Prospective evaluation of prognostic factors uPA/PAI-1 in node-negative breast cancer: Phase III NNBC3-Europe trial (AGO, GBG, EORTC-PBG) comparing 6 × FEC versus 3 × FEC/3 × Docetaxel
Prospective evaluation of prognostic factors uPA/PAI-1 in node-negative breast cancer: Phase III NNBC3-Europe trial (AGO, GBG, EORTC-PBG) comparing 6 FEC versus 3 FEC/3 Docetaxel
Eva J Kantelhardt 3
Martina Vetter 3
Marcus Schmidt 1
Corinne Veyret 0
Doris Augustin 7
Volker Hanf 6
Christoph Meisner 5
Daniela Paepke 4
Manfred Schmitt 4
Fred Sweep 9
Gunter von Minckwitz 8
Pierre-Marie Martin 2
Fritz Jaenicke 10
Christoph Thomssen 3
Nadia Harbeck 11
0 Centre Henri Becquerel, OB-GYN , Rouen , France
1 Klinik fur Gynakologie, Johannes Gutenberg-Universitat , Mainz , Germany
2 Laboratoire de Transfer en Oncologie Biologieque, l'AP-HM , Marseille , France
3 Klinik und Poliklinik fur Gynakologie, Martin-Luther Universitat , Halle Saale , Germany
4 Frauenklinik, Technische Universitat , Munchen , Germany
5 Eberhard Karls Universitat, IMB , Tubingen , Germany
6 Klinik fur Gynakologie, Klinikum Furth , Furth , Germany
7 Klinik fur Gynakologie, Klinikum Deggendorf , Deggendorf , Germany
8 German Breast Group GmbH, GBG , Neu-Isenburg , Germany
9 Department of Laboratory Medicine Radboud University Nijmegen Medical Cente , Nijmegen , the Netherlands
10 Klinik und Poliklinik fur Gynakologie. Universitatsklinikum Hamburg-Eppendorf , Hamburg , Germany
11 Brustzentrum, Frauenklinik, Universitat zu Koln , Cologne , Germany
Background: Today, more than 70% of patients with primary node-negative breast cancer are cured by local therapy alone. Many patients receive overtreatment by adjuvant chemotherapy due to inadequate risk assessment. So far, few clinical trials have prospectively evaluated tumor biology based prognostic factors. Risk assessment by a biological algorithm including invasion factors urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor type 1 (PAI-1) will assess up to 35-55% of node-negative patients as low-risk and thus avoid chemotherapy. In contrast, a clinical-pathological algorithm will only classify 20-40% of patients as lowrisk. High-risk node-negative patients should receive chemotherapy. Anthracycline-based regimens are accepted as a standard, the additional benefit of taxanes remains an open question. Methods/Design: The international NNBC3 ("Node Negative Breast Cancer 3-Europe) trial compares biological risk assessment (UP) using invasion factors uPA/PAI-1 with a clinical-pathological algorithm (CP). In this trial, the type of risk assessment (CP or UP) was chosen upfront by each center for its patients. Fresh frozen tissue was obtained to determine uPA/PAI-1 using an enzyme-linked immunosorbent assay (ELISA). Patients assessed as high-risk were stratified by human epidermal growth factor receptor 2 (HER2) status and then randomised to receive anthracycline-containing chemotherapy 5-Fluorouracil (F)/Epirubicin (E)/Cyclophosphymide (C) or an anthracyclinetaxane sequence (FE100C*6 versus FE100C*3 followed by Docetaxel100*3). Discussion: In this trial, 4,149 node-negative patients with operable breast cancer from 153 centers in Germany and France were included since 2002. Measurement of uPA/PAI-1 by ELISA was performed with standardised central quality assurance for 2,497 patients (60%) from 56 UP"-centers. The NNBC 3-Europe trial showed that inclusion of patients into a clinical phase III trial is feasible based on biological testing of fresh frozen tumor material. In addition, 2,661 patients were classified as high-risk and thus received chemotherapy. As adjuvant chemotherapy, 1,334 high-risk patients received FE100C-Docetaxel100, and 1,327 received French FE100C. No unexpected toxicities were observed. Chemotherapy efficacy and comparison of UP with CP will be evaluated after longer follow-up. Trial Registration: clinical Trials.gov NCT01222052.
Breast Cancer in Germany
In Germany, about 58,000 patients are newly diagnosed
with breast cancer every year. Today, approximately 80%
of patients can expect to be cured or to experience at
least long-term survival of more than 10 years. Due to
the activities of the national screening program, a
growing number of early tumors are detected. Most patients
have no or only a few (1-3) axillary lymph nodes
involved and therefore have a good chance of being
cured. Thus, overtreatment is increasingly becoming an
One of the major clinical questions is how to identify
those patients who may be able to avoid adjuvant
chemotherapy because of their low risk of recurrence.
Better prognostic factors are urgently needed to predict the
individual risk of recurrence.
Yet, patients with node-negative disease at high risk of
recurrence should receive adjuvant chemotherapy.
However, the most effective type of chemotherapy regimen is
uncertain. In order to avoid unnecessary side-effects,
prospective, randomised controlled comparisons of
regimens with and without taxanes are needed.
Prognostic factors in node-negative breast cancer
Clinical and pathological assessment
Most clinicians use grade of differentiation, age, tumor
size, steroid hormone receptor status, HER2 expression
and sometimes proliferation markers (e.g. Ki-67 or gene
signatures like OncotypeDX) in order to decide which
patient with node-negative disease should receive
adjuvant chemotherapy. In case of undifferentiated cancers
(grade 3), patients are truly at high-risk and may benefit
from chemotherapy, whereas in case of
well-differentiated grade 1 cancers, the risk of recurrence may be
rather low. However, in the heterogeneous group of
grade 2 tumors, it is essential to know for which
patients the benefits of chemotherapy will outweigh its
potential side effects. The widely used
clinical-pathological risk evaluation was defined at the consensus
meetings of St. Gallen .
uPA/PAI-1 for risk evaluation
There is an increasing focus on new biological factors to
further assess risk of recurrence in patients with grade 2
breast cancers in order to avoid unnecessary chemotherapy.
The uPA/PAI-1-algorithm gives promising results [2-4].
The capacity of breast cancer for invasion and early
hematogenic metastasis is closely related to the action
of receptor-bound, tumor-associated proteases and a
central role of the serine protease uPA (urokinase-type
plasminogen activator). In independent studies, several
groups have shown that the uPA antigen content in
tumor tissue is a strong and independent prognostic
factor in primary breast cancer . Particularly within the
node-negative group, the relative risk of relapse and
decreased survival was highest in patients with elevated
uPA-levels. In addition, the plasminogen activator
inhibitor-type 1 (PAI-1) content in tumour tissue is also
related to an increased risk of relapse and decreased
survival which becomes visible by PAI-1 being a strong
and independent prognostic factor in multivariate
analysis. Combining the two invasion factors, uPA and PAI-1,
by sequential selection (regression tree analysis) in
node-negative breast cancer patients, a high-risk group
can be identified comprising about 45% of all
nodenegative patients. Moreover, the remaining 55% of
node-negative patients have an extremely low risk of
relapse (93% disease-free survival after 3 years without
any adjuvant therapy), so that adjuvant chemotherapy
does not seem to be indicated [5,6,2].
In the first prospective, randomized, multicenter trial
(Chemo N0), these retrospective data were validated and
confirmed [3,7]. In long-term follow-up (10 years), uPA/
PAI-1 and tumor grade remained the only independent
prognostic factors with a hazard ratio of 3.2 and 2.8,
The Chemo N0 trial demonstrated that determination
of uPA and PAI-1 in tumor tissue by ELISA is easily
feasible in every laboratory. The international,
qualitycontrol system for uPA/PAI-1 determination showed
only non-relevant inter-laboratory variations (CV
1015%) . The clinically most important conclusion from
the Chemo N0 data is that at least 44% of all
nodenegative patients could potentially be spared from
adjuvant chemotherapy. Patients with high levels of uPA
and/or PAI-1 are at high risk for relapse and should
therefore receive optimal adjuvant chemotherapy [10,8]
A pooled analysis by the European Organisation for
Research and Treatment of Cancer Receptor and
Biomarker Group (EORTC-RBG) used the raw data of
most uPA and PAI-1 determinations available at the
time worldwide in numerous retrospective and
prospective breast cancer studies and confirmed the prognostic
value in more than 8,000 patients [2,11,12]. These new
prognostic factors, uPA/PAI-1, thus conform to the
requirements for clinical acceptance put forward by the
late W McGuire and GM Clark in the early nineties
. The American Society of Clinical Oncology
(ASCO) also added uPA/PAI-1 to the list of
recommended prognostic tumor markers for breast cancer
. The recommendations for diagnosis and treatment
of breast cancer issued by the Kommission Mamma
der Arbeitsgemeinschaft Gynkologische Onkologie
(AGO) e. V. in der Deutschen Gesellschaft fr
Gynkologie und Geburtshilfe e. V. sowie in der Deutschen
Krebsgesellschaft e. V. have even supported the use of
uPA/PAI-1 in node-negative breast cancer already since
Other biological factors for risk evaluation
The current ASCO guidelines also recommend the use
of the 21-Gene assay. This test is available for paraffin
embedded tissue. It is based on a reverse transcriptase
polymerase chain reaction (RT-PCR) determination of
the mRNA expression of 16 tumor-specific genes and 5
control genes. It is widely used in the USA for
nodenegative or node-positive (1-3 lymph nodes),
hormonereceptor positive patients. A high recurrence score is
associated with a high probability of recurrence and
benefit from adjuvant chemotherapy. This commercially
available test (OnkotypeDX) is currently evaluated in
a prospective randomised trial (Trial Assigning
IndividuaLized Options for Treatment (Rx), TAILORx)
comparing clinical-pathological versus biological risk
assessment by the 21-Gene assay. First results of
>10,000 patients are expected at the end of the trial in
2014 . In Germany, the prospective WSG Plan B
trial evaluating efficacy of anthracycline-free
chemotherapy in primary HER2-negative breast cancer after
molecular-based risk assessment according to Oncotype DX
and uPA/PAI-1 has already recruited almost 2,500
primary breast cancer patients by early 2011.
An additional test, the 70-gene array, is also based on
genetic profiling of the tumor. MRNA is prepared from
fresh tissue of node-negative and node-positive (1-3
lymph nodes) breast cancer and analyzed by a
multigene expression-array. This signature is also able to
predict an individual patients risk of recurrence and
survival (low vs. high). This commercially available test
(MammaPrint) is currently being evaluated in a
prospective randomized trial comparing
clinical-pathological versus biological risk assessment. The Microarray In
Node-negative and 1 to 3 positive lymph node Disease
may Avoid ChemoTherapy (MINDACT) trial started
2006 and will recruit 6,000 patients .
Another commercially available gene array to predict
the individual risk of recurrence is the 76-gene signature
(Rotterdam signature, Affymetrix) . In addition,
the HOXB13:IL17BR ratio index give prognostic
information for ER positive tumors and the molecular grade
index (five genes) (H/ISM and MGISM by
Biotheranostics, France) [20,21].
Many promising preclinical results have been
published for biomarkers giving not only prognostic
information but also predicting therapeutic response and
monitoring therapeutic interventions. Information may
be derived from the tumor or other patient specimens
as mRNA but also DNA, DNA-methylation status,
histone markers and miRNA .
Molecular intrinsic typing
Biologically meaningful breast-cancer tumor types have
been derived from expression array analyses [23,24]. This
molecular typing defines luminal (ER-positive),
HER2type (HER2 overexpressing) and basal-like (often ER and
PR and HER2-negative, so called triple-negative) breast
cancers, and thus tumor types that respond to specific
therapies such as endocrine therapy for luminal and
antiHER2 agents for HER2 type cancers. Luminal breast
cancers can be further divided into low-risk (luminal A) and
high-risk (luminal B) tumors. Luminal B tumors are
highly proliferating  and should be treated with
chemotherapy in addition to the endocrine therapy. Efforts
have been made to substitute gene-expression profiling
(requiring frozen tissue) by immunohistochemical
analysis of formalin fixed specimen. Since the results of these
two techniques do not correlate well so far, their results
should still be used with care.
Treatment of patients with node-negative breast cancer
In 2005, the Early Breast Cancer Trialists Collaborative
Group (EBCTCG) showed that anthracycline-containing
regimens account for up to a 38% (postmenopausal) and
20% (premenopausal) relative reduction in the cumulative
15-year mortality in addition to that attributable to
endocrine therapy . Several anthracycline-containing
regimens are currently used  and so far there is no direct
comparison supporting superiority of an individual
regimen provided that an adequate dose is used. The EORTC
Breast Cancer Group used two different
anthracyclinecontaining regimens as standard in their neo-adjuvant
chemotherapy trials. Study 10921 (closed 1996) used Canadian
FE120C . Analysis of the dose-intensity data, however,
demonstrated that the median dose-intensity delivered for
epirubicin was 100 mg/m for the standard arm because of
toxicity leading to early dose adjustment .
Consequently, the EORTC trial 10994 (p53 study) used the
FE100C regimen as its standard  - it also seems to be a
feasible standard in the adjuvant setting [30,15].
A recent meta-analysis demonstrated that Taxanes
(docetaxel (T), paclitaxel (Pac)) are very active agents in
the adjuvant treatment of all breast cancer patient
subgroups . There was one major trial particularly
comparing a taxane-containing regimen with a solely
anthracycline-based regimen in node-negative breast
cancer: The GEICAM 9805 trial recruited 1,059
highrisk, node-negative, breast-cancer patients.
DocetaxelA50C*6 (TAC) was compared to FA50C*6. The taxane
arm was significantly superior regarding disease-free
survival (DSF) (hazard ratio (HR) 67%). Overall survival
(OS) showed a non-significant trend (HR 0.70) favoring
TAC. As expected, side effects were more frequent in
the taxane-containing arm . Recently, Docetaxel has
also been approved in Europe for treatment of
nodenegative breast cancer.
The CALGB #9344 trial compared 4 courses of
standard AC with a sequence of 4x AC followed by 4 courses
of Pac in 3,000 node-positive patients. Patients treated
by ACPac had significantly fewer recurrences .
Yet, trial results were discussed controversially: patients
in the ACPac arm were on therapy twice as long as
those in the control arm; the beneficial chemotherapy
effect may thus be merely attributable to a longer
duration of chemotherapy. In addition, retrospective
subgroup analyses of this trial showed that the significant
advantage of adding paclitaxel was only present in
patients with steroid hormone receptor-negative tumors.
Consequently, in the NNBC 3-Europe trial, a balanced
comparison between an anthracycline-containing
(FE100C) and a sequential anthracycline-taxane
(FE100CDocetaxel sequence) therapy was chosen with identical
dose-intensity and chemotherapy duration in both trial
arms. For the taxane-containing sequence, safety data
and efficacy data for node-positive disease were already
Aim of the NNBC 3-Europe trial
The NNBC 3-Europe trial was aimed to compare
biological with clinical-pathological risk assessment in
patients with newly-diagnosed node-negative breast
cancer. In addition, the question should be answered
whether high-risk node-negative patients should receive
adjuvant chemotherapy with or without taxanes.
Design of the NNBC 3-Europe trial
In this international, multicenter,
prospectively-randomized, controlled trial, 4,149 node-negative patients
with operable breast cancer were included. Risk of
recurrence was assessed either by a clinical-pathological
(CP) or by a biological algorithm based on uPA/PAI-1
(UP). Type of risk assessment (CP or UP) was decided
up-front by each center for all of its patients and then
used as a stratification parameter for the trial. Patients
assessed as high-risk were stratified by HER2 status and
then randomized to receive anthracycline-containing
(FE100C*6) or anthracycline and taxane-containing
(FE100C*3 followed by Docetaxel100*3) chemotherapy.
A difference regarding disease-free survival between
the two chemotherapy arms is expected. Moreover,
discordance/concordance between the two methods of
prognostic assessment will be compared concerning
proportion and DFS of low-risk patients in each of the two
risk assessment groups.
The benefit of anthracycline/taxane-containing
chemotherapy compared to solely anthracyline-containing
chemotherapy will also be evaluated in HER2 positive
Comparison of the chemotherapy regimes
Comparison of clinical-pathological vs. biological
(uPA/PAI-1) risk assessment
Comparison of the chemotherapy regimes in HER2
over-expressing breast cancer
Ethical approval was obtained by the institutional
review board of the principle investigator as well as of
each participating center.
Patients age 18-65 were eligible for the trial if they had a
histological proven primary breast cancer (0.5-5 cm,
pN0, M0, R0). For centers using biological risk
assessment, frozen tissue had to be available from all patients
for uPA/PAI-1 testing. Patients needed to be of
adequate health in order to undergo the recommended
After informed consent, patients were registered for the
trial (figure 1). In biological centers working with UP,
frozen tissue was sent to designated laboratories for
uPA/PAI-1determination. After confirmation of
nodenegative disease, patients were classified by their risk
status as assessed by either clinical-pathological criteria,
or by biological criteria (Classification 1). Patients found
to be low-risk according to either CP or UP (see below)
were observed; patients found to be high-risk either by
CP or UP received adjuvant chemotherapy within the
trial (Classification 2). A stratification step was
performed according to HER2 status (Stratification). All
high-risk patients were randomly assigned to one of the
Patients with node-negative breast cancer
(NNBC); tumour size 0.55 cm; age 1865 yrs
C1: type of risk assessment
S: stratification by HER-2/neu expression
Endocrine treatment if Pg.-positive accorording to the latest standard recommendations of the AGO
Figure 1 Design of the NNBC 3-Europe trial. Stratification and
randomization schedule: C1 = stratification by type of risk
assessment based on choice of the centre (type of risk assessment);
C2 = stratification by risk status; S = Stratification by HER-2/neu-FISH
result; R = randomisation (FE100C: 5-Fluorouracil 500 mg/m,
Epirubicin 100 mg/m, Cyclophosphamide 500 mg/m, q3 wks; Doc:
Docetaxel 100 mg/m, q3 wks.). += fresh tumour tissue for patients
within the biological UP pathway, additionally in all patients
paraffin blocks for central review.
two adjuvant chemotherapy schedules (FEC or
FECDocetaxel) (Randomization). All patients received breast
radiotherapy if breast conserving surgery had been
performed. Radiotherapy, endocrine therapy, and trastuzumab
were given according to current AGO recommendations
at the time (http://www.ago-online.org).
The gold standard to prospectively compare risk
assessment procedures would have been
randomisation of each patient to either CP or UP risk
assessment. To avoid reduced accrual because of physicians
bias or patients not accepting such a rather
complicated procedure, consistent risk assessment within
each center was chosen as a clinically feasible
procedure for the trial. Moreover, not all centers were
logistically able to ascertain fresh frozen tissue for UP.
Thus, randomization of the centers for method of risk
assessment was not feasible. Accounting for individual
conditions and preferences, each centre was therefore
allowed to select the method of risk assessment for all
of their patients.
Risk assessment by clinical and pathological factors (CP)
The integrated clinical-pathological algorithm used to
select high-risk patients was derived from the St. Gallen
recommendations and the Nottingham Prognostic Index
(NPI) system Centers using CP classified all patients
younger than 35 years or with a G3 tumor, or with a
HER2 positive tumor or with progesterone
receptornegativity or vascular invasion as high-risk. In addition,
patients with G2 tumors were considered high-risk if
their tumor was 2 cm (figure 2).
Check for risk factors: age<35 yrs., G3, HER2-pos.,
PgR-neg., vascular invasion
Figure 2 Risk assessment using the clinical and pathological
algorithm (CP) adopted from St. Gallen and NPI. - all patients
G1 or G2 <2 cm were assessed as low-risk if no additional risk
factors were present. Patients showing an additional risk factor or
G2tumors 2 cm or G3 were assessed high-risk.
Risk assessment by biological factors (UP)
Centers using biological criteria classified all patients
with G3 tumors or <35 years as high-risk, and all
patients with G1 tumors as low-risk. Invasion factors
uPA/PAI-1 were determined in all G2 tumors. If either
factor was above the pre-defined cut-off, the patient was
classified as high-risk. The other clinical and
pathological factors did not influence biological risk assessment
Tissue sampling for biological risk assessment (UP) was
done either by excisional or by core-needle biopsy
(3 biopsies sent). Core-needle biopsy material has been
shown to allow reliable uPA/PAI-1 determination .
Regarding excisional biopsies, the pathologist confirmed
the diagnosis of breast cancer, excised a representative
piece of the tumor (optimum 100-300 mg) and
snapfroze it immediately in liquid nitrogen.
The centers received UP results within a maximum
duration of 10 days.
In the trial, uPA and PAI-1 concentrations were
measured in non-ionic, detergent-released, tumor-tissue
extracts (Triton X-100) using the FEMTELLE ELISA
kit #899 by American Diagnostica (cut-off values had
been previously validated for this kit ). Total protein
measurements were performed by the Bicinchoninic
acid (BCA) test (Pierce #23225). Tissue handling and
methods of measurement have been extensively
described elsewhere . Patients with an uPA
concentration of 3 ng/mg total protein and a PAI-1
concentration of 14 ng/mg total protein in the Triton X-100
tissue extract of their primary tumor have a very low
Figure 3 Risk assessment using the uPA/PAI-1 algorithm (UP)
all patients with G1 tumors were assessed low-risk, all patients with
G3 tumors or 35 years were assessed high-risk. Patients with G2
tumors were assessed high-risk if uPA and/or PAI-1 were elevated
above the validated cut-off levels.
risk of relapse and were consequently classified as
All laboratories participating in this multicenter trial
also participated in the EORTC PBG supported
international quality assurance program provided by the central
trial laboratory (FCGJ Sweep, Nijmegen, Netherlands).
The coefficient of variation showed a maximum of 12%
for both assays.
All patients received 3 cycles of FE100C every 3 weeks
consisting of 5-FU 500 mg/m2 IV, epirubicine 100 mg/
m2 IV infusion, and cyclophosphamide 500 mg/m2 IV
infusion. Prophylactic antiemetic treatment was given
according to each centers policy, no prophylactic
antibiotics or prophylactic granulocyte-colony stimulating
factors (G-CSF) were allowed but secondary prophylaxis
was allowed. Patients in the FEC arm then continued
for another 3 cycles of FEC. Patients in the docetaxel
arm continued with docetaxel 100 mg/m2 IV every 3
weeks. Oral steroids were given for 3 days. Prophylactic
G-CSF was recommended according to guidelines. Dose
modification was done for hematological or
non-hematological toxicity according to the protocol consistent
with standard recommendations (National Cancer
Radiotherapy, endocrine therapy and trastuzumab
All other adjuvant treatment was given according to
current AGO recommendations .
On-site monitoring was performed once for all centers
after the last patient was included into the trial. All
high-risk and low-risk patients are followed for 5 years.
Data is entered by the local centers into an electronic
data capture system.
Endpoints were chosen as follows:
1. For the population treated by chemotherapy:
Primary endpoint of the study is Disease-Free
Secondary endpoints are: Overall Survival (OS) and
side-effects of chemotherapy in each study arm
2. For the entire population of registered patients: DFS in each low-risk group (or in each patient group stratified for type of risk selection, CP or UP, respectively), and
proportion of node-negative breast cancer patients
grouped into each low-risk group
Sample size calculation
1. NNBC3-Europe is a randomized, multicenter,
open-label, phase III trial designed to detect - with
adequate power - a difference in efficacy between
two chemotherapy regimens in high-risk
node-negative, operable breast cancer.
2. It will also quantify the discordance/accordance of
clinical and pathological characteristics and of the
biological characteristics with regard to risk assessment in
all patients. Therefore, DFS of each low-risk group and
of each patient group classified according to type of
risk selection, respectively, will be compared as well as
the proportion of node-negative breast cancer patients
grouped into that particular low-risk group.
3. To investigate whether registered patients (low
risk patients according to their particular risk
assessment criterion) can still be discriminated with
respect to their risk of first recurrence and survival
if the other assessment criterion is used.
4. To investigate prospectively whether patients with
HER2 over-expression have a higher benefit by an
adjuvant anthracycline-taxane sequence than by a
solely anthracycline-based combination.
Ad question 1 - difference between chemotherapy regimens
The primary endpoint for the biometrical evaluation is
Assuming an event rate of 13% at 5 years follow-up in
the standard FE100C arm, a difference of 4% in the event
rate is considered to be clinically relevant.
When the sample size in each group is 1,286, an
exponential maximum-likelihood test of equality of survival
curves with a 0.050 two-sided significance level will
have 80% power to detect the difference between a
group 1 exponential parameter of 0.0023 (equivalent to
a 13% event rate at 5 years) and a group 2 exponential
parameter of 0.0016 (equivalent to a 9% event rate at 5
years) (constant hazard ratio of 1.477).
Ad question 2 - difference between risk assessment types
The primary endpoint for the biometrical evaluation is
DFS. The power was computed for the question whether
the two types of risk assessment are equivalent
regarding DSF. The statistical analysis was planned under the
condition that patient allocation to one of the two types
of risk assessment is not randomized but depends on
the policy of each center (see above). Therefore, it was
expected that the two groups formed by the risk
assessment methods are not homogeneously distributed
regarding all relevant prognostic factors.
There is no method described in the literature for an
exact power calculation for multivariate statistical
methods with event times as the endpoint and equivalence as
the main question of the analysis. As an approximation
for the power calculation, we used the known calculation
for simple proportions as the endpoint and equivalence
as the main question. In the final analysis, multivariate
methods using DFS as the endpoint will be used.
If sample sizes in the groups are 1,900 and 3,800, a
two-group large-sample normal approximation test of
proportions with a one-sided 0.010 significance level will
have 76% power to reject the null hypothesis that the
two groups are not equivalent (the difference in
proportions is 0.02 or farther from zero in the same direction)
in favor of the alternative hypothesis that the
proportions in the two groups are equivalent, assuming that
the expected difference in proportions is 0.000 and the
overall proportion of disease-free patients is 0.959.
Closure of recruitment after entering 4,149 patients
In December 2008, the steering committee decided to
close recruitment to the trial due to several reasons:
The estimated number of patients needed to
answer the chemotherapy question was definitely
The answer to the second question concerning the
difference between the risk assessment groups will
probably be answered since the sample size was
calculated rather generously due to lack of an exact
power calculation for multivariate statistical methods
with event times as the endpoint and equivalence as
the main question of the analysis (see above).
Since initially there was a rather slow inclusion of
patients, the actual recruitment fell behind the
planned recruitment and premature closure was a
Estrogen receptor status and HER2 expression are clear
predictive factors in breast cancer indicating who will
benefit from endocrine and anti-HER2 therapy. No such
factor has yet been found to predict response to
chemotherapy. So far, only some clinical and pathological
factors estimating prognosis have been identified. Since
chemotherapy is associated with side-effects and reduced
quality of life, the indications should be carefully decided.
Node-positive patients will most likely benefit from
adjuvant chemotherapy in a significant way. Node-negative
patients are a heterogeneous group where 70% of the
patients will be cured by loco-regional therapy alone.
Especially for patients with G2-tumours, there are no
good prognostic factors. Recently, numerous biological
tests have been suggested for risk estimation in such cases.
Only a few are currently recommended by international
guidelines (AGO, ASCO, National Comprehensive Cancer
Network). uPA/PAI-1 have been evaluated by a
prospective trial showing independent prognostic value even after
10 years of follow-up (Chemo N0 trial). A meta-analysis
including more than 8,000 patients validated this
independent prognostic value. This NNBC-3 Europe trial is the
confirmatory trial regarding the prognostic value of these
factors. Moreover, the important questions of optimal
chemotherapy in high-risk node-negative patients and of
identifying node-negative patients benefitting from
adjuvant taxane-containing therapy are addressed. 4,149
patients were included and the recruitment was closed in
January 2009. First results will be available in 2011 when
142 events have been observed.
The investigators are very grateful to all patients who participated in the
This manuscript is written on behalf of all the participating centers of the
NNBC 3-Europe study group.
Participating Centers: Aachen, Universittsklinikum Aachen, Frauenklinik fr
Gynkologie und Geburtshilfe; Aalen, Ostalb-Klinikum, ABC Brustzentrum,
Frauenklinik; Amberg, Klinikum St. Marien, Frauenklinik; Amberg, Oberpfalz,
Onkologische Schwerpunktpraxis; Aschaffenburg, Klinikum Aschaffenburg,
Frauenklinik; Augsburg, Klinikum Augsburg, Frauenklinik; Aurich, Ostfriesland,
Onkologische Praxis; Avignon, Institut Sainte-Catherine, France; Bergisch
Gladbach, Ev. Krankenhaus Bergisch Gladbach, Gynkologie; Berlin, DRK
Kliniken Kpenick, Frauenklinik; Bielefeld, Franziskus Hospital, Frauenklinik;
Bietigheim-Bissingen, Klinikum Ludwigsburg-Bietigheim, KH Bietigheim,
Gynkologie; Bblingen, Klinikum Sindelfingen-Bblingen/Kliniken Bblingen,
Frauenklinik; Bonn, Johanniter Krankenhaus, Gynkologie und Geburtshilfe;
Bonn, Universittsklinikum, Anstalt des ffentl. Rechts, Frauenklinik; Bordeaux,
Polyclinique Bordeaux Nord Aquitaine, France; Bottrop, Marienhospital
Bottrop gGmbH, Klinik f. Gynkologie u. Geburtshilfe; Bremen, Evangelisches
Diakonie-Krankenhaus, Frauenklinik; Bremen, Klinikum Bremen Nord,
Frauenklinik/Brustzentrum; Bremen, Onkologische Schwerpunktpraxis;
Bremerhaven, Klinikum Bremerhaven Reinkenheide, Frauenklinik; Brest Cedex,
Centre Hospitalier Universitaire de Brest/Hopital Morvan, Institute de
Cancerologie et dHematologie, France; Buchholz, Onkologische Praxis im KH
Buchholz; Celle, Allgm. Krankenhaus Celle, Frauenklinik; Chemnitz, Sachs,
Gynkologie/Onkologie, Praxis; Clermond-Ferrand, Centre Jean-Perrin, France;
Darmstadt, Klinikum Darmstadt, Frauenklinik; Deggendorf, Klinikum
Deggendorf, Abt. f. Senologie, Mammazentrum; Dortmund,
Knappschaftskrankenhaus Dortmund, Frauenklinik/Geburtshilfe; Duisburg, Ev.
Bethesda Krankenhaus, Frauenklinik II, Senologie; Dren, Rheinl, St. Marien
Hospital Dren, Gynkologisch-geburtshilfliche Abteilung; Dsseldorf,
Heinrich-Heine-Universitt Dsseldorf, Frauenklinik; Dsseldorf, Kaiserswerther
Diakonie - Florence-Nightingale-Krankenhaus, Onkologische Tagesklinik;
Ebersberg, Kreiskrankenhaus Ebersberg, Gynkologisch-onkologische
Abteilung; Erkelenz, Praxis Dr. Adhami, Facharzt fr Frauenheilkunde und
Geburtshilfe; Essen, Universittsklinikum Essen, Klinik fr Frauenheilkunde und
Geburtshilfe; Esslingen, Kliniken Esslingen, Gynkologie Onkologie,
Frauenklinik; Frankenthal, Pfalz, Stadtklinik Frankenthal, Gynkologie/
Geburtshilfe; Frankfurt, Klinikum der J. W. Goethe Universitt, Zentrum der
Frauenheilkunde und Geburtshilfe; Frankfurt/Hchst, Klinikum Frankfurt
Hchst GmbH, Klinik fr Gynkologie und Geburtshilfe; Freiburg im Breisgau,
Praxis fr Interdisziplinre Onkologie & Hmatologie, Frth, Brustzentrum
und Onkologische Gemeinschaftspraxis Frth; Gehrden,
Robert-KochKrankenhaus, Frauenklinik; Gelnhausen, Main-Kinzig-Kliniken, Frauenklinik;
Gifhorn, Kreiskrankenhaus Gifhorn, Interdisziplinres Brustzentrum; Grlitz,
Neie, Malteser Krankenhaus St. Carolus, Praxis fr Onkologie; Greifswald,
Universitt Greifswald, Klinik fr Frauenheilkunde und Geburtshilfe, alte
Frauenklinik; Grevenbroich, Kreiskrankenhaus Grevenbroich, Frauenklinik;
Halle/Saale, Krankenhaus St. Elisabeth und St. Barbara Halle, Klinik fr
Gynkologie und Geburtshilfe; Halle/Saale, Martin-Luther-Universitt Halle
Wittenberg, Universittsklinik u. Poliklinik f. Gynkologie; Hamburg, Asklepios
Klinik Barmbek, Brustzentrum; Hamburg, Krankenhaus Elim am
Diakonieklinikum Hamburg, Frauenklinik und Brustzentrum; Hamburg,
Onkologische Tagesklinik, Zentrum fr operative und onkologische
Gynkologie; Hamburg, Praxisklinik Harburger Ring; Hamburg,
Universittsklinikum Hamburg-Eppendorf, Klinik und Poliklinik fr
Gynkologie; Hanau, Klinikum Stadt Hanau, Frauenklinik; Hannover,
Gynkologisch-onkologische Praxis; Hannover, Henriettenstiftung,
Frauenklinik; Hannover, Medizinische Hochschule Hannover, Zentrum
Frauenheilkunde/Brustzentrum; Hannover, Vinzenzkrankenhaus, Gynkologie;
Heide, Westkstenklinikum Heide, Frauenklinik; Heppenheim, Bergstrae,
Kreiskrankenhaus Bergstrae, Gynkologie und Geburtshilfe; Hilden, St. Josefs
Krankenhaus Hilden, Abteilung fr Senologie/Brustzentrum; Hildesheim,
Gemeinschaftspraxis, Gynkologie; Ilsede, Frauenarztpraxis; Itzehoe, Klinikum
Itzehoe, Holsteinisches Brustzentrum; Jena, Universittsklinikum, Klinik fr
Frauenheilkunde und Geburtshilfe; Karlsruhe, Ev. Diakonissen Krankenhaus,
Frauenklinik; Karlsruhe, St. Vincentius Kliniken Karlsruhe, St. Marien - Klinik
gGmbH Fachklinik fr Frauenheilkunde und Geburtshilfe; Kassel,
Gemeinschaftspraxis am Rhnplatz, Standort Onkologie; Kassel,
Gemeinschaftspraxis, Hmatologie und Onkologie; Kassel, Klinikum Kassel
GmbH, Gynkologische Ambulanz; Kaufbeuren, Klinikum Kaufbeuren
Ostallgu, Frauenklinik; Koblenz am Rhein, Institut fr Versorgungsforschung
in der Onkologie, Praxisklinik fr Hmatologie und Onkologie; Kln, Praxis
Innere Medizin, Hmatologie; Kln, St. Elisabeth-KKH, Brustzentrum
KlnHohenlind; Kln, Unifrauenklinik Kln, Studienzentrum Gynkologie; La Roche
Sur Yon Cedex, Centre Hospitalier Departemental, Les Oudairies, France;
Landsberg a Lech, Klinikum Landsberg am Lech; Landshut,
Gemeinschaftspraxis Dres. Vehling-Kaiser/Greif, Hmatologie und
Internistische Onkologie; Landshut, Klinikum Landshut, Frauenklinik; Limburg,
St. Vincenz Krankenhaus, Frauenklinik; Limoges Cedex, CHU de Limoges/
Hopital Dupuytren, France; Lbeck, Universittsklinikum Schleswig-Holstein,
Frauenklinik; Ludwigsburg, Wrtt, Klinikum Ludwigsburg, Frauenklinik;
Magdeburg, Klinikum der Otto-v.-Guericke-Universitt, Frauenklinik;
Magdeburg, Klinikum Magdeburg gGmbH, Klinik f. Hmatologie/Onkologie;
Mainz, Katholisches Klinikum Mainz - St. Vincenz u. Elisabeth Hospital,
Frauenklinik; Mainz, Universittsmedizin Mainz, Klinik fr Gynkologie und
Frauenheilkunde; Mannheim, Universittsklinikum Mannheim, Frauenklinik;
Marktredwitz, Klinikum Fichtelgebirge, Frauenklinik; Meiningen, Klinikum
Meiningen GmbH, Frauenklinik; Memmingen, Klinikum Memmingen,
Onkologie; Meppen, Ludmillenstift Krankenhaus/Emslndisches Brustzentrum,
Gynkologie und Geburtshilfe; Minden, Klinikum Minden, Zentrum fr Innere
Medizin, Klinik fr Hmatologie/Onkologie; Montpellier cdex 5, Centre Val
dAurelle Paul Lamarque, France; Mnchen, Klinikum der Universitt
Mnchen/Klinikum Grohadern, Frauenklinik; Mnchen, Klinikum rechts der
Isar der Techn. Univ. Mnchen, Frauenklinik; Mnchen, Stdt. Klinikum
Mnchen Harlaching GmbH, Brustzentrum Sd; Mutlangen, Klinikum
Schwbisch-Gmnd, Gynkologie und Gynkolog. Onkologie;
Neubrandenburg, Dietrich-Bonhoeffer Klinikum, Frauenklinik; Neumarkt,
Klinikum Landkreis Neumarkt, Frauenklinik; Neumarkt, Praxis im Klinikum
Neumarkt; Neuss, Johanna Etienne Krankenhaus Neuss, Gynkologie;
Neuwied, Marienhaus Klinikum, St. Elisabeth Neuwied; Nordhausen, Sdharz
Krankenhaus, Frauenklinik; Nrnberg, Mittelfr, Klinikum Nrnberg Nord,
Medizinische Klinik 5, Schwerpunkt Hmatologie/Onkologie; Offenbach a.M.,
Klinikum Offenbach, Klinik fr Gynkologie und Geburtshilfe; Offenbach am
Main, Ketteler-Krankenhaus Offenbach, Abteilung fr Geburtshilfe und
Gykologie; Olpe, Biggesee, Martinus Hospital Siegen-Olpe, Brustzentrum;
Ostfildern, Paracelsus Krankenhaus Ruit, Brustzentrum; Paderborn, St.
VincenzKrankenhaus, Frauenklinik; Passau, Klinikum Passau, Gynkologische
Onkologie; Pforzheim, Krankenhaus Siloah, Gynkologie und Geburtshilfe;
Pinneberg, Onkologische Praxis Pinneberg, Innere Medizin, Hmatologie,
intern. Onkologie; Pttlingen, Knappschaftskrankenhaus Pttlingen,
Frauenklinik; Regensburg, Schwerpunktpraxis, Hmatologie, Onkologie;
Remagen, Praxis f. Innere Medizin, Hmatologie u. Onkologie; Remscheid,
Sana Klinikum, Frauenklinik; Reutlingen, Klinikum am Steinenberg,
Frauenklinik; Rheinfelden, Frauenklinik Rheinfelden Betriebs GmbH,
Geburtshilfe, Gynkologie, Brustzentrum; Rostock, Klinikum Sdstadt,
Universittsfrauenklinik; Rottweil, Kreiskrankenhaus Rottweil, Frauenklinik;
Rouen cdex 1, Centre Henri Becquerel, France; Saarlouis, DRK Krankenhaus
Saarlouis, Gynkologie; Saarlouis, St. Elisabeth-Klinik Saarlouis, Kooperatives
Brustzentrum Saarlouis; Salzwedel, Altmark-Klinikum gGmbH/Krankenhaus
Salzwedel, Klinik fr Frauenheilkunde/Brustzentrum Altmark; Schwandorf,
Gemeinschaftspraxis Gynkologie; Schweinfurt, Leopoldina-Krankenhaus der
Stadt Schweinfurt, Frauenklinik; Schwerin, Klinikum Schwerin, Frauenklinik;
Siegen, Ev. Jung-Stilling Krankenhaus, Frauenklinik; Siegen, St.
MarienKrankenhaus gGmbH, Brustzentrum Siegen/Olpe; Sigmaringen, Kliniken
Landkreis Sigmaringen GmbH, Gynkologie und Geburtshilfe; Simmern,
Hunsrck Klinik kreuznacher diakonie, Gynkologie/Geburtshilfe; Solingen,
Stdt. Klinikum Solingen, Frauenklinik; Stade, MVZ Onkologie/Hmatologie in
der Klinik; Stendal, Johanniter-Krankenhaus Genthin-Stendal, Klinik fr
Frauenheilkunde und Geburtshilfe; Stralsund, Klinikum Stralsund, Frauenklinik;
Straubing, Onkologische Schwerpunktpraxis Straubing; Suhl, Zentralklinikum
Suhl gGmbH, Frauenklinik; Tours Cedex, Centre Hospitalier Universitaire de
Tours/Hpital Bretonneau, France; Troisdorf, GOSPL - Gesellschaft fr
onkologische Studien, Hmatologie und Onkologie; Tbingen,
Universittsfrauenklinik Tbingen, Frauenklinik; Ulm, Universittsklinikum,
Frauenklinik; Vechta, St. Marienhospital Vechta, Frauenklinik/Perinatalzentrum;
Villingen-Schwenningen, Klinikum Villingen-Schwenningen, Frauenheilkunde
und Geburtshilfe; Weiden, Klinikum Weiden, Brustzentrum; Weinheim,
Krankenhaus Weinheim, Gynkologie und Geburtshilfe; Wernigerode,
HarzKlinikum GmbH, Frauenklinik; Westerstede, Ammerland Klinik GmbH,
Frauenklinik; Wiesbaden, Asklepios Paulinen Klinik, Frauenklinik; Wiesbaden,
Dr.-Horst-Schmidt-Kliniken, Klinik f. Gyn. und gyn. Onkologie; Wiesbaden, St.
Josefs-Hospital, Gynkologie und Geburtshilfe;
This trial was funded by unrestricted grants from Sanofi-Aventis Deutschland
GmbH Pfizer Pharma GmbH and Roche Pharma AG.
The trial was supported by NBL Program Martin-Luther-Universitaet
HalleWittenberg # FKZ 15/29 and 17/37.
EJK made substantial contributions to the concept and design of the study,
coordinates the study and has drafted the manuscript; MV made substantial
contributions to the concept and design of the study, coordinates laboratory
testing and has helped drafting the manuscript; MS made substantial
contributions to the concept and design of the study, coordinates the study
and critically revised the manuscript; CV coordinates the study and critically
revised the manuscript; DA coordinates the study and critically revised the
manuscript; VH coordinates the study and critically revised the manuscript;
CM made substantial contributions to the concept and design of the
statistical part of the study, coordinates the statistics of the study and
critically revised the manuscript; PD coordinates the study and critically
revised the manuscript; MS made substantial contributions to the concept
and design of the study, coordinates laboratory testing and critically revised
the manuscript; FS made substantial contributions to the concept and
design of the trial, coordinates laboratory testing and critically revised the
manuscript; GvM made substantial contributions to the concept and design
of the trial, coordinates the trial and critically revised the manuscript; PM
made substantial contributions to the concept and design of the trial,
coordinates the trial in France and critically revised the manuscript; FJ made
substantial contributions to the concept and design of the trial, coordinates
the trial and critically revised the manuscript; CT is responsible for concept
and design of the trial, is principal investigator of the trial and critically
revised the manuscript; NH is responsible for concept and design of the trial,
is principal investigator of the trial and critically revised the manuscript.
All authors read and approved the final manuscript.
The trial has received unrestricted funding for the trial from Sanofi-Aventis
Deutschland GmbH Pfizer Pharma GmbH and Roche Pharma AG. The trial
was supported by NBL Program Martin-Luther-University # FKZ 15/29. It is
possible that these organizations may gain or loose financially from the
publication of this manuscript. The authors declare that they have no other
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