Molecular Markers Predictive Response to Dose Dense Chemotherapy With Epirubicin and Docetaxel in Sequences for Locally Advanced Breast Cancer.
Phase: PHASE2
Conditions: Stage III Breast Cancer AJCC V7
Sponsor: University of Bergen

Protocol version 1.0, March 16, 2006 
Molecular markers predicting response to dose 
dense chemotherapy with epirubicin and docetaxel 
in sequence for locally advanced breast cancer 
 
 
 
Protocol summary. 
 
 
Principal Investigator 
Per E Lønning, Professor, Section of Oncology, Department of Medicine 
 
Collaborators. 
Dept of Surgery –  
 
 
 
Responsible: Turid Aas, Consultant Surgeon 
Dept of Molecular Biology –  
 
Responsible: Professor Johan Lillehaug 
Dept of Anatomy and Cellular Biology –  
Responsible: Professor Rolf Bjerkvig 
 
Participants. 
Dept of Oncology 
 
 
 
Gun Anker,    
Consultant Oncologist 
 
 
 
 
 
 
Stephanie Geisler,  
Consultant Oncologist 
 
 
 
 
 
 
Jurgen Geisler,  
Consultant Oncologist 
 
Type of Study 
Phase II, Translational research 
 
Scientific aims: Addressing factors predicting response to dose intensive epirubicin followed 
by docetaxel sequential therapy 
 
Treatment regimen: epirubicin 60 mg/m2 on a 2 weekly basis x 4 followed by docetaxel 100 
mg/m2  2-weekly x 4. 
 
Patients: Breast cancer patients below 65 years of age suffering from large (>4 cm largest 
diameter, non-inflammatory and / or N2-N3) primary breast cancer. 
 
. 
 
Clinical aim: Assessing responsiveness to this dose intensive regimen. 
 
Number of patients to be enrolled: 60 – 100 
 
 
 
 
 
 
 
 
 

 
2 
 
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.  
 
Clinical Therapy 
Our center was the first in Norway applying primary chemotherapy for locally advanced 
primary breast cancers back in 1991-92 (1). For the years to follow, primary medical therapy 
has become general clinical practice for large primary breast cancers in all Western countries. 
While there are no randomized data revealing improved long-term survival for primary 
medical treatment in comparison to ordinary adjuvant treatment (2), justification is based on 
improved local control. Thus, many inoperable tumors become fit for surgery following 
primary medical treatment. In addition, the number of local relapses, previously representing 
a major clinical problem in patients with stage III tumors, has been significantly reduced. 
Finally, the possibility of assessing tumor response to treatment allows the clinician to 
terminate non-effective therapy, implementing other drugs in exchange. 
 
While use of primary medical treatment is now considered general practice for locally 
advanced tumors in Western hospitals, many clinics also use it for primary operable tumors 
not suitable for local surgical therapy, aiming at reducing their size allowing breast 
conservation treatment. There is no standard regimen, but most treatments will include an 
anthracycline and/or taxane, similar to what is used for adjuvant therapy. 
 
Up to now, our practice has been to treat all tumors above 5 cm in diameter and/or T4 
(cutaneous or chess wall infiltration) with primary medical treatment before surgery. Many 
centers around the world now include patients with primary operable tumors suitable for 
mastectomy but not breast conservation, aiming at local down-staging to allow more limited 
surgical procedures. Notably, while such therapy in general implements chemotherapy, in 
many centers it has also become practice to treat postmenopausal women with receptor 
positive tumors with one of the novel aromatase inhibitors (3). We currently apply such 
therapy for older patients harboring receptor positive tumors. 
 
Considering chemotherapy for primary treatment, contemporary trend has been to treat these 
tumors more aggressively. High-dose therapy involving stem cell support is not advocated 
(4), 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. There are two main reasons for it: first, there are several studies now 
revealing pathological complete response to primary therapy to predict improved relapse-free 
survival (5, 6). While such a finding is consistent with the hypothesis that more aggressive 
therapy improves outcome, there are certain pitfalls, in as much as it could also mean that 
tumors harboring an intrinsic responsiveness to therapy for biological reasons may have an 
improved prognosis. Secondly, studies in the adjuvant setting have revealed implementation 
of taxanes in concert with anthracyclines to improve relapse-free- and overall survival (7, 8). 
 

 
3 
An important issue relates to drug dosing, in particular with respect to the anthracycline 
compounds. Following the seminal study by Muss et al (9) reporting optimal benefits to be 
achieved from a doxorubicin “normal dose regimen” except for patients with tumors 
overekspressing HER-2, the concept of a “threshold level” with respect to anthracycline 
dosing in HER-2 negative tumors has been confirmed by studies in the adjuvant as well as the 
primary chemotherapy setting (7, 10, 11).  
 
Based on theoretical modeling, an alternative approach, “dose-dense” therapy, has been 
advocated (12). 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 
(13). 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 (14). 
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. 
 
Implementing our first study on neoadjuvant chemotherapy , we used a “low-dose” regimen 
(weekly doxorubicin) based on current practice in advanced disease (15). For the next study, 
we implemented a regimen of conventional doses of epirubicin or paclitaxel (randomized), 
subsequently to be followed by the alternative drug in non-responders. While the results of 
that study are currently analyzed, we recognized the overall response-rate here was lower 
compared to some of the more intensive regimens currently applied. Thus, the clinical 
justification of the study outlined here is to implement a regimen resembling the recent dose 
dense regimen that revealed superiority in adjuvant disease (13) for neoadjuvant treatment of 
women with large primary breast cancers below 65 years of age. Based on clinical experience, 
we decided to use docetaxel instead of paclitaxel, although there are no randomized data 
comparing the two regimens. 
 
Rationale for regimen 
Much evidence now suggests treatment involving an anthracycline and a taxane either given 
in concert or sequentially to be optimal therapy in breast cancer. A potential exception is the 
recent unpublished study from the BCIRG, revealing a platinum compound with a taxane to 
provide similar efficacy to a taxane – anthracycline combination in HER2 positive tumors 
without amplification of the topo-II gene in concert (16, 17). Notably, in this group of patients 
the anthracycline – taxane approach was not inferior to the experimental arm, suggesting the 
two options may be of equal efficacy. Considering the fact that the number of tumors 
harboring HER-2 amplifications being Topo-II normal may account for probably 15% of the 
total population, we select not to develop a separate drug regimen for this sub-group.  . 
 
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 cyklophosphamide is to the 
effectiveness of such regimens (18), in particular not when a taxane is administered in 
sequence or concert (19).. Thus, the NSABP-group has abandoned use of 5-FU from their 
adjuvant regimen (20). Considering cyclophosphamide, this compound seems to add to the 
carcinogenetic effect of anthracyclines enhancing the risk of secondary leukemia (21), while  

 
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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 (22, 23). Considering docetaxel, the dose generally advocated for monotherapy 
is 100 mg/m2, while a dose of 75mg/m2 is recommended for combined use. (19). Thus, the 
potential exists that the dose for combined use may be sub-optimal in some patients. 
 
In the recent adjuvant study by Citron et al. (13) they evaluated a dose dense treatment with 
doxorubicin, paclitaxel and cyklophosphamide in sequence versus doxorubicin plus 
cyclophosphamide followed by paclitaxel. Each of the 2 regimens was administered at 2- or 
3-weekly intervals (2 X 2 factorial design). Total dose of each drug administered was similar 
in each arm. Here, they recorded similar relapse-free and overall survival between the 
sequential and combined regimen, but with a clear advantage for the two dose-dense 
regimens. Thus, the results from this study suggest 1) similar efficacy for sequential and 
combined treatment provided each drug is administered at similar doses, and 2) an advantage 
for dose-dense treatment. 
 
Justification for a sequential approach is further supported by studies in metastatic 
breast cancer (24-27). 
 
Scientific aims 
A major issue in breast cancer research (as well as research on other types of cancer) is to 
explore potential mechanism of drug resistance. Drug resistance is the major reason for 
therapy failure and subsequent death in cancer patients. In addition to characterizing 
molecular changes, we aim to identify and cultivate stem cells on an epithelial as well as 
mesenchymal background in culture to further characterize tumor biology with respect to 
treatment outcome. 
 
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. Apart from 
predicting sensitivity to treatment with trastuzumab, overekspression or amplification of the 
HER-2 gene was found associated with a dose-response to anthracyline therapy in breast 
cancer not recorded in HER-2 negative tumors (9, 11, 28). However, there is no evidence it 
may explain the bulk of drug resistance recorded. The reason for this differential sensitivity 
could be co-amplification of HER-2 with Topo-II (29, 30), a target for anthracycline action 
cytotoxicity. Notably, patients with tumors harboring Topo-II amplifications do not seem to 
have an inferior response to anthracyclines; indeed, there is evidence in the literature they 
may actually be more sensitive to such therapy provided administration at adequate doses (31-
33). Considering TP53, we (34) as well as others (35) have found a correlation between TP53 
mutations and lack of response to anthracycline therapy in breast cancer. However, TP53 
status alone could not fully explain sensitivity, suggesting other genes may interact as well 
(36).  
 
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 (37-41), 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 

 
5 
damage repair or apoptosis in relation to drug resistance (36). Thus, our primary aim is to 
explore potential gene disturbances based on functional hypotheses (36, 42). 
 
In an ongoing pilot study (REK 05/3013) vi aim at sampling and cultivating epithelial stem 
cells from the breast together with mesenchymal stem cells from the bone marrow. While we 
so far have been unsuccessful identifying an epithelial breast stem cell, we have been able to 
identify and cultivate mesenchymal stem cells from the bone marrow. Based on recent 
findings in the literature (43-49), we are hypothesizing these stem cells may play a vital role 
fueling growth of epithelial cells with a gene damage, suggesting they may be of critical 
importance to carcinogenesis as well as tumor progression and metastatic seeding. To take 
this approach forward, we aim at harvesting such stem cells both from the breast but from 
bone marrow in addition as part of this project. 
 
There are several arguments for choosing a primary medical treatment setting for such studies 
(50). Allowing direct assessment of tumor response to therapy, such studies provide valuable 
information from cohorts of less than 100 patients. In contrast, adjuvant studies, having 
relapse-free or overall survival as endpoints, may need enrollment of more than 1000 patients. 
Considering metastatic disease, results from ongoing studies by our team suggest a biological 
heterogeneity between different metastatic deposits from the same tumor making such a 
model inconvenient (S. Knappskog et al; Unpubl results).   
 
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 (50). 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. The reason for 
this is two-fold. First, anthracyclines are shown to work to a similar extent in HER-2 positive 
tumors compared to HER-2 negative ones, provided the doses administered are in the range of 
100mg/m2 on a 3-weekly basis (11).. Considering the regimen provided here, 60 mg/m2 on a 
2-weekly basis should approach the 100 mg/m2 3-weekly regimen. Second, administration of 
an anthracycline and trastuzumab in concert may enhance cardiotoxicity. 
 
Notably, while a  Finnish study has shown trastuzumab administered for 9 weeks to 
provide significant benefits in early breast cancer (51), this study enrolled a limited number of 
patients, and the results need further confirmation before being implemented in the routine 

 
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setting. Currently, general recommendation is to extend trastuzumab treatment on a 3-weekly 
basis for 51 weeks (52, 53), which is what will be done in this study. 
 
HER-2 status will be evaluated based on common routine criteria in our hospital 
(immunostaining with FISH /CISH when indicated). 
 
 
Inclusion Criteria. 
- 
Primary breast cancer >4cm in diameter and / or lymph node status N2-3. 
- 
Age 65 years or younger 
- 
“Limited” distant metastases allowed, but patients with massive distant metastases 
should be excluded 
- 
Willing to participate in the study 
 
 
Exclusion criteria. 
- 
Known allergy toward any of the cytotoxic compounds to be administered 
(epirubicin and doxorubicin) 
- 
Liver enzymes > 2 times upper normal limit or bilirubin > 3 times upper normal 
limit 
- 
Other medical conditions making them unfit for dose-dense therapy 
- 
Cardiac insufficiency; for patients not to receive trastuzumab, decision whether to 
exclude such patients will be at the physicians discretion. Considering patients 
with HER-2 positive tumors who should have trastuzumab, exclusion criteria will 
be according to the NBCG (Norwegian Breast Cancer Group) general guidelines 
(www.NBCG.net). 
 
 
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 (54). 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. By using such a criterion, it should mean that a tumor with a largest diameter 
of say 6 cm is allowed to expand to a size of 7.2 cm before considered “non-responsive”. 
Based on experience in our clinic, we believe the definition of progressive disease as an 

 
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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. 
 
Logistically, clinical response evaluation will be based on a log scheme recording 
tumor diameters with documentation of which physician performed the measurement. For 
correct assessment, measurements performed by the same doctor on different occasions are to 
be compared. 
 
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. This 
biopsy should contain between 150 – 500 mg of tissue, to be split into samples 
snap-frozen (liquid nitrogen) in the theatre, with a single piece for paraffin-
fixation. In addition, a small sample will be brought to the laboratory for stem cell 
culturing. 
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. 
Of particular interest is to explore changes in protein phosphorylation status (like 
for p53) 
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 endopoint 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 
 
 
Treatment regimen. 

 
8 
Epirubicin is administered as 60 mg/m2, while docetaxel is given as 100 mg/m2. Both 
regimens are administered on a 2-weekly basis. Each drug is administered as an i.v. infusion 
with anti-emetics (and for docetaxel glucocorticoids) in accordance to general treatment 
administration in our Department. 
 
Due to bone marrow suppression, each patient will be treated with pegfilgrastim 
(Neulasta) 6 mg injection 24 hours after each chemotherapy cycle. 
 
After each cycle, a blood sample is obtained on day 7-10 to assess for bone marrow 
toxicity. 
 
 
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.  
 
Previously, we identified mutations in the TP53 but also the CHEK2 gene to be associated 
with lack of responsiveness to anthracycline as well as mitomycin therapy in primary breast 
cancer (34, 55, 56). Current, we are analyzing other genes for mutations but also promoter 
hypermethylations aiming at identifying not individual genetic events but rather disturbances 
in “functional pathways” controlling drug sensitivity (36). Considering we may start the 
laboratory part of our program in 4-5 years from now, decisions which genes / molecular 
factors are of most relevance need to be based on contemporary results achived from our own 
research as well as reports from the literature. As such, we believe studies like this need an 
“open frame” with respect to individual molecular parameters and also which laboratory 
techniques are of most relevance. 
 
 
Considering laboratory experience, our team has the knowledge and experience with common 
methods in molecular biology including gene sequencing, promoter methylation status, 
analysis for splice variants and so on (Head of activity; Professor Johan Lillehaug).. In 
addition, we have collaborations with other teams working with micro-array techniques. With 
respect to proteomics, we aim at establishing collaborations with other Norwegian research 
teams with that type of knowledge here in Bergen or at other Norwegian institutions. 
 
While the stem cell research in breast cancer is at an early stage, our team has successfully 
identified mesenchymal bone marrow stem cells and is well on way characterizing their 
biology on a general basis. Thus, we have in hand most of the techniques needed for this 
research activity (Head; Professor Rolf Bjerkvig). 
 

 
9 
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. 
 
 
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