
DOI 10.1593/neo.10402
www.neoplasia.com
Volume 12 Number 8 August 2010 pp. 628636 628
Introduction
The role of adjuvant chemotherapy in locally advanced urothelial carci-
noma of the bladder is still a matter of debate. Whereas some random-
ized multicenter trials have demonstrated superior progression-free
survival after treatment with three to four courses of methotrexate, vin-
blastine, adriamycin or epirubicin, and cisplatin (M-VAC/M-VEC)
[15], no benefit in overall survival has been demonstrated. In addi-
tion, the M-VEC regimen is associated with significant toxicities,
which may outweigh its potential benefits especially in elderly patients.
Against this background, the AUO-AB 05/95 trial was designed to
explore a deescalated adjuvant chemotherapy regimen consisting of cis-
platin and methotrexate (CM). In this randomized multicenter phase 3
study, the anthracycline-containing M-VEC standard therapy failed to
outperform the less toxic CM regimen [6]. However, because of the lack
of an observation arm, it remains unclear whether adjuvant CM is
the standard for all patients with locally advanced bladder cancer. More-
over, the superior progression-free survival after adjuvant chemother-
apy in locally advanced bladder cancer could result from a strong
effect in a subgroup of patients, whereas others experience no benefit.
Hence, biomarkers predicting the relative risk reduction from adju-
vant therapy are needed to individualize treatment strategies in bladder
cancer patients.
Several gene products have been described to modify the cellular
response to chemotherapeutic agents in vitro and to correlate with clin-
ical outcome in vivo. Excision repair cross-complementing 1 (ERCC1)
is a component of the nucleotide excision repair pathway, a major re-
pair mechanism of DNA damage induced by platin compounds react-
ing with DNA and forming interstrand and intrastrand cross links.
The balance of DNA damage to DNA repair dictates tumor cell death
or survival after cisplatin therapy [7]. ERCC1 expression as detected by
immunohistochemistry as well as gene expression has been linked to
response and survival in many retrospective and some prospective
studies in nonsmall cell lung cancer, colorectal cancer, and bladder
cancer patients treated with platin-based therapies [810]. The multi-
drug resistance gene 1 (MDR1) encodes an integral membrane protein
denamed P-glycoprotein (Pgp) or an ATP-binding cassette subfamily B,
member 1, which acts as an energy-dependent cellular efflux pump.
Pgp was shown to reduce intracellular concentrations of a variety of
cytotoxic drugs, including anthracyclines, vinca alkaloids, and taxanes.
Under certain conditions, such as the presence of defective folate car-
rier transport proteins, methotrexate can also be a substrate of Pgp
[11]. Pgp activity results in blunted chemotherapy-induced cytotoxicity
in vitro and in vivo. Moreover, anticancer drugs were found to induce
MDR1 gene [12]. High Pgp levels were associated with inferior treat-
ment outcome in elderly patients with acute myeloid leukemia [13
15], breast cancer [16,17] sarcoma [18,19], and other entities.
Thus far, only a few chemotherapy response modifiers have been
assessed in small retrospective studies of bladder cancer, which failed
to produce unequivocal results [20]. More than a decade ago, it was re-
ported that M-VAC treatment of bladder cancer leads to transactiva-
tion and significantly increased expression of MDR1, although this
result was not obtained in an outcome-driven study [21]. Analyzing
tumor samples from bladder cancer patients receiving uniform adju-
vant chemotherapy in a large randomized multicenter trial should in-
crease the ability to identify truly predictive biomarkers. To this end,
we retrieved formalin-fixed paraffin-embedded (FFPE) tumor samples
from patients enrolled in the AUO-AB 05/95 trial [6], which com-
pared adjuvant CM to M-VEC in 327 patients with locally advanced
bladder cancer. Because both treatment arms were based on cisplatin,
we focused on ERCC1 expression. In addition, we assessed MDR1 ex-
pression because its gene product was shown to modulate the cytotoxic-
ity of epirubicin, vinblastine, and possibly methotrexate.
Materials and Methods
Study Population and Tumor Samples
The study population has been described previously (Table 1) [6].
Tumor staging was performed according to the criteria of the Inter-
national Union Against Cancer [22].
Table 1. PatientsDemographics.
Demographic AUO-AB 05/95 Trial Group Study Group
CM (n= 163) M-VEC (n= 164) Total (n= 327) CM (n= 56) M-VEC (n= 52) Total (n= 108)
n%n%n%n%n%n%
Tumor category
pTis/pT1 pN+ 7 4.3 4 2.4 11 3.4 4 7.1 0 0.0 4 3.7
pT2 pN+ 14 8.6 29 17.7 43 13.1 7 12.5 11 21.2 18 16.7
pT3 pN0 58 35.6 61 37.2 119 36.4 17 30.4 17 32.7 34 31.5
pT3 pN+ 56 34.3 44 26.8 100 30.6 18 32.1 16 30.8 34 31.5
pT4a pN0 13 8.0 10 6.1 23 7.0 5 8.9 3 5.8 8 7.4
pT4a pN+ 15 9.2 16 9.8 31 9.5 5 8.9 5 9.6 10 9.3
Nodal status
pN0 71 43.6 71 43.3 142 43.4 22 39.3 20 38.5 42 38.9
pN+ 92 56.4 93 56.7 185 56.6 34 60.7 32 61.5 66 61.1
1 lymph node 43 46.7 38 40.9 81 43.8 15 44.1 13 40.6 28 25.9
2-5 lymph nodes 37 40.2 48 51.6 85 45.9 14 41.2 17 53.1 31 28.7
>5 lymph nodes 12 13.1 7 7.5 19 10.3 5 14.7 2 6.3 7 6.5
Age, years
50 26 16.0 24 14.6 50 15.3 5 8.9 11 21.2 16 14.8
51-60 61 37.4 61 37.2 122 37.3 25 44.6 19 36.5 44 40.7
61-70 76 46.6 79 48.2 155 47.4 26 46.4 22 42.3 48 44.4
Median 60.2 60.7 60.5 59.5 59 59
Sex
Male 123 75.5 134 81.7 257 78.6 43 76.8 42 75.0 85 78.7
Female 40 24.5 30 18.3 70 21.4 13 23.2 10 17.9 23 21.3
Neoplasia Vol. 12, No. 8, 2010 MDR1 and ERCC1 in Bladder Cancer, AUO-AB 05/95 Hoffmann et al. 629
FFPE tissue samples were available for expression analysis from 108
of 327 study patients. The clinicopathologic characteristics of all pa-
tients were reviewed by one surgical pathologist (A.H.). Representative
hematoxylin and eosinstained slides of FFPE tissue blocks obtained
at cystectomy were reviewed to estimate the tumor load per sample.
For laser-captured microdissection (PALM Microlaser Technologies
AG, Munich, Germany), slides of 10-μm thickness were obtained.
All tumor slides were prepared as described previously [23].
Quantitative Real-time Polymerase Chain Reaction
RNA was isolated from microdissected tumor samples following
a proprietary procedure at Response Genetics, Inc (Los Angeles, CA;
US patent no. 6248,535). The resulting tumor RNA was reverse-
transcribed into complementary DNA (cDNA) as described previously
[23]. Expression of MDR1,ERCC1,andACTB (β-actin, endogenous
reference) was quantified by real-time fluorescence detection of ampli-
fied cDNA (ABI PRISM 7900 Sequence Detection System [TaqMan];
Perkin-Elmer Applied Biosystems, Foster City, CA). The reverse
transcriptionpolymerase chain reaction (RT-PCR) assay was imple-
mented as described previously [23]. All primers were selected using
the Gene Express software (Applied Biosystems) but were adapted
to the requirements of cDNA generated from RNA, which was ex-
tracted from the FFPE tissue. We used previously published sequences
of MDR1,ERCC1,andACTB [7,18,24]. All primers were validated
following a previously described protocol [25]. All genes were run on
all samples in triplicates, that is, one sample was run with each gene
three times on the same plate to identify potential outliers. The detec-
tion of amplified cDNA results in a cycle threshold (C
t
) value, which is
reciprocal to the amount of cDNA contained in the sample. Normal
colon, liver, and St. Universal Mix RNA (Stratagene, La Jolla, CA)
were used as control calibrators on each assay plate. Gene expression
levels were described as ratio between two absolute measurements (gene
of interest/endogenous reference gene, here beta-actin) to control for
intersample variation. Before statistical analysis, all ratios were logarith-
mically transformed including a multiplier, which accounted the aver-
age C
t
values obtained for each gene during the validation process. This
procedure facilitated the comparison samples, which were run on dif-
ferent assay plates. Depending on the used genes and mutlipliers, the
interplate variation is around 5%.
Statistical Analyses
Associations of gene expression levels and progression-free or overall
survival were tested for each gene by the Kaplan-Meier method. Sur-
vival differences between the high- and low-expression group were an-
alyzed by the log-rank test. To detect independent prognostic factors
associated with overall and progression-free survival, multivariate Cox
proportional hazards regression analysis with stepwise selection was ap-
plied. After adjusting for potential confounders, the following param-
eters were accounted for: pathologic tumor stage (pT), lymph node
involvement (pN), vascular invasion (V ), tumor grade (G), and the
gene set. In addition, receiver operating characteristic curve analysis
was performed to test the ability of the chosen cut points to discrim-
inate short survivors from long survivors [26,27].
The level of significance was set to P< .05. All Pvalues reported
were based on two-sided tests. All statistical analyses were performed
using the Software Packages SPSS for Windows (Version 16.0; SPSS,
Inc, Chicago, IL) and JMP 7.0 software (SAS, Cary, NC).
Results
Study Group and Tumor Samples
The AUO-AB 05/95 trial enrolled a total of 327 patients [6]. Tissue
blocks suitable for RNA extraction were retrieved from 108 patients
(33%) and subjected to further analysis. This subgroup was equally
balanced for clinicopathologic parameters compared with the entire
study population (Table 1). The Spearman coefficient of rank correla-
tion of 17 staging parameters of the trial and the study group was
0.987 (P= .0001, 95% confidence interval [CI] = 0.964-0.995).
Gene Expression and Survival
Kaplan-Meier analysis revealed that patients with MDR1 expression
below the 75th percentile (P= .0006, hazard ratio [HR] = 0.25, 95%
CI = 0.11-0.55) had a higher chance for prolonged survival. After
5 years, only 23% of patients with high MDR1 expression (>75th per-
centile) were still alive, whereas 62% of patients with low MDR1 ex-
pression (<75th percentile) survived 5 years. This association was still
significant, when each treatment arm, CM (P= .01, HR = 0.26, 95%
CI = 0.09-0.74; Figure 1) and M-VEC (P= .02, HR = 0.27, 95%
CI = 0.083-0.88; Figure 2) was analyzed separately. Furthermore, pa-
tients with low MDR1 expression had a lower risk for early progression
(P= .002, HR = 0.28, 95% CI = 0.13-0.62). After 2 years, only 25%
of patients with low MDR1 expression experienced disease progression,
whereas more than 65% of patients with high MDR1 expression had
progressed. When evaluating progression-free survival in relation to
MDR1 expression for each treatment arm, significant associations were
obtained (CM: P= .01, HR = 0.26, 95% CI = 0.09-0.76; M-VEC:
P= .05, HR = 0.34, 95% CI = 0.11-1.04). Next, we built a statistical
model for overall survival based on MDR1 expression, pT, pN, and
pV as covariates using Cox proportional hazards regression analysis
with stepwise selection (Table 2). Vascular invasion, which was appar-
ent in 7% (8/107 patients) of the study group, was revealed as the
strongest independent risk factor in this model, with a relative risk
of 3.09 (P= .02, 95% CI = 1.19-8.03) for reduced survival time.
The relative risks for high MDR1 expression and pN2 were 2.88
(P= .001, 95% CI = 1.52-5.48) and 2.87 (P= .001, 95% CI =
1.52-5.43), respectively. Comparable results were obtained in a model
for progression-free survival based on MDR1 expression, pT, pN, and
pV as covariates (Table 3).
Low ERCC1 expression (<75th percentile) was also associated with
prolonged progression-free survival (P= .03, HR = 0.52, 95% CI =
0.27-1.01; Figure 3). Within 5 years of follow-up, only 45% of pa-
tients with low ERCC1 expression had progressed, whereas almost
70% of patients with high ERCC1 expression (>75th percentile) ex-
perienced disease progression. Separate subgroup analyses of both
treatment arms revealed a trend for a reduced risk of progression in
patients with low ERCC1 expression (CM: P= .21, HR = 0.54,
95% CI = 0.20-1.42 [Figure 4]; M-VEC: P= .07, HR = 0.43,
95% CI = 0.17-1.10 [Figure 5]). A significant association of ERCC1
expression with progression-free survival (relative risk = 2.24; P=.01,
95% CI = 1.23-4.08) was revealed by Cox regression analysis (Table 4).
Median overall survival times were 72.4months for the low-ERCC1 ex-
pression group and 33.1 months for the high-ERCC1 expression group,
which failed to reach significance at Kaplan-Meier analysis (P= .19,
HR = 0.66, 95% CI = 0.35-1.24) or Cox regression analysis (relative
risk = 1.75, P= .10, 95% CI = 0.89-3.44; Table 5). Both genes were
tested together in multivariate regression models for their independent
630 MDR1 and ERCC1 in Bladder Cancer, AUO-AB 05/95 Hoffmann et al. Neoplasia Vol. 12, No. 8, 2010
association with both overall survival and progressions-free survival.
However, when both genes were included in the above-mentioned
model, only MDR1 remained as the most significant divisor of patients
with a longer or a shorter survival (Tables 6 and 7).
Performance of MDR1 and ERCC1 Expression in Relation to
Treatment Regimen
Kaplan-Meier plot analysis for overall survival revealed an early sep-
aration of the groups with high and low MDR1 expression in patients
Figure 2. Kaplan-Meier plot estimating the overall survival of patients in the M-VEC treatment arm. Differences in survival between the
high- and the low-MDR1 expression group were analyzed with the log-rank test. The upper black line represents the low-expression
group, whereas the lower broken line represents the high-expression group.
Figure 1. Kaplan-Meier plot estimating the overall survival of patients in the CM treatment arm. Differences in survival between the high-
and the low-MDR1 expression group were analyzed with the log-rank test. The upper black line represents the low-expression group,
whereas the lower broken line represents the high-expression group.
Neoplasia Vol. 12, No. 8, 2010 MDR1 and ERCC1 in Bladder Cancer, AUO-AB 05/95 Hoffmann et al. 631
treated with M-VEC compared with CM treatment (Figures 1 and
2). In contrast, no such difference was observed when separating for
ERCC1 expression (Figures 4 and 5). Receiver operating characteristic
curve analysis was applied to test whether there was a difference in the
sensitivity and specificity of MDR1 expression for discrimination of
short-term survivors from long-term survivors. In the M-VECtreated
patient group, high MDR1 expression exhibited significant (P=.008,
area under the curve = 0.71, 95% CI = 0.56-0.82) sensitivity of 69%
(true-positive rate) and specificity of 72% (true-negative rate) for dis-
crimination between patients surviving longer than 24 months and
those who died earlier. This level of significance was not observed in
the CM-treated patient group (P= .91, area under the curve = 0.5,
95% CI = 0.37-0.65), which revealed a sensitivity of only 46% and
a specificity of 56%.
Discussion
Patients experiencing bladder cancer growing beyond the lamina mus-
cularis propria [28] and/or metastasizing to the lymph nodes have a
Table 2. Cox Proportional Hazard Regression: Overall Survival, MDR1.
Method Stepwise
Enter variable if P<.05
Remove variable if P>.1
Sample size 107
Overall Model Fit
Null model 2 log likelihood 331.920
Full model 2 log likelihood 308.682
χ
2
23.238
df 3
Significance level P< .0001
Coefficients and SE
Covariate bSE PExp(b) 95% CI of Exp(b)
MDR1 > 75% 1.0588 0.3294 .001306 2.8829 1.5167-5.4797
pN = 2 1.0541 0.3267 .001254 2.8694 1.5174-5.4261
Vascular invasion 1.1277 0.4900 .02137 3.0886 1.1879-8.0306
Variables not included in the model
pT = 1
pT = 2
pT = 4
pN = 1
Table 3. Cox Proportional Hazard Regression: Progression-Free Survival, MDR1.
Method Stepwise
Enter variable if P<.05
Remove variable if P>.1
Sample size 105
Overall Model Fit
Null model 2 log likelihood 376.539
Full model 2 log likelihood 349.441
χ
2
27.097
df 3
Significance level P< .0001
Coefficients and SE
Covariate bSE PExp(b) 95% CI of Exp(b)
MDR1 > 75% 1.0478 0.3234 .001194 2.8514 1.5178-5.3567
pN = 2 1.0742 0.3071 .0004684 2.9277 1.6087-5.3283
Vascular invasion 1.3289 0.4580 .003715 3.7769 1.5461-9.2264
Variables not included in
the model
pT = 1
pT = 2
pT = 4
pN = 1
Figure 3. Kaplan-Meier plot estimating the progression-free survival of patients in both treatment arms. Differences in survival between
the high- and the low-ERCC1 expression group were analyzed with the log-rank test. The upper black line represents the low-expression
group, whereas the lower broken line represents the high-expression group.
632 MDR1 and ERCC1 in Bladder Cancer, AUO-AB 05/95 Hoffmann et al. Neoplasia Vol. 12, No. 8, 2010
high risk of relapse despite radical cystoprostatectomy and systematic
lymph node dissection. Chemotherapy has been proven efficacious in
patients experiencing metastatic bladder cancer, with cisplatin being
the most active agent. Accordingly, it was hypothesized that cisplatin-
based chemotherapy applied before or after surgery for locally advanced
bladder cancer would increase survival in this high-risk patient popula-
tion. Although several randomized trials have been conducted, none
of them conclusively demonstrated a significant survival benefit. How-
ever, improved progression-free survival was observed after adjuvant
M-VAC compared with observation [29]. Relatively low patient numbers
Figure 4. Kaplan-Meier plot estimating the progression-free survival of patients in the CM treatment arm. Differences in survival between
the high- and the low-ERCC1 expression group were analyzed with the log-rank test. The upper black line represents the low-expression
group, whereas the lower broken line represents the high-expression group.
Figure 5. Kaplan-Meier plot estimating the progression-free survival of patients in the M-VEC treatment arm. Differences in survival
between the high- and the low-ERCC1 expression group were analyzed with the log-rank test. The upper black line represents the
low-expression group, whereas the lower broken line represents the high-expression group.
Neoplasia Vol. 12, No. 8, 2010 MDR1 and ERCC1 in Bladder Cancer, AUO-AB 05/95 Hoffmann et al. 633
and brief follow-up in these studies could explain the lack of a mea-
surable survival benefit from adjuvant chemotherapy. Alternatively, it
is conceivable that only a subgroup of bladder cancer patients benefits
from adjuvant chemotherapy, whereas it is of no effect or even detri-
mental for other patients. Thus, the development of biomarkers that
are able to predict the presence or the absence of a benefit from ad-
juvant chemotherapy is of high importance for optimizing the care of
patients with locally advanced bladder cancer. To this end, the present
study was conducted to correlate MDR1 and ERCC1 gene expression
with the outcome of patients undergoing adjuvant chemotherapy for
muscle-invasive and/or nodal-metastasized urothelial bladder cancer
within the randomized, prospective AUO-AB 05/95 phase 3 trial
[6]. The MDR1 and ERCC1 genes were chosen for analysis because
their encoded gene products have been implied as modifiers of the tu-
mor cell response to the anticancer agents tested in AUO-AB 05/95.
The MDR1 gene product Pgp is an energy-dependent efflux
pump, which, among others, reduces intracellular concentrations of
epirubicine and vinblastine, both of which were administered in the
M-VEC arm of the trial. Moreover, methotrexate seems to be a sub-
strate of Pgp when cells show deficient carrier-mediated methotrexate
uptake [11]. Although cisplatin is not considered a de novo substrate
of Pgp, some studies have suggested an altered expression of MDR1
after cisplatin administration, possibly resulting in decreased cytotoxic
Table 4. Cox Proportional Hazards Regression: Overall Survival, ERCC1.
Method Stepwise
Enter variable if P<.05
Remove variable if P>.1
Sample size 107
Overall Model Fit
Null model 2 log likelihood 331.920
Full model 2 log likelihood 318.061
χ
2
13.859
df 2
Significance level P= .0010
Coefficients and SE
Covariate bSE PExp(b) 95% CI of Exp(b)
pN = 2 1.0649 0.3246 .001037 2.9006 1.5402-5.4627
Vascular invasion = 1 0.9975 0.4860 .04011 2.7115 1.0512-6.9945
Variables not included in
the model
ERCC1 > 75%
pT = 1
pT = 2
pT = 4
pN = 1
Table 5. Cox Proportional Hazards Regression: Progression-Free Survival, ERCC1.
Method Stepwise
Enter variable if P<.05
Remove variable if P>.1
Sample size 105
Overall Model Fit
Null model 2 log likelihood 376.539
Full model 2 log likelihood 352.487
χ
2
24.052
df 3
Significance level P< .0001
Coefficients and SE
Covariate bSE PExp(b) 95% CI of Exp(b)
ERCC1 > 75% 0.8054 0.3082 .008969 2.2377 1.2268-4.0815
pN = 2 1.1446 0.3077 .0001991 3.1411 1.7240-5.7231
Vascular invasion 1.1676 0.4514 .009690 3.2142 1.3330-7.7507
Variables not included in
the model
pT = 1
pT = 2
pT = 4
pN = 1
Table 6. Cox Proportional Hazards Regression: Overall Survival, MDR1 and ERCC1.
Method Stepwise
Enter variable if P<.05
Remove variable if P>.1
Sample size 107
Overall Model Fit
Null model 2 log likelihood 331.920
Full model 2 log likelihood 308.682
χ
2
23.238
df 3
Significance level P< .0001
Coefficients and SE
Covariate bSE PExp(b) 95% CI of Exp(b)
pN = 2 1.0541 0.3267 .001254 2.8694 1.5174-5.4261
Vascular invasion 1.1277 0.4900 .02137 3.0886 1.1879-8.0306
MDR1 > 75% 1.0588 0.3294 .001306 2.8829 1.5167-5.4797
Variables not included in
the model
pT = 1
pT = 2
pT = 4
pN = 1
ERCC1 > 75%
Table 7. Cox Proportional Hazards Regression: Progression-Free Survival, MDR1 and ERCC1.
Method Stepwise
Enter variable if P<.05
Remove variable if P>.1
Sample size 105
Overall Model Fit
Null model 2 log likelihood 376.539
Full model 2 log likelihood 349.441
χ
2
27.097
df 3
Significance level P< .0001
Coefficients and SE
Covariate bSE PExp(b) 95% CI of Exp(b)
pN = 2 1.0742 0.3071 .0004684 2.9277 1.6087-5.3283
Vascular invasion 1.3289 0.4580 .003715 3.7769 1.5461-9.2264
MDR1 > 75% 1.0478 0.3234 .001194 2.8514 1.5178-5.3567
Variables not included in the model
pT = 1
pT = 2
pT = 4
pN = 1
ERCC1 > 75%
634 MDR1 and ERCC1 in Bladder Cancer, AUO-AB 05/95 Hoffmann et al. Neoplasia Vol. 12, No. 8, 2010
efficacy [3033]. Whereas these studies might argue for a correlation
between MDR1 expression and resistance to platin compounds, addi-
tional reports failed to establish such an association [34]. Accordingly,
the positive correlation between high MDR1 expression and inferior
survival and progression-free survival after adjuvant cisplatin-based
chemotherapy as observed in our study does not automatically imply
a causative role of Pgp. Moreover, it is tempting to speculate that the
bulk of the prognostic or predictive value of MDR1 expression is based
on the inclusion of patients from the M-VEC arm. This hypothesis is
supported by our findings studying the biomarkers separately in both
treatment arms. MDR1 expression performed significantly better as a
discriminator of patient outcomes in the M-VEC arm than in the CM
arm. In contrast, ERCC1, which encodes a gene product primarily
modifying the cellular response to platin compounds and demonstrates
significant association with progression-free survival in the whole study
group, showed no difference between the two platin-based treatment
arms. Because of the low patient numbers per group, these findings
have to be interpreted with caution. However, they are in line with
a potential biologic explanation for the association of MDR1 expres-
sion and patient outcome after adjuvant chemotherapy.
As cisplatin is still regarded the main active drug in urothelial blad-
der cancer treatment, it is biologically plausible that the expression of
an established modifier of the cellular platin response correlates with
treatment efficacy. In our homogeneously defined and prospectively
collected patient cohort, ERCC1 expression was significantly and
independently associated with progression-free survival, thus sub-
stantiating its role as biomarker for chemotherapy response in blad-
der cancer.
Our present study has been retrospectively conducted in samples
collected from a completed clinical trial. Accordingly, the results may
have been influenced by confounders that have occurred during the
follow-up period but were not reported and by additional bias result-
ing from the fact that evaluable tissue blocks were only available from
one third of the patients. Importantly, clinicopathologic parameters
were equally balanced in the present study group and the entire trial
population. Because of the lack of an observation arm in AUO-AB
05/95, it is impossible to decide whether expressions of MDR1 and
ERCC1 are prognostic or predictive markers in this high-risk bladder
cancer population. Interestingly, the results obtained with the two
biomarkers applied in the present study can be corroborated by a bi-
ologic hypothesis, which is different from findings revealed by unse-
lected expression analysis of thousands of parallel genes. This provides
a strong rationale for implementing MDR1 and ERCC1 expression
analysis in future trials of biomarker development in bladder cancer.
To this end, the adjuvant setting is particularly suitable because tissue
availability is not an issue. Because RT-PCR is a feasible method to re-
trieve results even from small tissue fractions, expressions of MDR1
and ERCC1 may also be used to better estimate which patients could
benefit from neoadjuvant chemotherapy, even more because Hussain
et al. [35] recently pointed out that administering chemotherapy to
patients with resistant disease delays definitive local therapy while
the disease progresses. In two large randomized trials, neoadjuvant che-
motherapy with three courses of M-VAC before radical cystectomy
provided a significant survival benefit [3638]. However, this has
not entered clinical practice in many centers, in part because of the
substantial toxicities of the M-VAC regimen as well as the fear of tu-
mor progression because of delayed surgery. As new drugs, such as
gemcitabine and taxanes, have been introduced to the management
of urothelial cancer, biomarkers in addition to MDR1 and ERCC1
may be required to provide a broader basis for the selection of treat-
ment options for individualized patient care. This calls for further
exploratory studies comparable to this one before embarking on a pro-
spective biomarker trial. It will be of particular interest to our findings
to explore the prognostic value of MDR1 expression in a sufficiently
powered bladder cancer population treated without anthracyclines and
vinblastine. In conclusion, we have identified MDR1 and ERCC1 ex-
pressions as determined by real-time RT-PCR analysis as indepen-
dent markers, which significantly correlate with overall survival and
progression-free survival in patients undergoing cisplatin-based adju-
vant chemotherapy after resection of locally advanced urothelial blad-
der cancer. This defines two promising and robust biomarkers to be
prospectively validated toward the implementation of individualized
care for bladder cancer patients.
Acknowledgments
The authors thank the patients, investigators, and pathologists of
AUO-AB 05/95 who primarily diagnosed the tumor for contributing
materials and clinical data.
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636 MDR1 and ERCC1 in Bladder Cancer, AUO-AB 05/95 Hoffmann et al. Neoplasia Vol. 12, No. 8, 2010
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... Indeed, 45% of patients with ERCC1 low-expressing tumors reported disease progression within five years compared with 70% of patients with ERCC1 high-expressing tumors. Median overall survival was also longer for ERCC1 low-expressing patients (72.4 vs. 33.1 months), although not statistically significant (p = 0.19) [29]. The present study further established the role of ERCC1 as a predictive marker of platinum-based adjuvant chemotherapy. ...
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Introduction: As the understanding of molecular mechanisms of bladder cancer advances, molecularly-guided precision medicine becomes increasingly relevant. Biomarkers play a critical role in this setting, predicting treatment response and identifying candidates for targeted therapies. Areas covered: Current literature on biomarkers in their role in disease prognosis, and response to neoadjuvant and adjuvant therapies. In non-muscle invasive bladder cancer, particular focus is on markers of disease progression, and response to intravesical therapy. In muscle invasive and advanced bladder cancer, particular emphasis is on markers associated with neoadjuvant chemotherapy, as well as systemic immunotherapy. We discuss current shortcomings and pitfalls in contemporary markers, and future avenues of prospective research. Expert opinion: The focus on biomarkers has moved from immunohistochemical analysis and tumor-related phenotypic changes to examining genetic alterations. Single marker analysis has been shown to be insufficient in predicting both disease course and response to therapy, and studies have shifted towards examining marker combinations and genetic classifiers. Ultimately, significant progress in implementing biomarkers into clinical guidelines remains elusive, largely due to lack of prospective studies in well-defined patient cohorts and with clinically-meaningful endpoints. Until then, despite their promising value, tissue markers should be limited to experimental settings and clinical trials.
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This review aims to provide a practical update regarding the current role of tissue-based biomarkers in bladder cancer. Their prognostic and predictive role both in non-muscle-invasive (NMIBC) and in muscle-invasive disease (MIBC) has been reviewed with particular focus to their use in clinical practice. In summary, the literature on the prediction of disease recurrence in NMIBC is inconclusive, and there is little information on prediction of response to intravesical bacillus Calmette-Guerin (BCG). Concerning disease progression, external prospective validation studies suggest that FGFR3 mutation status and gene signatures may improve models that are based only on clinicopathologic information. In MIBC, tissue-based biomarkers are increasingly important, since they may predict the response to systemic chemotherapy and immunotherapy. In particular, the advent of molecular characterization promises to revolutionize the paradigm of decision-making in the treatment of MIBC. Molecular subtyping has been shown to improve the prediction of pathological stage at RC and to predict the response to systemic chemotherapy and immunotherapy. However, external and prospective validations are warranted to confirm these preliminary findings. Several different tissue-based biomarkers such as PD-1/PD-L1 expression, tumor mutational burden, and the analysis of tumor microenvironment, may in future play a role in selecting patients for systemic immunotherapy. However, to date, no pretreatment recommendations can be definitively made on the basis of any molecular predictors. In conclusion, despite the potential of tissue-based biomarkers, their use in bladder cancer should be limited to experimental settings.
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Patients with high-risk primary breast cancer remain at high risk for relapse. More precise prognostic and predictive tools are needed to improve treatment of such patients. Formalin-fixed, paraffin-embedded tumors from 239 high-risk breast cancer patients were examined for expression of human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), estrogen receptor, progesterone receptor, Ki-67, p16, p21, p27, and p53 by immunohistochemistry. Gene expression of EGFR, HER2, glutathione S-transferase-Pi (GSTP1), excision repair cross complementation1 (ERCC1), p21, beta-tubulin-3, multidurg resistance (MDR1), cyclooxygenase2 (COX2), and cyclin-E was measured by RT-PCR. Eighty percent of patients presented with locally advanced, or > or =10 axillary nodal metastasis, and 20% with inflammatory breast cancer. The median age was 46 years (26-62 years) and the median number of involved axillary lymph nodes was 12 (0-42). At a median follow-up of 86 months, relapse-free survival (RFS) and overall survival for the entire group were 50% (95% CI 43% to 57%) and 62% (95% CI 56% to 69%). Multivariate Cox stepwise analysis resulted in a simple model for RFS consisting only of p21 expression, EGFR expression assessed by RT-PCR, and number of axillary nodal metastases. A prognostic model on the basis of the expression of a limited number of proteins and genes may help to guide target-specific therapies in patients with high-risk breast cancer.
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P-glycoprotein (PGP), the product of the MDR1 gene, is a transmembrane active efflux pump for a variety of carcinogens and cytostatics. It has been suggested that MDR1 polymorphisms contribute to the variability in cancer risk and therapeutic outcome. We examined the relevance of C3435T polymorphism in relation to breast cancer susceptibility, clinical and pathological characteristics of breast carcinoma, the therapeutic response and hematologic toxicities after anthracycline-based chemotherapy. A significant association between allele frequencies and histological type, stage and histological grade was observed (P=0.024, 0.014, 0.006, respectively, chi(2)-test or Fisher's exact test). We also found significantly higher (P=0.019, chi(2)-test) T allele frequency in breast cancer patients (n=221) than in controls (n=113). A significantly enhanced therapeutic outcome after neoadjuvant therapy (n=38; P=0.021, Fisher's exact test) and longer time to progression after anthracycline-based chemotherapy (n=102; P=0.049, log-rank test) were observed in CC homozygotes. However, no significant association between hematologic toxicities and C3435T polymorphism was detectable.
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Finding markers or gene sets that would further classify patients into different risk categories and thus allow more individually adapted multimodality treatment regimens in soft tissue sarcomas is necessary. In this study, we investigated the prognostic values of hypoxia-inducible factor 1a (HIF1a), heparin-binding epidermal growth factor-like growth factor (HB-EGF), vascular endothelial growth factor (VEGF), and other angiogenesis-related gene expressions, as well as their interrelationships. Formalin-fixed paraffin-embedded tissue samples were obtained from 45 patients with soft tissue sarcoma (median age 57 years, range 16-85 years). After laser capture microdissection direct quantitative real-time reverse transcription-PCR (TaqMan) assays were done in triplicates to determine HIF1a, HB-EGF, VEGF, and other gene expression levels. Multivariate Cox [corrected] regression analysis revealed significant independent associations of HB-EGF, HIF1a, and VEGF-C gene expression to the overall survival (P < 0.0001). A combined factor of these three genes showed a relative risk for shorter survival of 5.5, more than twice higher as in an increasing International Union against Cancer Stage. Receiver operating characteristic curve analysis showed a significant sensitivity of 73% and specificity of 82% of this factor for the diagnosis of short (<3 years) versus long (3-9 years) survival (P = 0.0002). VEGF-A showed significant gender differences in the association to survival. Measuring HIF1a, HB-EGF, and VEGF-C expression may contribute to a better understanding of the prognosis of patients with soft tissue sarcoma and may even play a crucial role for the distribution of patients to multimodal therapeutic regimens. Prospective studies investigating the response to different adjuvant or palliative therapies seem to be warranted.
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PURPOSEWe have previously shown that relative thymidylate synthase (TS) mRNA levels in primary gastric adenocarcinomas treated with fluorouracil (5-FU) and cisplatin are inversely associated with response and survival. This is a presumed function of TS as a target for 5-FU activity. We now test the hypotheses that the relative mRNA level of the excision repair cross-complementing (ERCC1) gene is inversely associated with response and survival as an independent function of cisplatin efficacy.PATIENTS AND METHODS Patients had intact, untreated, primary gastric adenocarcinoma cancer and were evaluated for eligibility on a preoperative cisplatin infusion-5-FU protocol. cDNA, derived from primary gastric tumors before chemotherapy, was used to determine ERCC1 mRNA levels, expressed as the ratio of polymerase chain reaction (PCR) product of the ERCC1 gene and the beta-actin gene.RESULTSThe median ERCC1 mRNA level from 38 primary gastric cancers (33 assessable for response) was 5.8 x 10(-3) (range, 1.8 x 10(-3) t...
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In oncology patients and clinicians are confronted with the search for measures which could help to elicit the individual patient's risk of future outcome, such as recurrence of disease after primary treatment, response to chemo-therapy or a general outline on the aggressiveness of a given lesion. In patient counselling, the emerging role of evidence based treatment choices reveals with cumulative certainty that the available information is inconclusive. This review will focus on current investigations of determinants to predict response to chemotherapy in advanced bladder cancer or to define prognosis of patients prior to any definite treatment. It will discuss the current evidence for the current systemic treatment options and highlight the many promising approaches of implementing markers either as a basis for a clinical decision in combination with other prognosticators (to better detect individuals at risk or to avoid unnecessary invasive procedures) or as a possible part of relevant pathways to be targeted. It will also discuss the role of biological markers with regards to the relevant clinical question and provide the current evidence to each field. It will highlight the need to further harmonize terminology, approaches and circumstances under which markers are evaluated and will provide suggestions for general methodological principles and guidelines for design, conduct, analysis and reporting of marker studies. The exploration of the current aspects of marker research may outline why collaborative, multicentre, and multidisciplinary efforts should be an integral part of future studies.