Trial Information

Molecular Markers Predictive Response to Dose Dense Chemotherapy With Epirubicin and Docetaxel in Sequences for Locally Advanced Breast Cancer.

Aim The scientific aim of this study is to explore mechanisms of resistance to chemotherapy
in breast cancer. To do so, we explore molecular parameters predicting response to
chemotherapy administered prior to local therapy in large, primary breast cancers.

Background Neoadjuvant (primary medical) therapy has got wide acceptance as primary therapy
in breast cancer. In addition, this treatment provides an optimal setting studying the
mechanisms of drug resistance in human cancers.

Considering chemotherapy for primary breast cancer treatment, contemporary trend has been to
treat these tumors more aggressively. High-dose therapy involving stem cell support is not
advocated, as this has not been shown to improve long-term survival in early breast cancer.
However, the attitude in general has been toward a more aggressive approach within the frame
of "conventional" therapy.

Based on theoretical modeling, an alternative approach, "dose-dense" therapy, has been
advocated. Recently, that concept was brought to the test in two adjuvant trials. Thus,
Citron et al applying doxorubicin, paclitaxel and cyclofosfamide revealed an improved
outcome for dose-dense (2-weekly) administration compared to regular 3-weekly scheduling. In
contrast, the German GEPARDUO study reported doxorubicin plus cyclophosphamide and
docetaxel, given in sequence on a 3-weekly basis (8 cycles), to be superior to doxorubicin
and docetaxel given in concert on a 2-weekly basis for 4 cycles. However, the doses
administered (doxorubicin 50 versus 60 mg/m2; docetaxel 75 versus 100 mg/m2) was unequal,
meaning total drug dose exposure differed between the two treatment arms. While more data
are warranted, a reasonable interpretation of available data suggest sequential
administration of different compounds in a dose-density approach to be a suitable regimen
provided adequate total doses are given.

Rationale for regimen Considering anthracyclines, most regimens today combine either
epirubicin or doxorubicin in concert with 5-fluorouracil and cyclophosphamide. However,
based on the evidence in the literature, it is not clear what the contribution of 5-FU or
cyclophosphamide is to the effectiveness of such regimens, in particular not when a taxane
is administered in sequence or concert. Thus, the NSABP-group has abandoned use of 5-FU from
their adjuvant regimen. Considering cyclophosphamide, this compound seems to add to the
carcinogenetic effect of anthracyclines enhancing the risk of secondary leukemia, while the
contribution to the antitumor efficacy of the regimen remains uncertain. The taxanes are
known to have significant antitumor effects in breast cancer when administered as
monotherapy. Considering docetaxel, the dose generally advocated for monotherapy is 100
mg/m2, while a dose of 75mg/m2 is recommended for combined use. Thus, the potential exists
that the dose for combined use may be sub-optimal in some patients.

Justification for a sequential approach is further supported by studies in metastatic breast
cancer.

Scientific aims While many mechanisms of resistance have been identified in experimental
systems, we have little knowledge what may be the cause of drug resistance in human cancers.
The factors so far identified that have been found associated with resistance to
chemotherapy in breast cancer are amplification of HER-2 / Topo-II and mutations affecting
TP53.

Recently, several groups have explored responsiveness to chemotherapy using mRNA
microarrays. While these studies consistently identified different gene profiles correlating
to responsiveness to different regimens, the predictive value was too low for clinical
application. Moreover, the results did not add to our understanding of the biological
mechanisms causing drug resistance. In contrast, studies exploring different forms of cancer
have started to reveal specific gene alterations, in particular affecting pathways of DNA
damage repair or apoptosis in relation to drug resistance. Thus, our primary aim is to
explore potential gene disturbances based on functional hypotheses.

A major issue identifying mechanisms of drug resistance is to explore different compounds on
a monodrug basis. Clinically, the major reason for combining or administering sequentially
an anthracycline and a taxane is the efficacy of each compound combined with a significant
lack of cross-resistance. Administered as a combined schedule, this limits the possibility
of identifying the mechanisms of resistance / sensitivity to each compound individually.
Provided that a sequential regimen provides the same efficacy as a combined schedule, such
an approach is justified ethically. Thus, for a patient given 2 and 3 drugs in concert, the
potential may be they respond to one drug only, while having the toxicity of the other
compounds. In case of non-responsiveness to a single compound, this may be exchanged for an
alternative treatment option. This contrasts the possibilities provided in the adjuvant
setting. In adjuvant therapy, there is no way by which we may assess the response to
individual drugs; thus the different compounds have to administer in concert.

Based on what is said above, we consider sequential dose-dense treatment using epirubicin
and docetaxel sequentially to be a feasible treatment option.

Treatment regimen Each patient will receive 4 cycles of epirubicin 60 mg/m2 given at 2
weekly intervals together with G-CSF. Thereafter, docetaxel 100 mg/ m2 will be given at 2
weekly intervals for 4 cycles. Patients revealing positivity for HER2 status will have
trastuzumab (Herceptin) implemented together with docetaxel but not during anthracycline
treatment.

Staging at baseline

After given informed consent, patients will be staged as follows:

- MRI of both breasts

- Chest X-ray

- Liver ultrasound (in case metastases are suspected or verified; to be followed by CT
and / or MRI confirmation)

- Skeletal scintigram. Any positive findings to be confirmed by subsequent X-ray / CT and
/ or MRI

- ECG

- LVEF (Left Ventricular Ejection Fraction)

Response evaluation Response will be evaluated based on clinical examination and MRI, each
assessment to be done separately.

Clinical examination will be performed prior to commencing therapy (before surgical biopsy)
and subsequently at 4, 6 and 8 weeks on therapy. Response will be classified according to
the common "RECIST" criteria. An important exception is to be made. As argued in a previous
protocol, we consider the RECIST definition of "progressive disease" as a 20% increase in
the sum of the largest tumor diameters to be too liberal with respect to large primary
tumors. Based on experience in our clinic, we believe the definition of progressive disease
as an increase of > 25% in the product of the largest tumor diameter and its perpendicular
(the previous common UICC criteria) to be a more suitable definition, protecting patients
from undergoing deterioration of their clinical condition. This is in accordance to our
previous experience.

In case of "progressive disease" at any stage during epirubicin treatment, the patient will
terminate epirubicin immediately and go ahead with docetaxel treatment. In case of
progressive disease on docetaxel treatment, further therapy is left to the physician's
discretion.

Considering MRI assessment, this should be performed prior to commencing therapy, in the
interval following the 4th cycle of epirubicin (prior to commencing docetaxel) and after the
4th cycle of docetaxel, prior to surgery.

Tissue sampling

1. Each patient will undergo a surgical biopsy prior to commencing treatment.

2. A Tru-cut biopsy should be obtained 24 hours after administration of the first
epirubicin cycle to assess "immediate" alterations in response to cytotoxic damage.

3. Immediately prior to commencing treatment with docetaxel, a third sample (this time a
Tru-Cut biopsy) is obtained for snap-freezing.

4. A Tru-cut biopsy should be obtained 24 hours after administration of the first
docetaxel cycle to assess "immediate" alterations in response to cytotoxic damage.

5. Finally, tumor tissue is collected and snap-frozen at surgery following docetaxel
therapy.

Bone marrow aspiration A single unilateral bone marrow aspiration is performed prior to
commencing chemotherapy treatment.

Study Endpoint

- Primary endpoint is to correlate molecular parameters to objective response to each of
the 2 regimens applied.

- Secondary endpoint are

: - to correlate molecular parameters to relapse-free and overall survival

- to identify and explore characteristics of epithelial and mesenchymal stem cells
isolated in tumor tissue and bone marrow

Surgery While many centers practice breast conservative surgery for tumors successfully
downstaged by primary medical treatment, in general we have applied a conservative approach,
advocating mastectomy. However, downstaging for limited surgery is not a primary or
secondary endpoint of this study. In general, we will advocate mastectomy also for patients
with a clinical complete response. However, this practice may change based on contemporary
results from other centers, and the protocol allow limited surgery at the physicians
discretion in individual patients.

Laboratory Investigations The area of molecular biology is rapidly developing with respect
to biological knowledge as well as technical analytical methods. Thus, it is not possible to
predict upfront which genes may be of particular interest in 5 years from know; neither is
it possible to foresee completely which laboratory methods will be available. Our aim is to
explore potential genetic alterations explaining the mechanisms of drug resistance. While it
is not possible to predict in detail, the aim of the study and all analysis to be conducted
should aim at this major goal.

Number of patients to be enrolled The study is an exploratory translational study. Thus, we
do not know the number of patients expected to achieve a clinical or pathological complete
response. Currently, on average 20 patients are referred to our Department for a diagnosis
of locally advanced breast cancer on an annual basis; including patients with tumors
measuring between 4 and 5 cm in diameter, we estimate the total number may be somewhere
between 30 and 40 patients per year. From this cohort, we estimate an average number of 20
patients to be enrolled in the study on an annual basis. Our aim is to recruit 100 patients
with a minimum of 60.

Publication Our aim is to publish the results from this study in peer-reviewed international
journals with contributors from the clinic and laboratory as authors.

References; from the Trial Administrator upon request.