Conditions: Metastatic Breast Cancer Liver Metastases
Keywords: breast cancer liver metastases intrahepatic chemotherapy
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 38
Healthy volunteers: no

Brief summary:
This is a phase II trial evaluating intra-hepatic chemotherapy with oxaliplatin every second week in combination with systemic capecitabine and in patients with a HER2-positive tumour in combination with trastuzumab (Herceptin®) in patient with non-resectable liver metastases from breast cancer.

Only patients with limited extrahepatic disease are included.

Detailed description:

Arms:
arm 1: None

Interventions: intervention 1: oxaliplatin, capecitabine, trastuzumab
Intervention descriptions: intervention 1: Oxaliplatin intrahepatic capecitabine and trastuzumab systemic

Protocol document:
Prot_SAP_000.pdf: 
Intrahepatic chemotherapy in cancer mammae MA 0918+trastuzumab, protocol version 8, 20121108 _translated 
The trial has been approved by the Danish Medicines Agency Eudract No.: 2009-014821-17 and  
Research Ethics Committee No.: H-1-2009-088 Capital Region of Denmark  
1/66 
 
MA 0918 
 
Full Title: 
Intra-hepatic chemotherapy with oxaliplatin every 2 
weeks combined with systemic capecitabine and in 
patients with HER2-positive tumors additionally with 
trastuzumab (Herceptin®) in patients with non-
resectable liver metastases from breast cancer. 
 
A Phase II study of patients with limited extrahepatic 
disease. 
 
Brief title:  
Intra-hepatic Chemotherapy in Patients With Liver Metastases From Breast Cancer 
and Limited Extrahepatic Disease 
 
EudraCT: 2009-014821-17 
ClinicalTrials.gov: NCT01387295 
Protocol version 8  
dated  Nov, 8th 2012 
 
Protocol Committee and Clinical Investigators:  
Consultant, Dr. med. Dorte Nielsen, Department of Oncology 1) 
Consultant Benny Vittrup Jensen, Department of Oncology 1) 
Senior Project Nurse Birgitte Christiansen1) 
Consultant Bjørn Skjoldby, Surgical Section, Gastrounit 1) 
Consultant Henrik Nørgaard, Department of Radiology 1) 
Consultant, Professor, Dr. med. Julia S. Johansen, Department of Oncology1) 
Head of the Biobank, PhD. Estrid Høgdall, Department of Pathology1) 
Consultant Eva Balslev, Department of Pathology1) 
Consultant, PhD, Ann Knoop, Department of Oncology 2) 
Professor, Consultant, PhD, Per Pfeiffer, Department of Oncology 2) 
1) Herlev Hospital, 2730 Herlev, Denmark.   
2) Odense University Hospital, 5000 Odense, Denmark.  


Intrahepatic chemotherapy in cancer mammae MA 0918+trastuzumab, protocol version 8, 20121108 _translated 
The trial has been approved by the Danish Medicines Agency Eudract No.: 2009-014821-17 and  
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Coordinating Investigator:  
Consultant Dorte Nielsen, Department of Oncology, Herlev Hospital  
Sponsor: 
Consultant Dorte Nielsen, Department of Oncology, Herlev Hospital  
"Trial Office" 
Clinical Research Unit  
Department of Oncology 
Herlev Hospital 
Monitor: 
The University of Copenhagen's GCP unit is the coordinating GCP unit. Monitoring at the 
Department of Oncology, Aarhus and Odense will take place at the local GCP units.  


Intrahepatic chemotherapy in cancer mammae MA 0918+trastuzumab, protocol version 8, 20121108 _translated 
The trial has been approved by the Danish Medicines Agency Eudract No.: 2009-014821-17 and  
Research Ethics Committee No.: H-1-2009-088 Capital Region of Denmark  
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Signature page 
The study will be conducted in accordance with ICH-GCP (E6). All personnel responsible 
for the design and conduct of the study have undergone the GCP training. 
The signature below approves the Protocol and its Annexes and is intended to ensure that 
the trial is conducted in accordance with the Protocol. 
Having read and understood the requirements and obligations contained in the Protocol, I 
agree to conduct the clinical trial in accordance with national laws and international 
guidelines for good clinical practice. 
 
Sponsor 
Dorte Nielsen, Consultant, Dr. med. 
Department of Oncology  
Herlev Hospital 
Herlev Ringvej 75 
2730 Herlev 
Email: dornie01@heh.regionh.dk 
Tel: 44884000 82344----------------------------------------------- 
 
 
 
 
 
 
Date Signature 
 
 
Principal investigator 
Dorte Nielsen, Consultant, Dr. med. 
Department of Oncology  
Herlev Hospital 
Herlev Ringvej 75 
2730 Herlev 
Email: dornie01@heh.regionh.dk 
Tel: 44884000 82344----------------------------------------------- 
 
 
 
 
 
 
Date Signature 
 
 
Investigator 
xxxxxxxxxxxx 
Department of Oncology 
xxxxxxxxxx 
xxxxxxxxxxxxxx 
xxxxxxxxxxxxx 
Tel. xxxxxxxxxx----------------------------------------------- 
 
 
 
 
 
 
Date Signature  
 
 
 


Intrahepatic chemotherapy in cancer mammae MA 0918+trastuzumab, protocol version 8, 20121108 _translated 
The trial has been approved by the Danish Medicines Agency Eudract No.: 2009-014821-17 and  
Research Ethics Committee No.: H-1-2009-088 Capital Region of Denmark  
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1 
Protocol synopsis 
6 
1. Introduction 
9 
1.1 
Background 
9 
1.2 
Intrahepatic treatment 
9 
2 
Chemotherapy 
11 
2.1 
Capecitabine (Xeloda®) 
11 
2.2 
Oxaliplatin (Eloxatin®) 
11 
2.3 
Oxaliplatin intrahpatic 
12 
2.4 
Oxaliplatin intrahepatic in combination with capecitabine 
12 
2.5 
Trastuzumab 
13 
2.5.1 
Overview of clinical experience with continuation of trastuzumab after 
progression in metastatic breast cancer 
14 
2.5.2 
Overview of clinical experience with combination therapy with trastuzumab 
and capecitabine 
14 
2.5.3 
Administration of the drug 
15 
2.6 
DSM-TACE" (transarterial chemoembolisation) 
15 
2.7 
Local treatment 
17 
2.8 
HAI – XELOX 
17 
3 
Pharmacokinetics 
19 
4 
Tumor biology 
19 
5 
Endpoints 
20 
5.1 
Clinical endpoints 
20 
5.1.1 
Primary endpoint 
20 
5.2 
Paraclinical endpoints 
20 
6 
Trial design 
20 
6.1 
Expected number of patients 
21 
6.2 
Duration of treatment 
21 
7 
Selection of patients 
21 
7.1 
Inclusion criteria: 
21 
7.2 
Exclusion criteria: 
22 
8 
Treatment regimens and procedure 
23 
8.1 
Feasibility studies 
23 
8.1.1 
Caranatomy. 
23 
8.1.2 
Application of arterial Port A Cath. (HAI catheter) 
23 
8.1.3 
Side effects, as well as observation. 
24 
8.2 
HAI-XELOX 
24 
8.2.1 
Oxaliplatin 
24 
8.2.2 
Capecitabine (Xeloda®) 
24 
8.2.3 
EmboCeptS 
24 
8.3 
Visit schedule and observations 
24 
8.4 
Registration 
25 
8.4.1 
Before each treatment series 
26 
8.4.2 
Blood samples for the determination of biomarkers 
26 
8.5 
Assessment of response 
26 
8.5.1 
Definition of response 
26 
8.6 
Other concomitant medication 
26 
8.6.1 
Other medication 
27 
8.7 
Follow-up 
27 
9 
Storage, mixing and dispensing of investigational products 
27 
10 
Dose reductions 
27 
10.1 
Treatment delay 
28 


Intrahepatic chemotherapy in cancer mammae MA 0918+trastuzumab, protocol version 8, 20121108 _translated 
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10.2 
Dose reduction due to hematological toxicity 
28 
10.3 
Liver function 
29 
10.4 
Dose reduction in oxaliplatin related sensory polyneuropathy (sPNP) 
29 
10.5 
Hypersensitivity, allergies or laryngopharyngeal dysesthesia (LPD) 
30 
10.6 
Dose reduction for capecitabine 
30 
10.7 
Non-hematological toxicity other than sensory polyneuropathy 
31 
10.7.1 
Adjustments and postponements of trastuzumab dose due to toxicity 
31 
10.7.2 
Cardiac dysfunction 
32 
10.7.3 
Dose interruption due to reactions associated with infusion 
33 
10.8 
Treatment of complications 
34 
10.9 
Duration of treatment 
34 
10.9.1 
Causes of discontinuation of treatment. 
34 
10.10 Discontinuation of the study before scheduled time 
35 
11 
Patient safety 
35 
11.1 
Adverse events 
35 
11.1.1 
Definition 
35 
11.1.2 
NOT SAE 
36 
11.1.3 
Suspected, unexpected, serious adverse reaction – SUSAR 
37 
11.1.4 
Adverse Event Registration 
37 
11.1.5 
Reporting of serious adverse events (SAE/SAR/SUSAR) 
38 
11.2 
Early discontinuation of the study 
38 
12 
Data analysis and statistics 
38 
12.1 
Definition of populations 
38 
12.2 
Efficacy 
38 
12.2.1 
Primary analysis (response rate) 
39 
12.2.2 
Secondary analysis (PFS) 
39 
12.3 
Sample size 
39 
12.4 
Statistical methods 
39 
12.5 
Side effects 
40 
12.6 
Data management and archiving 
40 
13 
Ethics 
40 
13.1 
Ethical considerations 
40 
13.2 
Patient safety, ethics 
41 
14 
Subject identification 
42 
15 
Informed consent 
42 
16 
Insurance 
42 
17 
Timescale 
42 
18 
Sponsorship and finances 
43 
19 
Publication 
43 
20 
References 
44 
Appendix 1 
48 
APPENDIX 2 
49 
References 
57 


Intrahepatic chemotherapy in cancer mammae MA 0918+trastuzumab, protocol version 8, 20121108 _translated 
The trial has been approved by the Danish Medicines Agency Eudract No.: 2009-014821-17 and  
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1  Protocol synopsis 
Title  
 
Intrahepatic chemotherapy with oxaliplatin every 2 weeks 
combined with systemic capecitabine and in patients with 
HER2-positive tumors, in addition, trastuzumab 
(Herceptin®) in patients with non-resectable liver 
metastases from breast cancer 
Studio design 
Phase II study 
Primary objective 
Tumor response in the liver according to RECIST criteria 
version 1.1 
Secondary objectives 
a) Progression free survival (Intrahepatic)  
b) Progression free survival (Extrahepatic) 
c) Number of patients who can have the tumor burden 
reduced so much that they become suitable for local 
treatment (radiofrequency ablation (RF)) 
d) Survival 
(e) Toxicity 
Paraclinical endpoints 
Correlation between known markers in tissues (hormone 
receptor status, HER2 and topo IIα) and project biomarkers 
in blood and tissues in the form of gene profiles (e.g. 
mRNA and microRNA, SNP array profiles), proteins (e.g. 
p53, P1NP, P3NP, TIMP-1, IL-6, YKL-40, EGFR and 
VEGF), as well as metabolites and clinical parameters 
(response, time to progression, survival) 
Patient population 
 
 
Histologically or cytologically proven breast 
adenocarcinoma  
Liver metastases where local treatment (surgery/RF) is not 
indicated 
Patients with bone metastases or lymphnode metastases, 
without progression in the extrahepatic sites within 6 
months and whose tumor burden is predominantly hepatic 
(as assessed by PET-CT scan) 
Measurable disease according to RECIST criteria version 
1.1 
No progression on treatment with capecitabine 
Age 18 years or older 
Performance status <2 
Life expectancy over 3 months. 
Previous treatment with taxane either adjuvant or for 
metastatic disease. If treatment has been given for 
metastatic disease, treatment should have continued to the 
maximum response or cessation due to toxicity 


Intrahepatic chemotherapy in cancer mammae MA 0918+trastuzumab, protocol version 8, 20121108 _translated 
The trial has been approved by the Danish Medicines Agency Eudract No.: 2009-014821-17 and  
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Number of patients  
Up to 50 evaluable patients receiving a permanent catheter 
and up to 50 evaluable patients undergoing transarterial 
chemoembolism with Degradable Starch Microsperes 
(DSM-TACE) recruited over 3 years period. Initiated 
October 2009. Last patient included October 2012.  
 
Participating centers 
Herlev Hospital, Odense University Hospital 
Other departments in Denmark may refer to the 
participating centres for consideration to be treated within 
the trial. 
Treatment: 
 
 
Oxaliplatin 85 mg/m2 (max. 2 m2) intrahepatically over 1/2 
hour every 2 weeks  
 
Concomitant systemic treatment: capecitabine 1300 mg/m2 
divided into two daily doses continuously (max, 2 m2).  
Patients with HER-2 positive tumours: Trastuzumab 
(Herceptin®) 8mg/kg day 1 followed by 6 mg/kg every 3 
weeks. 
 
Due to anatomical conditions, in 1/2 of the patients it is not 
possible to apply a permanent catheter, in these patients 
DSM-TACE is performed, where chemotherapeutic 
followed by EmboCeptS is injicated in A.hepatica. 
 
Treatment given: 
Oxaliplatin intrahepatic with 70-85 mg/m2 over 10-30 
minutes followed by 0-7 ½ ml EmboCeptS (until flow stop) 
every 4 weeks. 
Oxaliplatin intravenously with 85 mg/m2 over 1/2 hour every 
4 weeks (alternating) 
Oral capecitabine 1300 mg/m2 daily divided into 2 doses 
continuously. 
In HER2-positive tumour, in combination with trastuzumab 
(Herceptin ®) 8 mg/kg day 1 followed by 6 mg/kg every 3 
weeks 
For 4 patients, pharmacokinetics are carried out, taking 2 
ml of blood on time 0, 15, 30, 60 min. and 2, 2½, 3, 4 and 6 
hours for determination of oxaliplatin concentration.  
  
 
Local treatment 
At each evaluation every 2 months, it is assessed whether 
patients are suitable for radiofrequency (RF) - ablation.  
 
Treatment after 
intrahepatic therapy 
At investigators discretion: Capecitabine 2000 mg/m2 for 
14 days every 3 weeks and if applicable with trastuzumab 
(Herceptin®) 6 mg/kg every 3 weeks in patients with HER-2 
positive tumours. Treatment is given for progression 
and/or unacceptable toxicity. 
 


Intrahepatic chemotherapy in cancer mammae MA 0918+trastuzumab, protocol version 8, 20121108 _translated 
The trial has been approved by the Danish Medicines Agency Eudract No.: 2009-014821-17 and  
Research Ethics Committee No.: H-1-2009-088 Capital Region of Denmark  
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EVALUATION 
Baseline 
 
 
 
 
 
 
 
 
 
 
 
 
Before each series 
 
 
 
 
 
After every 4th series in 
connection with treatment
then every 3 months 
Clinical evaluation, CT scan (possibly PET). If there are 
extrahepatic focuses in the bones, supplemented by MRI 
scans/x-ray (depending on the assessment at baseline). 
Hematological / biochemical status (hemoglobin, leucocyte 
count, neutrophil count, platelet count, sodium, potassium, 
creatinine, ASAT, LDH, alkaline phosphatase, billirubin),  
Project blood samples for the determination of biomarkers 
as above indicated in blood and described in Appendix 2. 
Tissue sample from the primary tumor is stored, and liver 
metastasis and possibly other metastases 
 
A biobank of blood and tissue from the patients is 
established. 
 
Hemoglobin, leucocyte count, neutrophil count, platelet 
count, sodium, potassium, creatinine, ASAT, LDH, alkaline 
phosphatase and bilirubin. 
 
Evaluation of side effects before each series. 
 
Tumor evaluation after every 4th series (every 7 weeks) 
according to RECIST criteria version 1.1 in during 
intrahepatic treatment thereafter every 3 months. 
 
At the time of each evaluation, project blood samples are 
taken to determine biomarkers as indicated in blood and 
described in Appendix 2. If biopsy is taken, tissue is stored 
from metastases. 
Disse samples are stored in the biobank. 
 
Duration of treatment 
 
Until disease progression 
Until unacceptable toxicity 
Until the patient wants the treatment discontinued 
A maximum of 12 series of intrahepatic treatment is given  
 
 
 


Intrahepatic chemotherapy in cancer mammae MA 0918+trastuzumab, protocol version 8, 20121108 _translated 
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1. Introduction 
 
1.1 Background 
 
Breast cancer is the most common cancer in women. The frequency has increased 
steadily in the last 50 years. In Europe, one in ten women will get breast cancer during 
their lives. Despite advances in diagnosis and treatment, breast cancer is the second 
leading cause of death in women. In the EU, around 179,000 new patients are diagnosed 
each year (28% of all cancers in women), of which around 74,000 patients die (15% of all 
deaths are related to cancer). The median survival rate for patients with metastasizing 
breast cancer (MBC) is 17-20 months, and the 5-year survival rate is about 15%. Patients 
in which the disease is confined to the bones may experience a more indolent course of 
the disease and have a greater chance of survival over a 5-year period (30-40%). Patients 
with visceral disease, especially liver metastases, have very poor prognosis and a median 
survival of 8-10 months. First-line treatment of metastasizing disease includes endocrine 
therapy or chemotherapy for hormone receptor positive tumor depending on localization of 
metastases, and chemotherapy for hormone receptor negative or endocrine non-
responding disease. Patients who have received adjuvant chemotherapy generally 
respond worse to first-line treatment in advanced disease, and the response to 
chemotherapy continues to decline with second and third-line therapy, both in terms of 
response rate and time to disease progression.  
 
 
1.2 Intrahepatic treatment 
 
Several Phase III multicenter trials have been published regarding hepatic arterial 
chemotherapy in patients with liver metastases from colorectal cancer. These articles 
describe higher response rates with intraarterial than with intravenous therapy. However, 
no difference in median survival has been described, which to some extent may be due to 
a high complication rate of intraarterial therapy. In contrast, Kemeny et al. (1999) showed 
a low complication rate with promising median survival compared to established 
chemotherapy regimens. Besides, Ricke et al. (2004) shown very low complication rate by 
the use of "a minimally invasive hepatic arterial port" established via arteria subclavia or on 
the thigh. In a subsequent report, Vogl et al. (2007) describe the efficacy of intraarterial 
chemotherapy with a combination of gemcitabine and mitomycin C in 12 patients with 
colorectal cancer and 12 patients with breast cancer. They observed 5 partial response 


Intrahepatic chemotherapy in cancer mammae MA 0918+trastuzumab, protocol version 8, 20121108 _translated 
The trial has been approved by the Danish Medicines Agency Eudract No.: 2009-014821-17 and  
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(PR) and 3 cases of stable disease (SD) in patients with colorectal cancer, one case with 
complete response (CR), 4 PR, and 6 SD in patients with breast cancer. The median 
survival was 10 months for patients with colorectal cancer and 11 months for patients with 
breast cancer. The authors concluded that treatment was safe with acceptable tumor 
control. Recently, four studies with promising survival data in colorectal cancer have been 
published. The studies suggest that combined locoregional and systemic chemotherapy 
may be a step towards improved survival in the treatment of metastatic colorectal cancer. 
If a patient with breast cancer develops liver metastases, all future treatment is considered 
palliative. Local tumor control in the form of hepatic resection and "radiofrequency" (RF) 
remains controversial treatment options, and there is no general consensus on whether 
patients benefit from these procedures. However, several authors have reported median 
survival of more than 30 months after such treatment approaches. These have been small 
patient populations that were probably highly selected. Hepatic intraarterial chemotherapy 
has so far only been very sparsely studied for the treatment of liver metastases in breast 
cancer. Thus, in most cases, only "case stories" (often in the media) have been reported. 
Only one Phase I/II trial has been reported in which a combination of adriamycin and 5-
fluorouracil was used (first and second line treatment, a total of 28 patients (26 evaluable), 
11 also had extrahepatic disease). The overall response rate was 63% with a remarkably 
long median survival of 25 months, in contrast to recent studies that reported a median 
survival of around 12 months. However, in some studies, there seems to be a trend 
towards increased survival for patients who have undergone regional therapy. To our 
knowledge, only 12 cases have been published where patients have been treated  with 
both intra- and extrahepatic disease (there are no independent reports for these patients)..  
Patients with bone and lymphatic metastases from breast cancer often have an indolent 
course, while patients with lever metastases have an exceedingly poor prognosis. Some of 
the latter patients could potentially benefit from intrahepatic treatment. 
In addition to the above, a retrospective report of 217 patients with liver metastases from c. 
mammae has been published as an abstract (it is not clear whether the patients had 
extrahepatic disease). Patients received mitomycin C alone, gemcitabine alone, or a 
combination of the two drugs, as well as embolization with lipiodol and starch 
microspheres. PR in the liver was achieved in 13%, SD in 50% and progressive disease in 
36%. One-year survival was 68% and 2-year survival 43%. Today, the treatment is offered 
in Germany, and many patients have a great desire to be able to receive the treatment in 


Intrahepatic chemotherapy in cancer mammae MA 0918+trastuzumab, protocol version 8, 20121108 _translated 
The trial has been approved by the Danish Medicines Agency Eudract No.: 2009-014821-17 and  
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Denmark. The protocol has been approved by the National Committee for Experimental 
Cancer Treatment.    
  
2 Chemotherapy 
2.1 Capecitabine (Xeloda®) 
Capecitabine is administered as a non-cytotoxic, systemic pro-drug of 5'-deoxy-5-
fluorouridine. After administration, it is largely absorbed unchanged from the 
gastrointestinal tract and is sequentially converted to the cytotoxic 5-fluorouracil (5-FU) via 
a series of metabolic steps. The final reaction in this signaling pathway is catalyzed by 
thymidine-phosphorylase (TP). 5-FU is preferably generated at the tumor site via utilization 
of the higher concentrations of TP found in tumor tissue compared to normal tissue. 
In MBC, capecitabine has been evaluated in clinical trials in combination with docetaxel 
and as monotherapy. Capecitabine in combination with docetaxel is indicated for the 
treatment of patients with MBC after lack of efficacy with anthracyclins. Capecitabine 
monotherapy is indicated for the treatment of patients with MBC who are resistant to both 
paclitaxel and a chemotherapy regimen containing anthracycline or who are resistant to 
paclitaxel and where further treatment with anthracycline is not indicated. 
Capecitabine has been studied in patients with liver metastases with mild to moderate liver 
dysfunction. There was no difference in the pharmacological parameters of the main 
metabolites from capecitabine in this group compared to patients with normal liver 
function.  
The dose-limiting side effects are especially diarrhea and hand and foot syndrome.  
 
2.2 Oxaliplatin (Eloxatin®) 
Oxaliplatin is a 3rd generation platinum derivative with mechanism of action such as cisplatin. 
Oxaliplatin forms cross-links between base pairs in DNA, which inhibits replication and 
transcription and results in cell death. After a 2-hour infusion, 15% of the given platinum is present 
in the systemic circulation while the remaining 85% is rapidly distributed in the tissues. The bulk of 
platinum is eliminated in the urine especially within the first 48 hours. After 5 days, 54% is excreted 
in the urine and 3% in the faeces. Oxaliplatin has an adverse reaction profile that is different from 
cisplatin in that no reports of renal toxicity, ototoxicity, hair loss or severe haematological toxicity 
have been reported. The dose-limiting side effect is a cumulative peripheral sensory 
polyneuropathy (pSPN), which is aggravated in low temperatures. 


Intrahepatic chemotherapy in cancer mammae MA 0918+trastuzumab, protocol version 8, 20121108 _translated 
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In several in vivo tumor models, the combination of oxaliplatin and 5-FU causes synergistic 
antiproliferative effect. 
Oxaliplatin was initially administered as an intravenous infusion over 2 hours. Studies have 
shown that the substance can be given over 1/2 hour with less local toxicity and 
unchanged systemic toxicity.  
Platinum derivatives have only been used to a limited extent by MBC. Previous studies 
have shown significant efficacy of cisplatin in combination with epirubicin and studies 
suggest that the platinums should be used in patients who have BRCA gene mutation. 
Oxaliplatin has only been used to a small extent but is considered one of the new 
promising substances. 
 
2.3 Oxaliplatin intrahpatic 
A Phase I study of intrahepatic infusion of oxaliplatin over 4 hours in combination with folic 
acid (200 mg/m2 over 1 h) and 5-FU (600 mg/m2 over 2 hours) was conducted in patients 
with colorectal cancer. Patients were treated every 3 weeks with increasing doses of 
oxaliplatin. 21 patients were evaluated, and all patients were evaluable. Dose-limiting 
toxicity (DLT) was observed in oxaliplatin 150 mg/m2/3rd week and consisted of 
leukopenia, obliteration of A. hepatica and acute pancreatitis. Toxicity consisted mostly of 
vomiting (16/21), anemia (16/21), upper abdominal pain (15/21), sensory neuropathia 
(10/21), diarrhea (9/21), and trombocytopenia (9/21). Ten of 18 evaluable patients (56%) 
achieved a complete or partial response. The authors recommended doses in a Phase II 
study for intrahepatic oxaliplatin 125 mg/m2 every 3 weeks.  
 
2.4 Oxaliplatin intrahepatic in combination with capecitabine 
The Department of Oncology, Herlev Hospital has considerable experience with 
intrahepatic infusion of oxaliplatin in combination with capecitabine for patients with 
colorectal cancer. The following regimen was used: oxaliplatin 100 mg/m2 intrahepatically 
over 2 hours day 1 every 2 weeks combined with Xeloda® up to 3500 mg/m2 daily divided 
into 2 doses for one week every 2 weeks. At present, 42 evalauble patients have been 
treated in a Phase II protocol. In connection with the treatments, the following side effects 
have been seen: hand-foot syndrome, diarrhea, thrombus formation at catheter tip (rarely), 
convulsive abdominal pain in connection with and after infusion of oxaliplatin, ulcers 
(rarely), fatigue, peripheral neuropathy, nausea, anorexia and vomiting. In all cases, the 


Intrahepatic chemotherapy in cancer mammae MA 0918+trastuzumab, protocol version 8, 20121108 _translated 
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side effects have been manageable. No patients have died as a result of treatment. The 
side effects have ledt to that almost all patients have received a reduced dose. We have 
therefore chosen to reduce the dose of oxaliplatin (85 mg/m2) and administer capecitabine 
continuously in a reduced dose (1300 mg/m2 daily divided into 2 doses) instead of over 1 
week (total dose of capecitabine is reduced). Continuous treatment with capecitabine is 
used in patients with ventricular cancer and the treatment is well tolerated. 
 
2.5 Trastuzumab 
Trastuzumab is a recombinant DNA-derived humanized IgG monoclonal antibody that 
selectively binds to the human epidermal growth factor receptor 2 protein (HER2).  
Trastuzumab used in combination with chemotherapy shows efficacy on all endpoints in a 
randomized study of 469 patients with MBC with overexpression of HER2 who had not 
previously been treated with chemotherapy for metastatic disease. Overall, the group of 
patients receiving combination therapy (trastuzumab and chemotherapy) achieved better 
response rates (50% versus 32%), longer time to disease progression (7.4 months versus 
4.6 months) and extended survival (25.1 months versus 20.3 months) compared to the 
group of patients treated with chemotherapy only. Den median survival was extended 
almost 5 months, despite the fact that approximately 70% of patients receiving 
chemotherapy only as first line therapy received trastuzumab upon progression. 
Trastuzumab added to the two chemotherapy regimens did not give rise to any more 
serious adverse events except for cardiac dysfunction, which was specifically increased in 
the group receiving both anthracyklin and trastuzumab. 
The most common side effects of trastuzumab are infusion-related symptoms such as 
fever and chills, usually after the first infusion of trastuzumab. Other side effects 
associated with the infusion may include nausea, vomiting, pain, headache, hypotension, 
rash, and asthenia. Symptoms rarely appear with subsequent trastuzumab infusions. 
Serious side effects of trastuzumab are rare, but may include ARDS and heart failure, 
such as dyspnea, increasing cough, peripheral edema or reduced left ventricle ejection 
fraction. Therefore, patients with severe dyspnea at rest due to advanced malignancy or 
patients requiring supplemental oxygen therapy should not be treated with trastuzumab. In 
addition, an increased incidence of anemia and leukopenia has been observed after 
administration of trastuzumab in combination with chemotherapy. Allergic reactions and 
hypersensitivity that occur during the first infusion of trastuzumab have rarely been 
reported. Today, the drug is routinely used in all patients with HER2 positive tumor. 


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2.5.1 
Overview of clinical experience with continuation of trastuzumab 
after progression in metastatic breast cancer 
More recently, a prospective, randomized Phase III trial has been closed prematurely 
following the recruitment of 156 of planned 482 patients. The trial was conducted by 
German Breast Group to evaluate the safety and efficacy of treatment with trastuzumab 
after progression. The primary purpose of this trial was to compare capecitabine alone or 
in combination with trastuzumab in patients with HER2-positive MBC and progression after 
prior treatment with trastuzumab (alone or in combination with chemotherapy drugs other 
than capecitabine). The majority of patients had received trastuzumab with chemotherapy 
as a 1st line treatment. An initial analysis indicates that continued treatment with 
trastuzumab after progression in addition to capecitabine had fewer cases of tumor 
progression (48 versus 53, hazard ratio 0.71 in favor of the trastuzumab arm) and fewer 
deaths (26 versus 31), and that continued treatment with trastuzumab combined with 
capecitabine achieved a response rate of 49% versus 25% achieved with capecitabine 
alone. The incidence of severe toxicities, including cardiac toxicities, did not differ 
significantly in the two arms. However, the final analysis has not yet been published. 
Several retrospective trials have also been conducted with continuation of treatment with 
trastuzumab after progression. The authors consistently conclude that data clearly support 
the clinical benefit of continued treatment with trastuzumab. 
2.5.2 
Overview of clinical experience with combination therapy with 
trastuzumab and capecitabine 
A recently published open-label Phase II trial of capecitabine plus trastuzumab recruited 
27 patients with MBC with overexpression of HER2 who had previously been treated with 
anthracylines and/or taxanes. Patients received 2500 mg/m2 of capecitabine for 14 days, 
followed by a 7-day break combined with trastuzumab 2 mg/kg weekly. Twelve patients 
(45%) achieved objective response, including CR in 4 patients (15%) and PR in 8 patients 
(35%). A further 9 (33%) of patients achieved stabilisation of the disease. Median survival 
(OS) was 28 months, time to progression (PFS) 6.7 months. The safety profile of the 
combination was favorable and predictable with a low incidence of grade 3 or 4 adverse 
events. The most common adverse events were pain (estimated primarily related to the 
disease), hand-foot syndrome (grade 1-2 in 17 patients, grade 3 in 4 patients), and 
gastrointestinal side effects (including grade 1-2 diarrhea in 13 patients). Severe 


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myelosuppression was rare (3 patients) and severe alopecia did not occur. One patient 
experienced trastuzumab-induced heart failure.  
Another trial investigated the efficacy of capecitabine combined with trastuzumab in breast 
cancer patients. Patients had previously received treatment with either anthracycycline 
and docetaxel or vinorelbine, and all patients had previously been treated with 
trastuzumab for metastatic disease. 40 patients were recruited. The median time to 
progression was 8 months, and the OS was 24 months. No significant difference was 
found between 2nd line treatment and treatment thereafter. The CR rate was 2.5% and the 
PR rate was 17.5%. The disease was stable in fifty percent of patients for at least 6 
months, resulting in a clinical benefit of 70%. Treatment-related adverse events of grade 
3/4 were diarrhea (5%) and hand-foot syndrome (15%). In addition, three patients (7.5%) 
developed brain metastases during treatment. 
For HER2 positive MBC, there is further evidence that capecitabine in combination with 
lapatinib is an effective treatment. Elevated liver enzymes are seen relatively frequently 
when treated with lapatinib, which is why the pharmaceutical company (GSK) advises 
against lapatinib being given in combination with intrahepatic therapy. Results regarding 
continued treatment with trastuzumab are comparable to results regarding lapatinib, which 
is why it is considered justifiable that patients are offered this treatment. 
2.5.3 
Administration of the drug 
Trastuzumab is administered according to the department's standard guidelines. In very 
rare cases, patients have experienced infusion symptoms or pulmonary symptoms 
occurring more than six hours after the start of the infusion. Patients are warned of the 
possibility of this and informed that the doctor should be contacted if these symptoms 
occur. 
2.6 DSM-TACE" (transarterial chemoembolisation) 
Our current experience has shown that the anatomy of the liver arteries in up to 1/2 of 
patients is of such a nature that it can prevent uncomplicated intrahepatic treatment. It can 
be a double liver artery supply or meandering vessels, which provide unstable catheter 
deposit. Although there are methods to address these challenges (internal fixation of the 
catheter, extensive liver artery embolization, catheter and porting via A.subclavia), these 
also carry the risk of new complications. An alternative method of regional chemoinfusion 
consists in repeated catheterizations of the hepatic artery, whereby one or more 


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chemotherapeutics are applied as bolus, and are followed by the injection of occluding 
material into the hepatic artery, so called TACE.  
The rationale of treatment is that the decreased perfusion achieved through embolization 
increases intracellular uptake of the chemotherapy. TACE for the treatment of 
hepatocellular carcinoma (HCC) is well described.  
TACE for the treatment of liver metastases has been less studied, but promising recent 
results are available from German centers. The experience from this suggests that the 
chemoembolization can be made less selective than when treating HCC, when the 
embolization is carried out gently and with resorbable material.  
This so-called DSM-TACE consists of intrahepatic infusion of chemotherapeutic followed 
by degradable glycogen particles (Degradable Starch Microsperes, DSM). The particles 
(EmboCeptS 450mg/7½ ml, Pharmacept Gmbh, Berlin) of about 50 μm cause hemostasis 
in the hepatic arteries for 1-2 hours, thereby exposing the tumor cells to a greater 
concentration of chemotherapeutics than with the usual intravenous or intraarterial 
infusion. The particles dissolve with a half-life of 50 min.  
The administration takes place in the radiological department by a radiologist, first 
performing selective arteriography of A. hepaticae to identify any lateral branches that 
need to be occluded before chemoembolization, in order to avoid chemoembolization of 
the stomach and intestines. After any coil embolization of the side branches, 
chemotherapeutics are injected. Optionally, several liver artery branches are to be treated 
separately. Experienced nurse with chemo course will be present. 
Side effects of TACE are predominantly described in the treatment of HCC and consist in 
abdominal pain, nausea and vomiting for 2-7 days. Further, fever appears immediately 
after treatment. Further, cases of intraperitonal hemorrhage have been described. The risk 
of side effects is correlated to the disease status, liver function (cirrhosis of the liver) and 
the volume treated. In the protocol, for this reason, the first 3 patients are treated at the 
lowest recommended dose. If none of these patients experience serious side effects, the 
dose will be increased. 
In cases where a permanent catheter cannot be installed (Herlev and Aarhus) or in all 
cases in Odense, the patient is offered DMS-TACE. As a total of 6 treatments with DMS-
TACE will be given, the treatment regimen will be:  
DMS-TACE with oxaliplatin:  
 
Oxaliplatin 70 mg/m2 with infusion over 1/2 hour followed by EmboCeptS til flow stop, max 
3 ml (3 patients). 


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Oxaliplatin 85 mg/m2 with infusion over 1/2 h followed by EmboCeptS til flow stop, max 7½ 
ml (3 patients). 
 
Oxaliplatin 85 mg/m2 with infusion over 10 minutes followed by EmboCeptS til flow stop, 
max 7½ ml (3 patients).  
 
 
If this treatment is tolerated in the latter patients, this treatment regimen will be continued. 
Treatment will be evaluated closely. 
Each time, treatment will be evaluated after 3 patients who have each received at least 1 
treatment. If Grade 4 (NCI CTC version 3.0) adverse events related to intrahepatic therapy 
are observed, this part of the trial will be discontinued immediately. In the event of death, 
the trial is immediately discontinued. If no grade 3 side effects are seen that cannot be 
treated with standard supportive treatment, the dose will be increased to: Oxaliplatin 85 
mg/m2 followed by a maximum of 7½ ml of EmboCeptS intrahepatically every 4 weeks.  
Oxaliplatin 85 mg/m2 intravenously over 1/2 hour every 4 weeks (alternating) (6 times). 
Capecitabine 1300 mg/m2 continuously. 
After catheter insertion/TACE: 
Patients are observed according to the instructions of the wards.  
Pain management, supportive treatment and observation in general will be done in 
accordance with the departments' instructions for intrahepatic treatment.  
Due to little experience with DMS-TACE, the treatment will initially only be offered at 
Herlev Hospital. 
 
2.7 Local treatment 
As part of the evaluation of tumor response, all patients will be assessed for indication for 
local treatment (radiofrequency ablation (RFA) = microwave destruction). These 
treatments are outside of current protocol and are therefore not described, but the 
combination of chemotherapy-treated liver metastases and surgery/RFA is well known 
from the treatment of colorectal cancer. Patients found suitable for local treatment will be 
treated according to the standard procedures of the wards, and patientinformation will be 
dispensed accordingly.  
 
2.8 HAI – XELOX 


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Based on the above pharmacological data, clinical studies and previous experience with 
intrahepatic chemotherapy, we will use the following regimen:     
By permanent catheter: 
1) Oxaliplatin intrahepatic with 85 mg/m2 over 1/2 hour every 2 weeks. 
2) Peroral capecitabine (Xeloda ®) 1300 mg/m2 daily divided into 2 doses continuously. 
3) In HER2-positive tumour in combination with trastuzumab (Herceptin ®) 8 mg/kg day 1 
followed by 6 mg/kg every 3 weeks. 
4) Evaluation after every 4. treatment according to RECIST 1.1. 
5) After discontinuation of intrahepatic chemotherapy and possibly local treatment, 
capecitabine 2000 mg/m2 day 1-14 every 3 weeks, and in HER2-positive disease in 
combination with trastuzumab until progression. 
6) Collection of tissue from primary tumor, liver metastasis and other metastases, blood 
cells, serum and plasma at the start of treatment. 
7) Collection of blood cells, serum and plasma at the evaluation times. 
8) Collection of tissue from metastasis at a possible later operation. 
 
 
DMS-TACE/treatment in non-permanent catheter 
1) Oxaliplatin 70 mg/m2 with infusion over 1/2 hour followed by EmboCeptS til flow stop, 
max 3 ml (3 patients). 
 
Oxaliplatin 85 mg/m2 with infusion over 1/2 h followed by EmboCeptS til flow stop, max 7½ 
ml (3 patients). 
 
Oxaliplatin 85 mg/m2 with infusion over 10 minutes followed by EmboCeptS til flow stop, 
max 7½ ml (3 patients).  
 
If this treatment is tolerated in the latter patients, this treatment regimen will be continued. 
Treatment will be evaluated closely. 
Oxaliplatin is given in the latter case at a concentration of 5mg/ml. 
Or: 
Treatment is given intrahepatically over 1/2 hour (not followed by microspheres). This 
treatment modality will be used in Odense and Herlev until it is demonstrated that it is safe 
to give oxaliplatin as a bolus. Patients (at Herlev Hospital) who do not wish to receive 
EmboCeptS in the initial phase of the study will also be offered this treatment 
2) Oxaliplatin intravenously with 85 mg/m2 over 1/2 hour every 4 weeks. 
3) Peroral capecitabine (Xeloda ®) 1300 mg/m2 daily divided into 2 doses continuously. 


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4) In HER2-positive tumour, in combination with trastuzumab (Herceptin ®) 8 mg/kg day 1 
followed by 6 mg/kg every 3 weeks. 
5) Evaluation after every 4th treatment according to RECIST 1.1. 
6) After discontinuation of intrahepatic chemotherapy and possibly local treatment, 
capecitabine 2000 mg/m2 day 1-14 every 3 weeks, and in HER2-positive disease in 
combination with trastuzumab until progression. 
7) Collection of tissue from primary tumor, liver metastasis and other metastases blood 
cells, serum and plasma at the start of treatment. 
8) Collection of blood cells, serum and plasma at the evaluation times. 
9) Collection of tissue from metastases at a possible later operation. 
 
3 Pharmacokinetics 
In order to investigate the difference between intrahepatic and intravenous treatment, 4 
patients will also  carry out pharmacokinetics, taking 2 ml of blood on timepoints 0, 15, 30, 
60 min, 2, 2½, 3, 4, and 6 hours, for the purpose of determining oxaliplatin concentration 
(this determination will be done in collaboration with Anders Johnsson, Lund Sweden).  
Each patient will have the analysis performed a total of 3 times (for IV treatment and for 
intrahepatic treatment with and without EmboCeptS). A total of 54 ml of blood will be 
taken, which is of no significance for the health of the patient. 
Once the first samples have been assessed, the timing of pharmacokinetics may be 
changed, no more samples will be taken.  
   
4 Tumor biology 
It is essential to find the patients who could benefit from intrahepatic chemotherapy, which 
is why material (tumor tissue, whole blood, serum and plasma) will be collected for studies 
for new and future potential prognostic and predictive biomarkers.  
A better understanding of the biology of the tumours will eventually lead to better cancer 
treatment and probably better selection and individualisation of the treatment. It is not yet 
clear which prognostic or predictive biomarkers in tumor tissue, the surrounding normal 
tissue and in blood tests in patients with breast cancer can predict the course of the 
disease or the effect of the chosen treatment.  
A biobank of blood and tissues will be established. The biological material will only be 
used for a new research project after obtaining approval from the Research Ethics 


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Committee. The material will be stored for 15 years, and thereafter it will be destroyed.  A 
code is kept for the identification of the biological material that is therefore personally 
identifiable. Together with the protocol itself, the research biobank has been notified to the 
Danish Data Protection Agency.  
See Appendix 2. 
 
5 Endpoints 
 
5.1 Clinical endpoints 
5.1.1 Primary endpoint 
 
Response rate. 
Fraction of patients who achieve complete or partial response in the liver according to 
RECIST 1.1. 
Secondary endpoints 
a) Time to Intrahepatic progression (PFS). 
b) Time to Extrahepatic progression (PFS). 
c) Number of patients who can have the tumor burden reduced so much that they become 
suitable for local treatment (radiofrequency ablation). 
d) Survival. 
e) Toxicity. 
 
5.2 Paraclinical endpoints 
Exploring the interaction between a number of biomarkers in cancer tissue and blood and 
their relation to relevant clinical parameters such as treatment effect (response), toxicity, 
time to progression and survival. 
 
6 Trial design 
Phase II study. A maximum of 12 intrahepatic treatments are given. The patient may then, 
at the discretion of the investigator, continue with systemic treatment.  Treatment is 


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continued to progression, unacceptable toxicity or until the patient does not want to 
continue treatment.  
    
6.1 Expected number of patients 
Up to 50 evaluable patients with a permanent catheter and up to 50 evaluable patients 
with microcathether, recrutied over 3 years period (see statistical calculations).  
Initiated October 2009 it is planned that the last patient will begin treatment in October 
2012.  
 
6.2 Duration of treatment 
Intrahepatic: Maximally 12 series of intrahepatic chemotherapy (equivalent to ½ year of 
treatment).  
Discontinuation: Patients discontinue treatment in case of progressive disease (PD) or 
unacceptable toxicity or until the patient does not wish to continue treatment. 
Cardiotoxicity: If during treatment the development of cardiomegaly, heart failure (CHF) 
or arrhythmia occurs that require treatment, treatment with trastuzumab should be 
stopped. A decrease in LVEF > 0.20 (absolute units) and/or decrease in LVEF > 0.10 to < 
0.50 will result in the patient's discontinuation of med trastuzumab. 
 
7 Selection of patients 
7.1 Inclusion criteria: 
• Informed written and oral consent. 
• Age over 18 years. 
• Performance status 0-1 and life expectancy ≥ 3 months.  
• Histologically or cytologically documented breast adenocarcinoma 
• Liver metastases where local treatment is is assessed as not suitable. 
• Patients with bone metastases or lymphnode metastases where there is no 
progression in the extrahepatic sites within 6 months and whose tumor burden is 
predominantly hepatic. 
• The presence of liver metastases and limited extrahepatic disease must be 
documented by PET-CT scan. 
• No progression on treatment with capecitabine. 


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• Previous treatment with taxane either adjuvant or for metastatic disease. If 
treatment has been given for metastatic disease, treatment should have continued 
until maximum response or cessation due to toxicity. 
• Metastases < 70 % of the liver 
• Neutrophil granulocytes 1.5 x 109/l and platelets 100 x 109/l  
• Bilirubin 2.0 x UNL (upper normal limit). 
• Creatinine clearance 30 ml/min. 
• INR < 2. 
• It is possible to perfuse the metastases through A. hepatica (possibly angiography). 
• The patient is approved by the Second Opinion Committee. 
If the patient is HER2 positive: 
• Baseline LVEF ≥ 50 % measured by either echocardiography or MUGA. 
 
7.2 Exclusion criteria: 
• Chemotherapy with drugs other than capecitabine within 4 weeks. 
• Concomitant or other prior malignant disease other than basal cell carcinoma and 
carcinoma in situ cervicis uteri. 
• Previous treatment with oxaliplatin. 
• Cytotoxic treatment or experimental treatment within 14 days before inclusion. 
• The patient may not participate in other clinical studies. 
• Pre-existing polyneuropathi more than or equal to grade 2 NCI CTC version 3. 
• Serious medical conditions that are assessed to prevent treatment. 
• Other serious medical illness (eg. severe heart disease or AMI within 1 year). 
• Physical or mental illnesses that can prevent the administration of oral treatment. 
• Patients with uncontrolled infection. 
• Pregnant or lactating women. In fertile women, this is ensured with a negative 
pregnancy test. 
• Female patients of childbearing age who do not use contraceptive (not hormonal) or 
breastfeed. The Danish Medicines Agency considers spiral to be adequate 
contraceptives. 
• Signs of active CNS metastases. If there is clinical suspicion of brain metastases, a 
CT scan or MRI of the brain should be performed within 4 weeks prior to inclusion. 


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• Patients who, for linguistic, intellectual or cultural reasons, will not be able to fully 
understand the treatment concept and respond to any complications. 
• Previous severe and unexpected reactions to treatment with fluoropyrimidine. 
• Hypersensitivity to one or more of the known active substances, excipients or 
fluorouracil. 
If the patient is HER2 positive: 
• Dyspnea at rest due to complications of advanced malignancy (e.g., pulmonary 
metastases with lymphangitis) or other conditions requiring supportive oxygen 
therapy.   
8  Treatment regimens and procedure 
Patients are referred to EFEK/EEK, Department of Oncology.   
Patients who meet the inclusion criteria and are approved at the multi-discipline 
conference ("HAI-konf"), are referred to Radiological Department for the purpose of 
establishment of port-a-kath. with catheter in A. hepatica.  
 
 
8.1 Feasibility studies 
8.1.1 Caranatomy. 
For the purpose to localise of the arteries in the liver an angiography of the hepatic and 
abdominal vessels may be performed. The arterial and venous portal anatomy of the liver 
is also clarified by the reconstruction of thin slices from CT scans. 
Variations in anatomy can change the surgical strategy for catheter placement.  
 
8.1.2  Application of arterial Port A Cath. (HAI catheter) 
The patient must meet the inclusion criteria and the anatomy of arteria hepatica must be 
detemined. The patient is referred to the Radiological department for X-ray guided catheter 
insertion through a peripheral artery branch, most frequently A. femoralis. An arterial 
catheter system (Port A Cath.) is implanted in A. hepatica. The aim is to place the catheter 
tip in A. hepatica without sticking into the lumen of A. gatroduodenalis A. hepatica. 5000 
units of heparin are given in the catheter before each closure and after each infusion.  
 


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8.1.3 Side effects, as well as observation. 
Patients should be informed of the general, albeit very small, risk of fatal outcome 
associated with catheter insertion, e.g. due to hemorrhagic shock. At the pump pocket, a 
hematoma/seroma or inflammation/infection, erosion or ulcers can be seen, or it can pop 
up. Incision pain may occur. At the catheter can be seen crack or dislocation such that one 
cannot draw or inject into the catheter port. Complete or partial catheterocclusion may 
occur. In addition, the patient should be observed for systemic side effects from 
chemotherapy specially elevated bilirubin, pancreatitis (amylase), cholecystitis and 
bleeding from the gastrointestinal tract. The patient is observed for 1 day with 
hospitalization after the first catheter insertion (permanent catheter), moreover, according 
to the procedure of the department. 
 
8.2 HAI-XELOX 
Treatment can be started from the day after catheter placement. Dosing is made at a 
maximum equivalent to 2 m2. 
8.2.1 
Oxaliplatin 
Catheter placement is checked before each treatment according to the procedure of the 
department. Through port-a-cath to A. hepatica is given oxaliplatin 85 mg/m2 over 1/2 hour 
day 1 every 2 weeks. Pre-treatment and supportive treatment for nausea are given 
according to the department's standard procedure for intrahepatic therapy. 
8.2.2 
 Capecitabine (Xeloda®) 
Xeloda® is dispensed as tablets in dosing boxes. The tablets are taken in conjunction with 
a meal, or no later than 30 minutes after. The dose is distributed over 2 roughly equal 
doses. The first dose is taken in the evening day 1. Daily dose is 1300 mg/m2.  
 
8.2.3 
EmboCeptS 
EmboCeptS are microspheres with a diameter of 50 μm. The substance has a half-life of 
50 minutes. Manufactured by PharmaCept Gmbh, Berlin, distributed by Nextpharma. The 
substance is routinely used to treat hepatocellular carcinoma. EmboCeptS is CE certified 
and is used on the approved indication. 
 
8.3 Visit schedule and observations 


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Assessment 
Screening 
(number of 
days before 
inclusion) 
During 
treatment 
End of 
treatme
nt 
Follow-up 
until 
progres-
sion 
Anamnesis and 
physical 
examination (+ PS) 
8 days 
Before each treatment 
(every 2 weeks) (physical 
examination may be omitted at the 
discretion of the investigator) 
X 
If indicated 
Hematology* 
8 days 
Before each treatment 
(every 2 weeks) 
X 
If indicated 
Biochemistry** 
 8 days 
every 2 weeks 
X 
If indicated 
Blood tests to 
determine 
biomarkers 
(Appendix 2)  
8 days 
For all evaluations up to progression X 
For all 
evaluations 
for 
progression 
ECG 
8 days 
 
 
 
Tumor 
assessment¤ 
For PET/CT 
followed by CT 
28 days 
After every 4th HAI-XELOX  
Then every 3 months 
Assessment of extrahepatic 
disease of the bones after every 
6th treatment (MRI or x-ray 
depending on baseline 
examination) 
X 
Until  
PD 
Ultrasound of the 
liver with biopsy  
28 days 
 
 
 
Side Effects/ 
Adverse Events#  
(SAE within 24 
hours) 
 
 
Before each treatment 
(every 2 weeks) 
and in case of the SAE 
X 
Followed 
until 
recovery 
MUGA, ECG (HER2 
positive disease) 
28 days 
every 3 months 
 
According to 
department 
standard 
* Hematology: leukocytes, neutrophil granulocytes, thrombocytes, hemoglobin. 
** Biochemistry: INR, alkaline phosphatase, bilirubin, LDH, ALT/AST, amylase, serum creatinine, sodium, 
potassium and s-Ca ion.  
Hematology and biochemistry are assessed before treatment series to ensure satisfactory values and to allow 
for necessary dose modifications if indicated. Side effects are reported in CRF. 
# Side effects are recorded and graded according to NCI CTC version 3.0. If no relevant NCI CTC is found, the 
adverse reaction should be recorded as 0 = none, 1 = mild, 2 = moderate, 3 = severe, 4 = life-threatening. 
Biomarkers are described in Appendix 2.  
 
 
8.4 Registration 
After recording the anamnesis, patient information is provided. The investigator conducts 
clinical, radiological and laboratory assessments to confirm that the patient meets all 
inclusion criteria. For patient information and ICF procedure, see Appendix 1.   
 


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If the patient meets the eligibility criteria, data will be faxed to the Clinical Research Unit, 
Department of Oncology, Herlev Hospital Fax. no.  4453 3076.  The phone number for this 
is 4488 4000 + 89438. 
 
8.4.1 
Before each treatment series 
The patient is assessed for the following parameters within 72 hours before each 
treatment series.  
• 
Performance status 
• 
Clinical assessment 
• 
Assessment of side effects 
• 
Blood tests according to the schedule. 
• 
Any reporting of SAE is carried out accordingly. 
 
8.4.2 
Blood samples for the determination of biomarkers 
• 
Before starting treatment og in connection with each evaluation.  See Appendix 2. 
 
8.5 Assessment of response 
Tumor evaluation is done after every 4th series (every 8 weeks) 
Thoraco-abdominal CT scan, clinical assessment and biochemical status as at baseline. 
8.5.1 
Definition of response 
Made according to RECIST version 1.1. 
8.5.1.1 Biomarkers  
Blood tests to determine tumor markers (Appendix 2). 
The samples will be stored in a biobank, in anonymized form.  See also point 3. 
 
8.6 Other concomitant medication 
Patients on oral anticoalgulation therapy should be monitored regularly with INR due to 
possible interaction with Xeloda®.  
Vitamin B6 (Pyridoxine) must not be used to treat hand-food syndrome, as there is a risk 
of reduced efficacy of oxaliplatin.  


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Prophylactic antiemetics including corticosteroids are allowed. Prophylactic antibiotics are 
allowed if it is deemed beneficial for the patient. G-CSF is not recommended as a 
prophylaxis but can be given if it is found beneficial for the patient. 
8.6.1 
Other medication 
All other symptomatic treatment of the patient to perform optimal care is allowed as long 
as the name, route of administration and duration of treatment are documented in the 
patient's medical record.  
No other antineoplastic treatment is allowed during treatment.  
Patients are allowed to be given bisphosphonate. 
 
8.7 Follow-up 
To determine any timepoint of progression, patients will be followed with clinical 
assessment and CT scan every 3 months after treatment cessation until progression.  At 
the same time, relevant routine blood samples are taken on indication and blood for 
project biomarkers are taken at each evaluation up to progression (Appendix 2). 
After the finding of progression, treatment/control continues according to local guidelines, 
but subsequent treatment and any date of death must be documented. 
 
9 Storage, mixing and dispensing of investigational 
products 
In all cases, these are medications that are used as standard in the department. Storage 
and mixing as well as handling in the department are carried out on the principles of 
current standard guidelines. All dispensed investigational product will be labeled and 
handled according to ICH-GCP. Patients return unused medication and keep a diary. 
 
10 Dose reductions 
 
Adverse reactions are graded according to NCIC-CTC criteria (version 3.0), except for 
neurological adverse events, which are assessed according to Table 2.  
As a rule, side effects of capecitabine and oxaliplatin are handled separately.  
In the case of DMS-TACE, any dose reductions should always be made based on the 
dose given. 
In the event of a reduction in the dose, the dose should not be increased again later.  


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Treatment postponement should be based on the day 15 observation (= the day of the 
scheduled day 1 of the following treatment) and the prescription should be based on the 
worst toxicity observed during the previous treatment. If hematological and non-
haematological toxicity of the same degree coincides, dose reduction shall be based on 
the non-haematological rules for dose reduction. 
  
10.1 
Treatment delay 
The next treatment with capecitabine and oxaliplatin is postponed until  
1: Neutrophil leukocytes (ANC) > 1.5 x 109/l and or platelets > 100 x109/l  
2: Any non-haematological toxicity such as mucositis, diarrhoea and PPE has decreased 
to ≤ Grade 2 or baseline toxicity level.   
 
10.2 
Dose reduction due to hematological toxicity 
Three consecutive dose reductions are allowed. If, at the 3rd dose reduction, grade 3/4 
haematological toxicity is still observed, the patient should stop treatment.  
Dose reductions for subsequent treatments are based on the worst side effects.  
The dose of capecitabine and oxaliplatin shall be reduced by 25% (see table) in all of the 
following series in the case of:  
• 
febril neutropenia (temperature ≥ 38,5 °C and ANC < 1.0 x 109/l). 
• 
neutropenia grade 4 (ANC < 0.5 x 109/l). 
• 
thrombocytopenia grade 4 (platelets < 25 x 109/L). 
• 
thrombocytopenia grade 3 and concomitant hemorrhage grade 2. 
• 
if treatment is postponed more than 2 weeks due to haematological toxicity.  
• 
grad 3 or 4 mucositis, diarrhea or nausea and vomiting despite appropriate medical 
treatment. 
 
An additional 25% dose reduction can be made if the side effects persist. 
 
For hematological toxicity, three consecutive dose reductions are allowed (only in isolated 
hematological toxicity): 
Dose reduction: 
Dose reduction: oxaliplatin 
Dose reduction: 
capecitabine 
Step 1 
25 % 
No dose reduction 


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Step 2 
25 % 
25 % 
Step 3 
50 % 
25 % 
 
In the case of chemotherapy related anemia grade 1-2 either transfusion of red blood cells 
or erythropoetin may be used to correct this side effect but dose reduction is not allowed.   
 
10.3 
Liver function 
At the beginning of a series, serum bilirubin should be  2.0 x UNL. If an elevated bilirubin 
persists 2 weeks after postponed treatment, treatment is stopped. In the case of a bilirubin 
increase of ≥ 2.0 x UNL that is not due to disease progression or obstructive icterus, 
treatment with corticosteroids is allowed (e.g. 100 mg daily for 5 days). If bilirubin returns 
to  2.0 x UNL within 2 weeks of postponed treatment, treatment may be continued at a 
lower level according to the rules of dose reduction in case of non-haematological toxicity.   
 
10.4 
Dose reduction in oxaliplatin related sensory 
polyneuropathy (sPNP) 
 
Any neurological side effects should be assessed according to Table 1 before each 
oxaliplatin treatment. 
 
Table 1 Grading neurological side effects 
Grade 
Symptoms 
Grade 0 
None 
Grade 1 
Short-duration paresthesias/dysesthesias with complete recovery 
before the next treatment 
Grade 2 
Paresthesias/dysesthesias, which persist between 2 treatment series, 
but without affecting function 
Grade 3 
Functional impairment (eg. reduced force) 
 
Table 2 
Dose of oxaliplatin and neurological side effects 
 
Side effects 
Duration of side effects Persistent side effects at the next 
treatment 
14 days 
1-7 days 
 >7 days 
1) Cold-triggered 
dysesthesias 
 
100 % 
100 % 
100 % 
2) Paresthesias 
100 % 
100 % 
1st time 75% 


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2nd time 50% 
3rd time stop 
3) Paresthesias + pain 
100 % 
1st time 75% 
2nd time 50% 
3rd time stop 
 
Stop 
4) Paresthesias with 
functional impairment 
100 % 
1st time 50% 
2nd time stop 
Stop 
 
No reduction 
For dysesthesias regardless of whether they are painful or not or are cold-related or not 
after an oxaliplatin infusion, the dose should not be modified. In case of persistent non-
painful paresthesias (other than due to cold exposure) or in painful paresthesias only in 
case of cold exposure without functional influence, the dose should not be reduced.  
 
10.5 
Hypersensitivity, allergies or laryngopharyngeal 
dysesthesia (LPD) 
Allergic or hypersensitive reactions (e.g. rash, hypotension, flushing, dyspnea) are seen, 
albeit rarely, in patients treated with platinum derivatives. If hypersensitivity cannot be 
ruled out, pre-treatment with steroid is recommended at the next series of chemotherapy. 
In case of suspicion of allergies or similar reactions, pre-treatment before the next series of 
chemotherapy is advised according to the department's standard for re-treatment.  
If the oxaliplatin dose is reduced due to side effects, the dose should not be increased 
again.  
If intrahepatic treatment is discontinued due to side effects or in cases where a catheter 
cannot be used, the patient is withdrawn from the protocold treatment and the patient can 
continue treatment in accordance with the department's usual guidelines.   
 
10.6 
Dose reduction for capecitabine 
• In the case of febril neutropenia (temperature ≥ 38.5 degrees and ANC < 1.0 x 
109/L), interuption of capecitabine treatment until the next scheduled series. 
 
Table 4 
Dose of capecitabine and non-hematological side effects (diarrhea and PPE) 


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Grade 2 
Grade 3 
Grade 4 
1st time 
Pause until  
grade 0-1 
Continue with 100% 
Pause until  
grade 0-1 
Continue at 75% 
Pause until  
grade 0-1 
Continue at 75% 
2nd time 
Pause until  
grade 0-1 
Continue at 75% 
Pause until  
grade 0-1 
Continue at 50% 
Stop* 
3rd time 
Pause until  
grade 0-1 
Continue at 50% 
Stop* 
 
4th time 
Stop* 
 
 
 
• Treatment is discontinued unless the investigator considers it in the patient's 
interest to continue with reduced-dose treatment.  
• If the investigator finds it in the patient's interest to switch from capecitabine to 
infusion therapy with 5-FU/LV as continuous pump therapy in the de Gramont 
regimen, this can be done. The investigator may deem it in the patient's interest to 
continue intrahepatic treatment with oxaliplatin, but the patient will be withdrawn 
from the study.     
 
10.7 
Non-hematological toxicity other than sensory 
polyneuropathy 
Side effects will be graded using CTC. If side effects occur that are not described in the 
CTC, they should be graded on the following scale:  
1 = mild, 2 = moderate, 3 = severe and 4 = life-threatening. 
 
In cases of non-hematological toxicity grade 3 or 4, the following consecutive dose 
reductions may be used:  
Dose reduction: 
Dose reduction: oxaliplatin 
Dose reduction capecitabine 
Level 1 
25 % 
No dose reduction 
Level 2 
25 % 
25 % 
Level 3 
50 % 
25 % 
A maximum of two consecutive dose reductions are allowed. 
 
10.7.1 
Adjustments and postponements of trastuzumab dose due to toxicity 
There are no dose modifications for trastuzumab and it is not necessary to interrupt 
trastuzumab due to haematological toxicity. Treatment with trastuzumab should be hold for 


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related non-hematological toxicity (other than cardiac dysfunktion) of Grade 3 or 4 until 
recovery to  Grade 2. If the same non-haematological toxicity again occurs with a grade 3 
or 4, treatment with trastuzumab should be permanently discontinued. 
Postponements due to trastuzumab-related toxicity is unusual. If a dose is postponed by 
less than one week, the usual dose of 6 mg/kg should be given as soon as possible and 
subsequent dosing continued according to the original dosing schedule. 
If a dose is postponed by more than one week, treatment should be restarted as soon as 
possible with a loading dose of 8 mg/kg, followed by a dosage of 6 mg/kg.  
  
10.7.2 
Cardiac dysfunction 
Treatment with trastuzumab should be discontinued if the patient develops clinical heart 
failure, i.e. is in NYHA grade II, III or IV.  
All patients should have a baseline LVEF of ≥50%. LVEF is regularly monitored according 
to the department's standard. Trastuzumab is discontinued in any patient who develops 
clinical signs and symptoms suggestive of CHF (to be confirmed by LVEF).  
CHF is treated and monitored according to medical standards. 
There are currently insufficient data to assess the prognostic significance of asymptomatic 
declines in LVEF. However, in order to ensure the safety of patients in the trial, 
trastuzumab should be discontinued for all patients where a decrease in LVEF to below 
40% is documented and confirmed with a new assessment within 4 weeks of initial 
assessment using the same assessment method. 
For patients whose LVEF falls to values between 40% and 50%, the decision to 
discontinue or discontinue trastuzumab is based on the algorithm in the figure below.  
Patients who develop symptom-giving cardiotoxicity will be referred to the cardiology 
department. 
  
Figure.    Algorithm for handling treatment with trastuzumab, based on LVEF 
assessments 
 


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10.7.3 
Dose interruption due to reactions associated with infusion 
Patients who experience a life-threatening infusion reaction at the first dose (e.g. 
tacypnea, bronchospasm, hypotension, hypoxia) should discontinue treatment. 
Patients experiencing severe or moderate infusion symptoms can be treated by lowering 
the infusion rate or stopping the trastuzumab infusion, or by treatment with, for example, 
oxygen, beta agonists, antihistamines or corticosteroids. 
Patients who experience mild, moderate or severe infusion reactions at the first dose may 
be re-treated with trastuzumab. Premedication with corticosteroids, antihistamines and 
antipyretics may be used before subsequent trastuzumab infusions.  
Withhold trastuzumab, and consult with a  
 cardiologist 
LVEF < 40% 
Monitor LVEF every month 
LVEF > 40% 
LVEF < 40% 
LVEF > 40% 
LVEF < 40% 
Continue with trastuzumab, monitor 
LVEF every 3 months, and consider 
Intervension to the cardiologist's 
choice 
Discontinue trastuzumab  
Proceed with heightened awareness and monitoring 
Continue with extremely heightened attention 
LVEF 40-50 % 
Continue with trastuzumab 
 
 
Monitor LVEF every month. 
LVEF decrease of > 15 % or LVEF decrease of > 10 % and below LLN (LLN = 50 %) 


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If the starting dose with trastuzumab is discontinued due to an infusion reaction, the 
following guidelines apply: 
• 
If the patient has received ≥ 75% of the starting dose, an additional load is unlikely 
to be necessary. However, the investigator may choose to administer the remainder 
of the load dose, either at any time before the next scheduled dose or together with 
the next scheduled dose. 
• 
If the patient has received ≥ 50% and < 75% of the dose, the patient should receive 
the remainder of the dose before the next scheduled dose and preferably within the 
first 2 weeks after the interrupted load dose. 
• 
If the patient has received < 50% of the starting dose, the patient should preferably 
receive the remainder of the starting dose within 1 week of the interrupted starting 
dose. 
10.8 
Treatment of complications 
All complications will be treated according to the standard procedures of the wards. 
Patients are informed to contact the wards in case of side effects. 
 
10.9 
Duration of treatment 
A maximum of 12 series of chemotherapy are planned unless there is progression or 
unacceptable side effects. 
Patients are evaluated after every 4th series by CT scans; MRI/x-ray of extrahepatic foci is 
performed after 6 and 9 treatments.  In case of response, the evaluation scans will be 
assessed in consultation with the Gastrounit, Department of Surgery  to evaluate for 
possible local treatment. 
10.9.1 
Causes of discontinuation of treatment. 
Disease progression at any stage during treatment. 
 
Complications of treatment either from the catheter, or unexpectedly severe adverse 
reaction to the chemotherapy, or by signs of development of hepatic insufficiency. 
 
The patient's own wish: the patient can at any time, at his own request, discontinue 
treatment. 


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10.10 
 Discontinuation of the study before scheduled time 
The study will be stopped for the following reasons: 
• 
If arrising adverse events are of such a serious nature that the continuation of the 
investigation becomes unacceptable 
• 
If the recruitment rate is too low to expect completion of the trial in its current form within an 
acceptable period of time 
• 
If, for administrative reasons, the number of drop-outs is too high and this situation cannot 
be improved 
11  Patient safety 
11.1 
Adverse events 
11.1.1 
Definition 
An adverse event is any symptom, sign or disease that occurs or worsens while the patient 
is participating in the study. The investigator shall assess whether there is a causal 
relationship between adverse events and investigational medicines.  
 
Serious adverse events (SAE) is any adverse event that regardless of dose meeting one 
of the following:  
• Results in death. 
• Any life-threatening event - the patient, at the discretion of the investigator, was at 
immediate risk of dying from the adverse event when it occurred. 
• Hospitalization or prolongation of existing hospitalization.  
• Results in permanent or significant disability/incapacity for work. 
• Congenital anomaly/malformation.  
• Any significant medical event. 
 


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11.1.2 
NOT SAE 
The following will not be considered serious adverse event for the purposes of this study: 
An event that results in hospitalization or prolongs an existing hospitalization if the sole 
reason for admission or extension is for the following: 
 
1. Death as caused by the progression of the patient's cancer. 
2.  Hospitalization is secondary to expected morbidity due to chemotherapy: 
- chills and temperature elevation 
- nausea and vomiting  
- bone marrow suppression 
– fever 
– anemia 
- obstipation 
- diarrhea 
– abdominal pain related to treatment 
 
3.  Hospitalization is secondary to expected morbidity due to cancer: 
– weight loss 
– fatigue  
– electrolyte disorders 
– pain management 
– uneasiness/anxiety 
– admission due to palliative care 
- administering chemotherapy 
- Catheter problems 
– transfusion with blood products 
– in conjunction with any of the study procedure 
– placement of a permanent intravenous catheter 
- hospice stays for terminal care; 
 
All admissions are recorded on an excel sheet. 
Any patient death must be recorded on the CRF. 
Usual side effects to chemotherapy, progressive disease and events secondary to 
progressive disease should not be reported.  


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11.1.3 
Suspected, unexpected, serious adverse reaction – SUSAR 
Serious and unexpected side effects, i.e. a reaction that has not been described previously 
(in the Investigator's Brochure or in the Summary of Product characteristics) and where a 
probable or possible causal relationship with the medicinal product is assessed. There is a 
special obligation on the investigator to report these, cf. section 11.1.5. 
11.1.4 
Adverse Event Registration 
All adverse events (AEs) occurring during the study period shall be recorded and 
documented in the relevant section of the CRF. 
Patients are followed with adverse event registration until discontinuation of intrahepatic 
therapy (maximum 12 series). The side effects will be followed until 1 month after the last 
treatment, or until the side effects are ≤ grade 1, or as at baseline or assessed as 
stationary. 
The events are described, graded and assessed for causality. 
The grading is assessed by the investigator according to the definitions in NCI-CTC, 
version 3.0: grade 1 – 5. If the AE is not listed, graded according to the following 
description: 
Grade 1 = mild 
Grade 2 = moderate 
Grade 3 = severe 
Grade 4 = life-threatening or disabling  
Grade 5 = death related to AE  
 
Causality in relation to the investigational treatment is assessed by the investigator. The 
decisive factor in the documentation is the temporally dependent possible relationship 
between AE and the investigational product (IP). The following assessments of a causal 
relationship to the IP or trial procedure should be used: 
Unrelated: There is no temporal relationship with the administration of the IP (pre-
administration, too late, or the patient has not received the IP), or there is a reasonable 
causal relationship between another drug, underlying disease, other conditions, and AE. 
Not likely: There is a temporal relationship to the administration of the IP, but there is not a 
reasonable causal relationship between the IP and the AE.  
Possible: There is a reasonable causal relationship between the IP and AE.  


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Probable: There is a reasonable causal relationship between IP and AE. Upon stopping IP, 
the incident ceases. There is no need for re-introduction of IP. 
Sure/definitive: There is a reasonable causal link between IP and AE. The event responds 
to the cessation of IP and is recurred upon re-introduction of IP (when clinically possible).  
11.1.5 
Reporting of serious adverse events (SAE/SAR/SUSAR) 
All SAEs must be reported to the sponsor on the SAE form within 24 hours of the 
investigator's knowledge of the event (however, see section 11.1.2 for exceptions). 
It is then the responsibility of the sponsor to assess whether the SAE is unexpectedly and 
presumably related and thereby = SUSAR and then further report to the Danish Medicines 
Agency. 
In cases where SUSAR is life-threatening, the Danish Medicines Agency is informed within 
7 days, otherwise within 15 days. 
It is also the responsibility of the sponsor to prepare annual lists of all SAEs and SARs in 
the trial for the Research Ethics Committee and the Danish Medicines Agency, in 
accordance with the applicable rules and at the end of the investigation. 
 
11.2  Early discontinuation of the study 
The patient is withdrawn from the examination in case of death, progression, unacceptable 
side effects, other severe medical disorder or if the patient wishes. The date and reason 
are given in the journal and CRF. 
12  Data analysis and statistics 
12.1 Definition of populations 
Intention-to-treat population: All patients who have received at least 1 series of intrahepatic 
chemotherapy. Patients with missing and/or illegitimate data will be included in the intent-
to-treat population.   
   
Evaluable-for-response population: All patients who have received at least 4 series of 
chemotherapy unless progression has been established prior to that.  
 
12.2 Efficacy  


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12.2.1 
Primary analysis (response rate) 
The response rate with confidence interval will be calculated on all evaluable patients. The 
response rate will be based on RECIST 1.1. Criteria. 
12.2.2 
Secondary analysis (PFS) 
Time to progression or death will be calculated based on the date of start of chemotherapy 
to the date of documented progression or death. Median time to progression or death with 
confidence interval will be calculated. 
 
12.3 Sample size 
 
Patients will be stratified according to treatment regimen i.e. with permanent 
catheter/DMS-TACE.  The two groups will analysed seperately.  
Rationale for sample size for endpoint response rate for which treatment is considered to 
have sufficient clinical efficacy (The analysis was performed according to "Flemming's 
Multiple Testing Procedure" (Ref. "One-Sample Multiple Testing Procedure for phase II 
clinical trials" T.R. Flemming, Biometrics, March 1982): 
Ho: p=5%. Ineffective rate, response rate for which treatment is considered to have 
insufficient clinical efficacy.  
H1: p=20%.  Minimum aim of effective rate, response rate above which treatment is 
considered to have sufficient clinical efficacy.  
With alpha = 5 % (one-sided test) and power = 92 %. 
If 5 or more patients out of 40 have clinical efficacy, the treatment regimen should be 
further investigated.   
As 40 evaluable patients are targeted, i.e. patients who have received at least 4 series of 
chemotherapy, 50 patients will be included, as it is to be expected that 20% (10 patients) 
will not meet the criteria for evaluability.  
Changes to the original statitistical plan will be notified to the Research Ethics Committee 
and the Danish Medicines Agency.  
 
12.4 Statistical methods 


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PFS and survival will be estimated with the Kaplan-Meier method and compared with a 
log-rank test. Categorical variables are indicated by median followed by the range. A 
significance level of 5% will be used.  The response rate is assessed in the evaluable 
population. 
   
12.5 Side effects 
Side effects are recorded among patients who have received at least 1 series of 
intrahepatic chemotherapy. 
Patients are given pain diary for the registration of pain with intensity and time, as well as 
treatment of these. 
 
12.6  Data management and archiving 
Conducted by the Clinical Research Unit, Department of Oncology at Herlev University 
Hospital.  
Monitoring is carried out by the University of Copenhagen's GCP unit. 
The trial has been notified to the Danish Data Protection Agency.  and the subjects are 
protected under the Act on the Processing of Personal Data and the Health Act (Section 3 
on the Legal Status of Patients).   
 
13  Ethics 
This study is carried out in accordance with the globally accepted standardes of GCP 
(Good Clinical Practice) and in accordance with the latest revision of the Helsinki 
Declaration (version 8) and according to the national regulations. All patients will be 
informed both orally and in writing about the purpose of the study and after a reflection 
period, patients must give both oral and written consent before inclusion. The patient's 
consent form will be kept in the medical record. The study is submitted to the Research 
Ethics Committee and the Danish Medicines Agency and it must be approved before the 
start of the study. At the end of the study, the study report is prepared. The trial will be 
registered in Clinicaltrial.gov. 
 
13.1  Ethical considerations 
In this study, incurable patients with breast cancer are treated.  In breast cancer, the 
consensus is that as soon as patients have developed liver metastases, treatment can 


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only be palliative. Hepatic intraarterial chemotherapy has so far only been very sparsely 
studied for the treatment of liver metastases in breast cancer. Thus, in most cases, only 
"case stories" (often in the media) have been reported. Only one phase I/II trial has been 
reported, using a combination of adriamycin and 5-fluorouracil. The overall response rate 
was 63% with a remarkably long median survival of 25 months.  However, since patients 
with breast cancer bone and lymphatic metastases often have an indolent course, while 
patients with living metastases have an exceedingly poor prognosis, a number of these 
patients will potentially benefit from intrahepatic treatment. Today, the treatment is offered 
in Germany and many patients have a great desire to be able to receive the treatment in 
Denmark. The protocol has been approved by the National Committee for Experimental 
Cancer Treatment.    
The primary purpose of this study is to investigate whether combining the injection of -
oxaliplatin directly into the hepatic artery that supplies the liver metastases with blood and 
concomitant tablet treatment with capecitabine can cause the metastases in the liver to 
dwindle. At the same time as the patient still has routine blood tests, extra blood samples 
will be taken to determine various project biomarkers, which hopefully in the future can be 
used to individualize the treatment or designate patients who would benefit from such 
treatment (the patient can waive this part of the study, see patient information). The 
possible side effects are, in our own experience and what has been previously reported 
from similar studies, moderate and tolerable and will probably be outweighed by the 
possible beneficial effect of the treatment. 
There is no experience with oxaliplatin as bolus intrahepatic. Treatment will therefore be 
evaluated after 3 patients who have each received at least 1 treatment. If Grade 4 (NCI 
CTC version 3.0) adverse events related to intrahepatic therapy are observed, this part of 
the trial will be discontinued immediately. In the event of death, the trial is immediately 
discontinued.  
 
13.2  Patient safety, ethics 
The responsible investigator will ensure that the study is conducted in accordance with the 
Helsinki Declaration and the laws and statutes of the country. The protocol will be 
approved by the local Research Ethics Committee, the Danish Medicines Agency and the 
Danish Data Protection Agency. The leaflet "The subject's rights in a biomedical research 
project" is handed out to each participating patient. Guidelines for providing oral 


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information and obtaining consent, see Appendiks 1.  As this appendix also states how the 
first contact with the subject will take place.  The patient's consent statement will be kept in 
the medical record. If a patient does not want to participate in the study, she will be treated 
according to the usual standards at the departments (systemic chemotherapy). 
 
14 Subject identification 
The patient's name should not be disclosed, nor will it be noted at the data center. Each 
patient will automatically be assigned an identification number when the patient is included 
in the study. This number will identify the patient, and the number should be noted on all 
Case Report Forms. To avoid identification problems, the patient's initials (maximum 4 
letters), date of birth, as well as hospital number will also be noted on each Case Report 
Form. 
 
15 Informed consent 
All patients will be informed orally and in writing about the purpose of this study, possible 
side effects, the methods and possible risks to which she will be exposed. Patients will be 
informed of the strict protection measures on their data, but they will also be informed that 
their medical information may be viewed by other auhtorised persons in addition to the 
attending physician. 
 
It will be emphasized that participation in the study is voluntary, as well as that the patient 
can withdraw from further treatment in the protocol at any time. This will not affect the 
further treatment of the patient. If the patient does not want treatment according to the 
protocol, she will receive treatment according to the department's usual guidelines. The 
investigation is therefore found to be ethically sound. 
 
A signed informed consent form must be received from all patients included in the study, 
and consent must be received prior to registration at the data center.  
 
16 Insurance 
Patients participating in the study are covered by the hospital's liability insurance. 
 
 
17 Timescale 


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Inclusion of the first patient is scheduled for 1.10.2009. The recruitment period is 3 years.  
Last patient will be included October 2012. 
 
18 Sponsorship and finances 
The study was initiated by the investigators. The Danish Cancer Society has given DKK 
1.2 million for expenses for project nurse, database and data processing as well as for 
bioanalyst or nurse associated with the blood sampling/piping, data recording and data 
calculation. Additional  financial support is being sought from the Danish Cancer Society, 
the Danish Health Science Research Council and private Danish foundations for salaries 
for bioanalysts, for the analysis of the biomarkers and for the analysis of the 
immunohistochemical examination, expenses for ELISA kits and immunohistochemical 
kits.  Roche has supported the project with DKK 20,000 for the analysis of VEGF. There is 
no financial gain for the departments or staff in connection with this trial. 
 
 
19 Publication 
Upon completion of the study, publications on the study will be prepared as well as study 
reports of the various potential biomarkers (singly or in combination), which will be 
published in international jounals. Both positive and negative results will be published. 
Coordinating principal investigator prepares manuscript (in case of Congress: abstract) 
and is 1st author on the primary publication. Co-authorship as well as author order are 
determined according to Vancouver rules.  
Articles emanating from the project with predominantly basic-medical, radiological or 
nuclear medicine results can be extracted from the person/department responsible for the 
majority of the work and the project group then discusses co-authorship at all clinical 
departments. 
 
 


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20  References 
 
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Bartsch R, Wenzel C, Altorjai G, et al. Capecitabine and trastuzumab in heavily pretreated patients with 
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Pohlen U, Mansmann U, Berger G, et al. Multicenter pilot study of 5-fluorouracil, folinic acid, interferon alpha-
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Pohlen U, Rieger H, Mansmann U, Berger G, Buhr HJ.  Hepatic arterial infusion (HAI). Comparison of 5-
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RECIST guideline (version1.1).  Eur J Cancer 2009; 45: 228-47.  


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Appendix 1 
First contact with the subject and guidelines for oral 
information and obtaining consent 
First contact 
The patient will be referred to an Experimental Unit in the appropriate departments for 
intrahepatic treatment. Prior to referral, the patient will be approved by the Second Opinion 
Committee. This means that even before the information session, the patient is informed 
about the treatment at the referring oncology department. 
 
Information and consent process 
1. Before the information interview 
• an agreement must be made on the time and place of the conversation. 
• information shall be provided that it is a request to participate in a scientific trial 
• information must be provided about the right to a period of reflection after information 
and the possibility of bringing a family member/friend/other relevant person to the 
interview. 
 
2. The Information Conversation 
• must be carefully planned 
• must take place in an undisturbed setting 
• the participant must be given sufficient time to read the written information, listen to the 
oral information and ask questions (the written information is provided after the oral 
information is provided) 
• the investigator must inform the participant of the right to waive knowledge of his or her 
own health conditions 
• the information is provided by the investigator responsible for the study or by the 
person authorised to do so (sub-investigator = doctor) associated with the trial; 
 
3. Obtaining consent 
• the patient's consent to trial participation is given as soon as possible after the 
information interview, taking into account the necessary reflection period, which is at 
least one day  
 
  
 


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APPENDIX 2 
Biomarkers background 
The strategy in the fight against cancer is currently undergoing a paradigm shift. The goal 
is no longer to find a universal treatment, but to develop methods and treatments that, 
based on the latest technology and knowledge, focus on the individual patient. There is a 
great desire for better and more individualized treatments "personalized medicine" - via so-
called translational research. The individual patient will thus benefit the maximum from the 
research by bridging the gap between the laboratory and the clinic.  
A "Biomarker" is defined as "a character trait that can be objectively measured and 
evaluated as an indicator of a normal biological process, a pathological process, or a 
pharmacological response to a therapeutic intervention". Biomarkers include a spectrum of 
molecules with different characteristics but that share association with diseases and can 
be used for clinical detection of diseases (screening, diagnosis) and/or clinical follow-up 
(prognosis, treatment effect, monitoring) for early signs of disease worsening in patients 
and individualised treatment.  
Genetic variation is thought to be a major cause of difference in treatment effect of a given 
medication in the individual. Predictive and prognostic genetic biomarker profiles have 
gained ground in recent years in, among other things, the treatment of haematological and 
oncological patients, where it is currently possible for several diseases to identify patient 
groups that will respond to a given treatment. Genetic biomarkers can be defined as an 
objectively measurable characteristic e.g. DNA, single nucleotide polymorphisms (SNPs) 
and copy number variation (CNV), RNA, and micro(mi)RNA that is related to normal 
biological processes, pathological processes, and/or effect on treatment 
(www.fda.gov/cder/guidance/6400fnl.htm/ September 2004). Prognostic biomarkers can 
predict disease worsening independent of treatment, while predictive biomarkers can 
predict treatment response. Some biomarkers can be both prognostic and predictive. 
New analytical methods make it possible to examine patients' genome profile and thus 
obtain information specific to the individual patient. There is great potential in using the 
information from these advanced gene and miRNA analyses to tailor a treatment plan for 
the individual patient.  
New research has further demonstrated a complex interaction between a number of 
growth factors and their receptors, cytokines, metalloproteinases and their inhibitors, as 
well as matrix proteins produced by cancer cells and by the inflammatory cells and 
fibroblasts/myofibroblasts localized around the cancer cells. This interaction is partly 
responsible for cancer cell growth, differentiation, migration, and metastasis and is of great 
importance for patient survival. In recent years, there has been a significant development 
of biochemical analyses, which now enable serum/plasma concentration determinations of 
a number of potential biomarkers reflecting angiogenesis, growth factors and their 
receptors, inflammation, and the metabolism of extracellular tissue.  
It is likely that disease severity, treatment efficacy and prognosis of patients with breast 
cancer and spread to the liver before, during and after treatment with various forms of 
chemotherapy (e.g. HAI treatment) and new biologic treatment (e.g. Herceptin) can be 
better assessed by analysing a combination of a number of potential tumor biomarkers 
than by using only conventional blood tests (haematology and liver counts),  X-ray, 
Ultrasound, MRI and CT scans.  
There is no consensus yet on the use of new biomarkers in these patients. Hayes et al. 
(1996, 1998) have described a number of guidelines "Tumor Marker Utility Grading 
System" (TMUGS) to facilitate the implementation of potential new cancer biomarkers from 
the laboratory to daily clinical practice. It remains open whether the knowledge of these 
potential biomarkers, which may reflect mechanisms of breast cancer with spread to the 


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liver, has a real clinical applicability in the individual patient, so that clinical decisions can 
be made that will ultimately improve the prognosis and quality of life of the individual 
patient compared to not having been known about the biomarker. All new biomarkers are 
on a "Utility scale +" (defined as: "Assay probably associated with process or end point, 
but additional confirmatory studies are required") or a "Utility scale +/-" (defined as: 
"Preliminary data are suggestive that assay correlates with process, or with end point, but 
substantially more definitive studies are required").  No marker is on a "Utility scale ++" 
(defined as: "Definitive studies demonstrate that assay reflects process or end point").  
According to "TMUGS", a number of validation requirements must be met for a biomarker 
before it can be assumed to have achieved "Level of evidence I (LOE I)", after which 
clinical implementation is feasible. "LOE I" studies are either large prospective studies that 
specifically ask the question whether a biomarker has a clinical applicability to, say: 1) 
predict prognosis for the patient; 2) to predict the treatment effect of, for example, surgery, 
chemotherapy, or new biological treatment; or 3) that the biomarker provides better 
information about disease activity than the methods currently used. "LOE I" studies may 
also involve a review of meta-analyses of biomarker studies. The published studies on 
potential biomarkers in patients with breast cancer are on "LOE III". In particular, these are 
retrospective studies in which the blood samples are not primarily collected from patients 
with the intention of testing the value of the marker as a prognostic marker of time to 
progression of the disease or death, or to test their predictive value to assess the efficacy 
of treatment. Individual studies are at the middle level "LOE II", which consists of 
accompanying studies with prospectively collected blood samples as part of a therapeutic 
study, with pre-established endpoints and an evaluation of both the biomarker and the 
therapeutic treatment. 
In the current project, blood samples (whole blood, blood in PAXgeneRNA tubes, serums 
and various types of plasma) are stored for later analysis of known potential new 
biomarkers and for later analysis of future biomarkers of breast cancer with spread to the 
liver. Tissue biopsies of primary tumor as well as liver metastases and other metastases 
will also be stored for later relevant in situ, immunohistochemical, miRNA and gene 
analyses. 
 
The following tumor markers are today established in cancer mammae: 
 
Estrogen and progesterone receptor: Expression of these steroid hormone receptors 
predicts response to endocrine therapy and the receptors are well-established predictive 
markers that are currently used routinely. Studies have shown that the expression can be 
altered so that the primary tumor and metastasis do not have the same expression and it 
is recommended today internationally to repeat the study in patients who have developed 
relapse and have a focus available for biopsy (standard). 
HER2 (Human epithelial growth factor receptor, Neu, c-erbB2) is a transmembranous 
glycoproteine with an extracellular ligand-binding domain, a transmembranous part, and 
an intracellular domain with tyrosine kinase activity. Is a Tyrosine kinase receptor 
belonging to the IGF family. These receptors are an integral part of the signaling pathways 
that regulate cell growth, and they are closely related to the development of cancer. The 
expression of HER2 in breast cancer tissue is today routinely used as a predictive and 
prognostic marker, and can predict response to treatment with trastuzumab. The 
expression of HER2 in breast cancer tissue can be altered so that primary tumor and 
metastasis do not have the same expression and it is recommended today internationally 
to repeat the analyses in patients who have developed relapse and have a focus available 
for biopsy (standard). Further, there seems to be an interaction between HER2 and the 
hormone receptors, so that the receptors interact (cross talk). 


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Topo IIα: Topoisomerase II, a nuclear enzyme involved in cell division. Overexpression of 
the enzyme causes increased sensitivity to anthracyclines. The enzyme is localized on the 
same amplicon as HER2 and is often overexpressed in cells with overexpression of HER2. 
  
In addition to the above biomarkers, the following will be analyzed in the current 
project: 
 
Gene and microRNAarray expression: The use of gene arrays to measure gene 
expression profiles has great potential in cancer research. For example, cluster algorithms 
can be used to group tumors on the basis of genprofiles and statistical methods to identify 
genes based on their relevance in relation to different clinical characteristics and treatment 
responses. With these methods, new classes of hematological diseases and solid tumors 
have been identified. The development of gene signatures for prognosis, progression and 
respons to treatment (chemotherapeutics and antibodies) has also advanced but is still 
only used in a few cases in daily clinical practice. Genarray profiles can discriminate 
between normal and cancerous tissue, or can distinguish between different 
histopathological stages, and is likely to become useful in identifying patients with a poor 
prognosis and different treatment responses. 
MiRNAs are small non-coding 19-25 nucleotides that regulate gene expression post-
transcriptionally by binding to 3'UTR on mRNA. MiRNA is not translated into protein, but 
instead the primary product, pri-miRNA, is processed into short structures called pre-
miRNA and finally into mature miRNA. The mature miRNA, together with the protein 
complex RISC, has a regulatory function on protein synthesis. In cancer, altered 
expression of miRNA is observed. Dysregulation of miRNA in the cell is associated with 
cancer development and changes in miRNA are related to survival. Currently, about 700 
different human miRNAs are known, which regulate genes that are important for cancer, 
angiogenesis, stem cell differentiation, and inflammation. Several studies have recently 
been published on interesting microRNAs (e.g. mir-21, mir-22, mir-155, mir-191, mir-196, 
mir-200, mir-205, mir-221, mir-342, mir-375, mir-520) in patients with breast cancer, and 
hopefully it will be possible to find specific gene and miRNA array profiles in these patients 
to assess diagnosis, disease severity, treatment effect and prognosis.  
p53: a Tumor suppressor gene. Experimental studies suggest that mutations in this gene 
may be predictive of resistance to anthracyclines. A few studies have shown that mutated 
p53 is correlated to a poor prognosis. 
Thymidine Phosphylase (TP): TP is a key enzyme in the metabolism of 5-FU and 
capecitabine. TP is also known as tumor-associated angiogenic factor in that it has a high 
vascular formation potential and has anti apoptotic properties. The concentration is 3.2 
times higher in tumor tissue than in normal tissue. High concentration of TP is correlated to 
rapid malignant growth, aggressive invasive potential, and a poor prognosis. TP is 
upregulated by radiotherapy as well as by a variety of chemotherapeutics (e.g. paclitaxel, 
docetaxel, mitomycin C, gemcitabine and vinorelbin) as well as oxaliplatin. The modulation 
of TP during chemotherapy varies among patients. Studies are ongoing on whether, on the 
basis of TP status in the tumor and its modulation by chemotherapy, it will be possible to 
design specific treatment measures for different patients.     
EGFR: The epidermal growth factor receptor (EGFR) is a central transmembranous 
growth factor receptor that is commonly found in many normal human tissues. 
Immunohistochemical analysis of EGFR protein in cancer tissue biopsis is highly 
dependent on fixation and time for immunohistochemical staining, and a validated scoring 
system has not yet been established regarding the degree of EGFR protein occurrence in 
cancer biopsis. EGFR can be determined in serum by ELISA, but the method has not been 


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validated. In non-small-cell lung cancer treated with the EGFR inhibitor Tarceva, response 
has been found almost exclusively in never smokers, and it is postulated that smoking can 
induce a mutation in EGFR that causes this difference. It is unknown whether mutations in 
EGFR in breast cancer patients have predictive or prognostic value.  
PTEN: The PI3K-Akt signaling pathway is one of the signaling pathways activated by the 
HER family and is important for, among other things, cell survival. PTEN (Phosphatase 
and tensin homologue deleted on chromosome 10) is an enzyme that has an inhibitory 
effect on this particular signaling pathway. PTEN was first clarified as being a tumor 
suppressor for a particular brain tumor, but it has since been seen impaired function of this 
enzyme in several other cancers and it has an important regulatory function in cancer 
development. PTEN expression can be examined immunohistochemically from tissue 
samples. 
Interleukin-6: Interleukin-6 (IL-6), an immunomodulatory cytokine, is produced both by 
cancer cells (including breast cancer cells) and normal cells (pmacrophages, lymphocytes, 
fibroblasts, and endothelial cells). IL-6 plays an important role in the immune system, in the 
acute phase response, acts as a paracrine and autocrine growth factor for cancer cells, 
increases tumor growth and angiogenesis in vivo, and inhibits radiation and chemotherapy 
induced apoptosis and response. IL-6 expression in breast cancer is elevated compared to 
normal tissue and some patients with metastatic breast cancer have elevated plasma IL-6 
compared to healthy individuals, and high plasma IL-6 is associated with poor prognosis. 
Plasma IL-6 is an independent predictor of treatment response.  
C-reactive proteine: C-reactive proteine (CRP) is an acute phase reactant produced by 
hepatocytes and upregulated by pro-inflammatory cytokines, including IL-6 in association 
with inflammation, particularly infection. Serum CRP is correlated to IL-6 protein 
expression in the tissues of cancer patients as well as to serum IL-6. Preoperative serum 
CRP in patients with cancer has been found in some studies to be significantly related to 
tumor size, stage, and detected lymphatic and liver metastases. It has been found that 
elevated CRP is significantly related to relapse after curative resection of gastrointestinal 
cancer. A high pre- and postoperative serum CRP is associated with shorter survival in 
patients with many types of cancer, including breast cancer.  
Vascular endothelial growth factor: Vascular endothelial growth factor A (VEGF-A, et 45 
kDa glycoproteine) has a key role in the formation and maintenance of cancerous tissue 
blood vessels (tumor angiogenesis). VEGF stimulates the proliferation and migration of 
endothelial cells to form new vessels, protects endothelial cells from apoptosis, increases 
the vascular permeability of plasmaroteins and is stimulated, among other things, by 
hypoxia. VEGF is formed by cancer cells, macrophages, monocytes, leukocytes, 
thrombocytes, lymphocytes and fibroblasts. Overexpression of VEGF and its receptors has 
been found in most solid tumors (including breast cancer). High VEGF-A expression in 
tumor tissue is in some studies related to poor prognosis. In normal tissue, expression of 
VEGF is observed in epithelial tissues in areas with hypoxia. In patients with several 
different types of cancer, a high expression of VEGF-A is related to advanced disease 
stage and poor prognosis, and patients with high tumor burden have higher serum VEGF 
than patients with low tumor burden. After curative resection of primary colon cancer, 
serum VEGF decreases to normal values, whereas no changes in serum VEGF have been 
found in patients with colorectal cancer who have only had palliative resection. High serum 
VEGF in cancer patients is associated with a poor response to chemotherapy, rapid 
relapse and short survival, and serum VEGF is an independent prognostic variable. Due to 
the release of VEGF from leukocytes and thrombocytes, VEGF should be measured in 
plasma.  
YKL-40: YKL-40 (CHI3L1, a 40 kDa glycoprotein) is formed by various cancer cells 
(including breast cancer) and may have implications for cancer cell proliferation and 


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survival, angiogenesis, and metastasis/invasive growth.  In vitro studies of glioblastoma 
cell lines have shown increased YKL-40 production by stress influences, such as hypoxia 
and radiotherapy. High expression of YKL-40 protein in breast cancer is not associated 
with the prognosis. A high serum YKL-40 (compared to healthy persons) is related to a 
short time to disease progression and death in patients with localized or metastatic breast, 
colorectal, small cell lung, prostata, ovarian, head-neck, cervix, and renal cell cancer, 
malignant melanoma, and acute myeloid leukemia. Serum YKL-40 is an independnet 
biomarker that is independent of serum HER2, estrogen receptor status, CEA, CA125, 
PSA, and LDH. A relationship has been found between high serum YKL-40 and 
chemoresistance in patients with metastatic breast and ovarian cancer. 
PINP and PIIINP: Cancer cells are involved in extracellular matrix remodeling processes 
and regulate both the degradation and formation of the surrounding extracellular matrix. 
Tye I and III collagen, which are the most abundant collagens in the connective tissue, are 
synthesized as prokollagens with proprptides at both ends (PINP and PIIINP, 
respectively). After cleavage, these are released into the blood. Serum PINP and PIIINP 
concentrations therefore reflect the synthesis of type I and III collagen. There is an 
increased amount of type I and III collagen in the connective tissue around the cancer 
cells, the desmoplastic reaction, and this has an impact on the malignant phenotye of the 
cancer cells. A relationship has been found between serum PINP and chemoresistence 
and aggressiveness of the disease in patients with metastatic breast cancer, and in 
patients with colorectal cancer, serum PIIINP was an early prognostician for postoperative 
recurrence.  
TIMP-1: Tissue inhibitor of metallorpoteinase (TIMP-1) regulates metalloproteinase activity 
and is a growth factor for cancer cells in vitro, stimulates angiogenesis and inhibits 
apoptosis. The expression of TIMP-1 mRNA and protein in various cancerp tissues such 
as breast, colorectal, ventricular and pulmonary cancer, as well as malignant melanoma is 
elevated relative to normal tissue, and high expression is associated with distant 
metastases and poor prognosis. Cancer cells stimulate surrounding connective tissue cells 
to produce TIMP-1, and genarray has shown several times increased exression of TIMP-1 
in cancer tissue compared to normal tissue. TIMP-1 is localized in the connective tissue 
and basal membranes, primary at the invasive edge of the tumor. Plasma TIMP-1 
concentration is elevated in patients with various solid tumors such as metastatic breast, 
colorectal, bladder, prostata, and renal cancer and related to localization, stage, and 
prognosis. In patients with colorectal cancer, plasma TIMP-1 is also a sensitive biomarker 
for identifying both early stages (Dukes' A and B) and right-sided colon cancer, and in 
combination with serum CEA, the sensitivity of plasma TIMP-1 for the identification of 
patients with colorectal cancer increased. High pre-operative  plasma TIMP-1 in these 
patients is the strongest prognostic biomarker for short survival and independent of Dukes' 
stages and other prognostic biomarkers.  
Proteases: Plaminogen activator system, plays an important role in remodulation of 
extracellular matrix as well as metastasis and invasive processes. Preliminary studies 
have shown that high expression of the uPA receptor (uPAR) may be a poor prognostic 
factor. Further, urokinase-type plasminogen activator (uPA) and plasminogen activator 
inhibitor type-1 (PAI-1) are key components of the tissue-degrading processes associated 
with cancer, and their expression likewise correlated with poor prognosis in several cancer 
types. 
Protein array: Only a few% of the proteins circulating in the blood are known today, and it 
is likely that several new biomarkers will be identified in the near future. Serum, plasma 
and blood cells will therefore be collected and stored for the determination of future as yet 
unidentified biomarkers that could later be studied with protein array.  


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Metabolomic: There are many thousands of metabolites (molecules with molecular weight 
<1500 Da) in humans. "Metabolomics" is the study of metabolites in biological fluids, 
tissues, and isolated cells. "Metabolome" represents a dynamic unit that is related to the 
genome of a particular organism and reflects the sustained interaction of metabolic and 
signaling pathways in relation to interaction with the environment, including diet, 
medication, pathophysiological characteristics, etc. Metabolomic studies result in complex 
data sets that require statistical methods for understanding.  
Nuclear Magnetic Resonance (NMR) spectroscopy is an accurate method based on the 
spin of atomic nuclei. NMR can be used to investigate the structure of molecules and their 
spatial and electronic structure and requires minimal sample preparation. This is of 
importance when examining biological fluids from patients. NMR spectra (normal 1H NMR 
spectra) of biological samples contains signals of many small molecules present in the 
sample at a concentration of > 1 M. NMR spectra or profiles of biological samples (blood 
samples, tissues) have been found informative in terms of diagnosis and prognosis of 
various diseases, including cancer diseases, allowing the division of patients into different 
categories (healthy vs. diseased,  patients who either have vs. do not have the effect of 
the treatment). The purpose of these procedures in the current study is to find 
relationships between metabolic parameters and disease status as well as efficacy of 
treatment in patients with breast cancer and liver metastases treated with intrahepatic 
chemotherapy and in some additionally in combination with trastuzumab.  
 
Material 
A biobank will be established with blood from patients before, during and after treatment 
and with tissues from both primary tumor and metastases. 
Blood samples: Whole blood is collected in PAXgene™ Blood RNA Tubes (Qiagen) and 
stored at -80oC. Serum, EDTA, citrate and heparin plasma are collected and stored at -
80oC. Cells (Buffycoat) from the EDTA glass are stored at -80oC. Extraction of DNA, 
mRNA and miRNA is carried out using Tri-X, which is a method where DNA, mRNA and 
miRNA can be isolated simultaneously from a PAXgen tube. miRNA is further purified from 
EDTA plasma and/or serum. 
Tissue samples: Tissues from primary tumour (formalin fixed/parafine-embedded) and 
tissues from liver metastases, and other metastases (fresh frozen tissue and formalin 
fixed/parafine-embedded) are stored for examination of any changes in the expression of 
the biomarkers. This is considered standard study, as several studies have shown that 
biomarkers can change in the course and the treatment offer (e.g. +/- trastuzumab) will be 
dependent on the expression of HER2 and partly for the purpose of biobanking.  Patients 
can also be included in the protocol if they do not want a biopsy from the liver.  
The biological material will only be used for a new research project after obtaining 
permission from the Research Ethics Committee. The material will be stored for 15 years.  
 
 
Methods 
Routine methods: The expression of estrogen receptor, progesterone receptor, HER2, 
topoIIα, p53, VEGF, EGFR, YKL-40, TIMP-1 and PTEN in tumor biopsies is determined 
with routine methods at the pathological department, Herlev Hospital.  
ELISA: Comercial ELISA is used to determine plasma VEGF (R&D Systems, UK), plasma 
IL-6 (R&D Systems, UK), serum EGFR (R&D Systems, UK), serum YKL-40 (Quidel 
Corporation, USA), serum HER2 (Dako, Denmark), plasma TIMP-1 (Abbott, USA), and 
serum P1NP (in-house). Serum P3NP is determined with RIA (Orion Diagnostica, Espoo, 
Finland). It applies to all these biomarkers that the validity of the methods is well 


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researched. CRP is measured as a routine blood test at the Department of Clinical 
Biochemistry. 
Genarray profiles are determined with Affymetrix U133 plus 2.0 Gene Chip System at 
Mogens Kruhøffer, AROS Applied Biotechnology A/S in Aarhus. 
MiRNAarray profiles are determined with Low Density Array (LDA) assays (Applied 
Biosystems) at Mogens Kruhøffer, AROS Applied Biotechnology A/S in Aarhus.  If there is 
a better miRNA array method in the future, it will be used.  
Metabolomic: The studies are carried out at Mogens Kruhøffer's collaborators at the NMR 
laboratory at CIRMMP, CERM, Italy (www.cerm.unifi.it), where there is a 600 MHz NMR 
spectrometer (Bruker BioSpin) with a 5 mm  1H-31P-15N cryoprobe and include an 
automatic tuning-matching (ATM) and an automatic sample switching and used primarily 
for metabolomic analyses.  A 900 MHz NMR spectrometer (Bruker BioSpin) equipped with 
a 5 mm TXI cryoprobe also exists and can be used for experiments needed for the 
identification of biomarkers. AROS and CERM have the necessary statistical competence 
to assess these analyses.  
 
 
Statistical methods 
The statistical analyses are carried out in collaboration with professional statisticians, 
among others, at AROS and CERM.  
Relevant demographic, clinical, histo-pathological and clinical chemical data, metastasis 
localization, medication, co-morbidity, as well as treatment repons, time to disease 
progression and time to death will be recorded in a data bank.  
The level (high versus low/normal) of potential biomarkers will relate to time to progression 
of disease and survival and will be estimated with the Kaplan-Meier method and compared 
with log-rank testing. Catagory variables are indicated by median followed by the span. 
The response rate is assessed in eluable population. Univariate and multivariate analyses 
will be made. 
The calculations of the gene and microRNA array studies will use, among other things, 
two-dimensional unsupervised hierarchial clustering, "The prediction Analysis of 
Microarrays(PAM)" version 2.1, R-software package (www.r-project.org) and Plink 
software (http://pngu.mgh.harvard.edu/~purcell/plink/), and "The Significance Analysis of 
Microarrays (SAM)" version 3.0.  
 
Risk assessment 
Project blood samples will only be taken in conjunction with other scheduled routine blood 
sampling. Likewise, the tissue samples necessary for the project will be taken at the same 
time as the patient has a biopsy taken to determine changes in conventional biomarkers. 
Thus, there is no independent risk in the project. 
After the biopsy, there may be discomfort in the form of soreness due to blood 
accumulation and there may be a risk of bleeding. Performing a biopsy is a routine 
function. 
Before treatment and at the planned scans for disease evaluation after every 4th series, 
63.5 ml of blood will be taken for project blood samples along with 10 ml of blood for 
routine blood tests. Project blood samples will be taken up to and including the time of 
disease progression. In most patients, a total of between 254 ml and 381 ml of blood will 
be taken over a period of 6 to 10 months, which does not pose a risk to the patient. 
 
Ethical considerations 


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One of the aims of the project is to investigate whether determining a number of new 
potential biomarkers can improve the determination of treatment response and prognosis, 
as well as the monitoring (for early signs of disease progression) of patients with 
metastatic breast cancer. The patients will have the extra blood samples taken at the 
same time as the patient will still have routine blood tests. 63.5 ml of extra blood will be 
taken, which does not pose a risk to the patient. Examination of the primary tumor does 
not pose any risk to the patient. Patients included in this study will not be informed of the 
results of the studies and the study results will not have therapeutic consequences. The 
basis for this decision is that the sensitivity and specificity of the analytical techniques 
used, as well as the significance of any findings, have not been clarified and that the 
average life expectancy for this patient group is 2 years. In addition, the analyses will not 
necessarily be carried out in the immediate aftermath of the sampling. After the first 2 
years, in connection with the evaluation of the methods, a decision will be made as to 
whether the methods should be established as standard analyses. At this stage, it will also 
be assessed whether future subsequent patients should be informed of the results.  


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Appendix 2.  
Guidelines for blood sample forms for 
HAI Mammae  
 
Upon inclusion, a patient specific blood sample 
form is prepared 
 
 
Flowchart of blood sample form 1 
The patient is registered on form 1 with Hospital, Patient 
Initials, and Patient No. as well as date of inclusion.  
In the form, the number of greiner tubes and Paxgen RNA 
tubes stored in the freezer are noted. 
The patient is given his own chartek, in which the blood 
sample forms are stored together with a copy of informed 
consent. 
 
Freezer boxes  
Each visit has its box. 
Each sample type has its box. 
It is noted in scheme 1 in which box (no.), where the greiner 
tubes are placed. 
 
 
If it is difficult to retrieve all blood samples from the patient, 
priority is given as follows: 
PURPLE EDTA glass 
RED dry glass  
PAXGEN tube  
BLUE citrate glass 
GREEN heparin glass 


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HAI Mammae contact names for blood sample handling 
 
 
 
…………………. 
Bioanalyst 
Clinical Research Unit 65C8 
Herlev Hospital 
Herlev Ringvej 75 
2730 Herlev 
…………………. 
……………………………..…. 
 
 
 
……………………….. 
Bioanalyst 
Clinical Research Unit 65c8 
Herlev Hospital 
Herlev Ringvej 75 
2730 Herlev 
…………………. 
……………………………..…. 
 
 
 
……………………….. 
Consultant 
Oncology Dept.  
Herlev Hospital 
Herlev Ringvej 75 
2730 Herlev 
…………………. 
……………………………..…. 


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HAI Mammae flowchart for blood samples 
Hospital: 
Patient initials:    
Patient number: 
For blood sample management – see Laboratory folder   
 
 
 
 
 
Table 1 
VISIT 
A 
B1 
B2 
B3 
B4 
B5 
B6 
B7 
Before 
treatment 
1. Scan 
after 4.  
series 
2. Scan 
after 8.  
series 
3. Scan 
after the 12. 
series 
4. Scan 
follow-up 
5. Scan 
follow-up 
6. Scan 
follow-up 
7. Scan 
follow-up 
Blood sample taken 
Date                   
 
 
 
 
 
 
 
 
3 X 6 ml Red/Black  
Dry glass for SERUM 
Greiner tube 5 pcs RED 
 
 
 
 
 
 
 
 
3 X 6 ml Purple/Black  
EDTA glass for EDTA plasma 
Greiner tube 5 pcs BLUE 
 
 
 
 
 
 
 
 
3 X 3.5 ml Light Blue/Black 
Citrate Glass for CITRATE 
Plasma 
Greiner tube 5 pcs WHITE 
 
 
 
 
 
 
 
 
3 X 4 ml Green/Black Heparin 
Glass for HEPARIN Plasma 
Greiner pipe 3 pcs GREEN 
 
 
 
 
 
 
 
 
Buffycoat from 3 EDTA 
glasses 
Greiner tube 3 pcs 
YELLOW 
 
 
 
 
 
 
 
 
 
 
2 Paxgene RNA tubes 
 
 
 
 
 
 
 
 
 
 
               
Signature 
 
 
 
 
 
 
 
 
 


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HAI Mammae handling blood samples 
 
   
 
Sampling 
ALL VISITS 
3 X 6 ml RED 
DRY GLASS 
3 X 6 ml 
PURPLE 
EDTA GLASS 
3 X 3.5 ML 
BLUE 
CITRATE 
GLASS 
3 X 4 ml 
GREEN 
HEPARIN 
GLASS 
3 tubes with  
Buffy coat 
 
2 X PAXGEN RNA 
tube 
 
PREPARING 
 
At least 30 min.  
 
Centrifuged at 
2330g  
10 min  
 
4° C 
 
Serum is 
transferred to   
5 x cryo tube 
with red lid 
minimum 500 μl 
At least 30 min.  
 
Centrifuged at 
2330g  
10 min  
 
4° C 
 
Plasma is 
transferred to  
5 x cryo tube 
with blue lid 
minimum 500 μl 
 
At least 30 min.  
 
Centrifuged at 
2330g  
10 min  
 
4° C 
 
Plasma is 
transferred to   
5 x cryo tube 
with white lid 
minimum 500 μl 
At least 30 min.  
 
Centrifuged at 
2330g  
10 min  
 
4° C 
 
Plasma is 
transferred to   
3 x cryo tube 
with green lid 
minimum 500 μl 
Use the glass from 
the EDTA plasma 
 
Remove plasma to 
approximately.  over 
cell layers.5 mm 
 
Take all the white 
blood cells from 1 
glass and put them 
in 1 cryo tube with 
yellow lid 
1 glass = 1 cryo 
tube 
So you have a total 
of 3 cryo tubes. 
Should be turned 
over gently 8-10 
times, 
IMMEDIATELY after 
blood sampling! 
 
 
Left for 2 - 48 hours 
at room temperature. 
 
Turn over before 
freezing the pipe. 
 
STORAGE 
 
-80°C freezes 
 
-80°C freezes 
 
-80°C freezes 
 
-80°C freezes 
 
-80°C freezes 
 
Freeze at -20° C for 
24 - 72 hours. 
Move to -80°C 
freeze. 


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Appendiks 3. 
New York Heart Association (NYHA) Classification System 
 
Class I: 
Patients with heart disease but without accompanying restrictions on physical activity. 
Ordinary physical activity does not cause excessive fatigue, palpitations, dyspnea or 
anginas pain. 
Class II: 
Patients with heart disease, which results in slight restriction in physical activity. They 
feel good at rest. Regular physical activity causes fatigue, palpitations, dyspnea or 
anginas pain. 
Class III: 
Patients with heart disease, which results in marked restriction in physical activity. 
They feel good at rest. Less than regular physical activity causes fatigue, palpitations, 
dyspnea or anginas pain. 
Class IV: 
Patients with heart disease that results in the inability to perform any physical activity 
without discomfort. Symptoms of heart failure or angina may also be present at rest. 
Discomfort increases with any physical activity. 
Ref.:  Textbook Of Medicine. Vol 2, pp2228, Oxford University Press, 1997.Oxford