Does Anesthesia Technique Affect the Presence of Circulating Tumor Cells in Primary Breast Carcinoma? A Randomised Controlled Trial.
Phase: PHASE4
Conditions: Female Breast Carcinoma
Sponsor: University of Zurich

Clinical Study Protocol 
Does anesthesia technique affect the presence of circulating 
tumor cells in primary breast carcinoma? 
A phase 4, randomized, controlled, double blinded, multicentric trial investigating the 
effect of anesthetics (sevoflurane versus propofol) on circulating tumor cells positivity 
in primary breast cancer patients 
CONFIDENTIAL 
The information contained in this document is confidential and the property of the Institute of 
Anesthesiology of the University Hospital Zurich. The information may not - in full or in part - be 
transmitted, reproduced, published, or disclosed to others than the applicable Independent 
Ethics Committee(s) and Regulatory Authority(ies) without prior written authorization from the 
Institute of Anesthesiology of the University Hospital Zurich, except to the extent necessary to 
obtain informed consent from those subjects who will participate in the study. 
Sponsor-Investigator and Principal 
Investigator: 
Prof. Dr. med. Beatrice Beck 
Schimmer 
Co-Investigator: 
Dr. med. Frédérique Hovaguimian 
Local Principal Investigator 
Prof. Dr. med. Manfred Seeberger 
Study Product: 
sevoflurane versus propofol  
Protocol Version and Date: 
Version 1.4, Date: 20.03.2017 
Short title: 
CTC_BREAST Study 

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STUDY SYNOPSIS 
 
 
Sponsor-
Investigator 
Prof. Dr. med. Beatrice Beck-Schimmer 
Study Title: 
“Does anesthesia technique affect the presence of circulating 
tumor cells in primary breast carcinoma?” 
A phase 4, randomized, controlled, double blind,multicentric 
 trial investigating the effect of anesthetics (sevoflurane 
versus Propofol) on the presence of circulating tumor cells in 
primary breast cancer patients 
Short Title/Study 
ID: 
CTC_ Breast Study 
Protocol Version 
and Date: 
Version 1.4, Date: 20.03.2017   
Clinical Phase: 
Clinical study phase 4 
Methodology: 
Prospective, randomized, double blind, controlled trial 
Study Duration: 
March 2014 to July 2018 
Study Center(s): 
Multicentric study,  
University Hospital Zurich 
Klinik Hirslanden Zurich 
Sponsor 
Investigator: 
Prof. Dr. med. Beatrice Beck-Schimmer  
Institute of Anesthesiology 
Raemistrasse 100 
CH-8091 Zurich 
Email: beatrice.beck@usz.ch 
Telefon: +41 44 255 20 32- Fax: +41 44 255 44 09 
Local Principal 
Investigator 
Prof. Dr.med Manfred Seeberger 
Institute of Physicians for Anesthesia and Intensive Care 
Klinik Hirslanden 
Wittelikerstr. 40 
CH-8032 Zurich 
Email: manfred.seeberger@hirslanden.ch 
Phone: +41 44 387 38 80 
Fax: +41 44 387 38 85 
Objective(s)/ 
Outcome(s): 
> Primary outcome: to determine if there is a difference in 
the mean of blood CTC levels over time among patients 
exposed to volatile anesthetics (i.e. receiving sevoflurane) 
compared to patients in the control group (i.e. receiving 
intravenous anesthesia with propofol) 
> Secondary outcomes:  
A) to determine if anesthesia interferes with the number and 
activity of blood natural killer (NK) cells 
 

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B) to examine if CTC levels correlates with the presence of  
extracapsular spread of tumor cells (ECS) in the 
histopathological findings in axillary lymph nodes 
Number of 
Subjects: 
Total number of 232 patients. 
Diagnosis and  
 
 
Main Inclusion 
Criteria: 
 
Diagnosis: primary breast cancer  
 
Inclusion Criteria:  
> Female 
> Age 18 to 85 
> ASA I-III 
> Primary breast cancer (TNM stage = T1-3, N0-2, M0) 
> Primary surgery  
> Written informed consent 
Main Exclusion 
Criteria: 
> Metastatic breast cancer 
> Other than primary surgery (recurrence, reconstruction) 
> Pre-operative chemotherapy or radiotherapy 
> Auto-immune disease, HIV, other active cancer, age>85, 
ASA IV or V 
> Concomitant regional anesthesia 
> Chronic opioids medication 
> Any systemic immunosuppressive therapy 
> Known hypersensitivity or suspected allergy to propofol, 
soya or egg proteins 
> Known hypersensitivity to volatile anesthetics (malignant 
hyperthermia) 
> Pregnancy 
> Breast feeding 
> Non German-speaking patients 
> Enrollment in any other clinical trial during the course of 
this trial, 30 days prior to its beginning or 30 days after its 
completion 
Study Product, 
Dose, Route, 
Regimen: 
Sevoflurane will be used in its inhalational form via an 
endotracheal tube for maintenance of anesthesia. The dose 
and regimen will be adjusted to keep an adequate depth of 
anesthesia (MAC 0.8-1.2), according to BIS values between 
40 and 60 
Duration of 
administration: 
Administration will be limited to the length of the surgical 
procedure 
Reference 
therapy, Dose, 
Route, Regimen: 
Propofol will be intravenously administered via a target-
controlled infusion (TCI) device providing a drug dose 
ranging from 3.0 to 5.0 mcg/ml. The dose and regimen will 
be adjusted to keep an adequate depth of anesthesia, 
attested by BIS values between 40 and 60. 
Study Schedule: 
March 2014 to July 2018  

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Statistical 
Methodology: 
Primary analysis : the number of CTC in peripheral blood will 
be measured before and after exposition to general 
anesthesia using a linear mixed model with random effects 
matrix, which accounts for within-subject treatment factor (i.e. 
anesthesia regimen) 
Secondary analyses: since most reports refer to CTC levels 
as a binary outcome (i.e. positive versus negative endpoints 
using a cut-off value of ≥5CTC/7.5 ml blood), data 
transformation from continuous into a binary variable will be 
performed using the same cut-off value. This new data set 
will be analyzed using a logistic regression mixed model with 
random effects matrix. 
All primary and secondary analyses will be performed 
following an intention-to-treat (IIT) model. This analysis will 
subsequently compared to a per-protocol analysis to ensure 
stability of results. Results will be reported as OR, 95% 
confidence intervals and their associated p-values. A p-value 
< 0.05 will be considered statistically significant 
GCP Statement: 
This study will be conducted in compliance with the protocol, 
the current version of the Declaration of Helsinki, and ICH-
GCP as well as all national legal and regulatory 
requirements.  
 
 

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STUDY SCHEDULE 
 
Study Periods 
Screening 
Treatment Period 
Follow-up 
Visit 
1 
2 
3 
4 
5 
Postoperative day 
-1 
0a 
0b 
2 
3 
Medical history 
x 
 
 
 
 
Physical Examination 
x 
 
 
 
 
Vital signs 
 
x 
x 
x 
x 
Concomitant therapy 
x 
x 
x 
x 
x 
In-/Exclusion Criteria 
x 
 
 
 
 
Subject information 
and informed consent 
x 
 
 
 
 
Pregnancy test 
x 
 
 
 
 
Randomization 
 
x 
 
 
 
Study medication 
(sevoflurane or 
propofol) 
 
 
x 
 
 
1° variable: CTC level 
 
x 
x 
x 
x 
2° variable A 
 
x 
x 
x 
x 
2° variable B* 
 
 
x 
 
 
2° variable C 
 
 
 
 
 
Perioperative data 
x 
x 
x 
x 
x 
Safety variables: AE 
  
  
x 
x 
x 
 
 
Visit 1: 24h before surgery 
Visit 2 (0a): before induction of anesthesia    
Visit 3 (0b): end of surgery, before extubation 
Visit 4: 48h after surgery 
Visit 5: 72h postoperative, before hospital discharge 
 
 
Primary variable: assessment of circulating tumor cells in peripheral blood 
Secondary variable A: assessment of natural killer (NK) cells (number and activity)  
Secondary variable B: extracapsular tumor spread in axillary nodes 
* Data will be collected after completion of the study  
Perioperative data: demographics and confounding factors  

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TABLE OF CONTENTS 
 
1. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ....................................... 8 
2. 
INVESTIGATORS AND STUDY ADMINISTRATIVE STRUCTURE.............................. 9 
3. 
ETHICS .......................................................................................................................10 
3.1 
Independent Ethics Committee (IEC) and Regulatory Authorities .......................10 
3.2 
Ethical Conduct of the Study ..............................................................................10 
3.3 
Subject Information and Informed Consent ........................................................10 
4. 
INTRODUCTION .........................................................................................................11 
4.1 
Background and Rationale .................................................................................11 
4.2 
Pre-clinical data and evidence from clinical research .........................................12 
4.2.1 Effects of anesthetics on tumor cells ...........................................................................13 
4.2.2 Effect of propofol and sevoflurane – clinical evidence .................................................15 
4.2.3 Secondary Outcomes: .................................................................................................15 
4.3 
Investigational Product and Dose Rationale .......................................................17 
4.4 
Risk/Benefits and Ethical Considerations ...........................................................18 
5. 
STUDY OBJECTIVES AND OUTCOMES ...................................................................19 
5.1 
Primary Variable: circulating tumor cells (CTC) ..................................................19 
5.2 
Secondary Variables ..........................................................................................19 
5.2.1 Secondary variable A: NK cells (number and activity) .................................................19 
5.2.2 Secondary variable B: extracapsular tumor spread (ECS) ...........................................19 
5.2.3 Other study variables ..................................................................................................20 
5.2.4 Safety variables ...........................................................................................................20 
6. 
STUDY DESIGN .........................................................................................................21 
6.1 
General Design ..................................................................................................21 
6.2 
Primary Study Endpoint: blood CTC levels .........................................................22 
6.3 
Secondary Endpoints .........................................................................................22 
6.3.1 Anesthesia, NK cells and tumor spreading ..................................................................22 
6.3.2 CTC positivity and extracapsular tumor spreading.......................................................22 
6.3.3 Safety Endpoints .........................................................................................................22 
6.4 
Study procedures (fig.1) .....................................................................................22 
6.4.1 Visit 1 (preoperative day) .............................................................................................22 
6.4.2 Visit 2 (day of surgery – before induction of general anesthesia) .................................23 
6.4.3 Visit 3 (day of surgery – end of surgery, before extubation) .........................................23 
6.4.4 Visit 4 and 5 (48h and 72h postoperative) ...................................................................23 
7. 
SUBJECT SELECTION AND WITHDRAWAL .............................................................25 
7.1 
Inclusion Criteria ................................................................................................25 
7.2 
Exclusion Criteria ...............................................................................................25 
7.3 
Subject Recruitment and Screening ...................................................................26 
7.4 
Early Withdrawal of Subjects ..............................................................................26 
7.4.1 Criteria for Early Withdrawal of Subjects .....................................................................26 
7.4.2 Data Collection and Follow-up for Withdrawn Subjects................................................26 
7.5 
Early Termination of Study .................................................................................27 
8. 
TREATMENTS ............................................................................................................28 

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8.1 
Treatments to be Administered ..........................................................................28 
8.2 
Method of Assigning Subjects to Treatment Groups ...........................................28 
8.3 
Treatment Compliance .......................................................................................28 
8.3.1 Study drugs  ...............................................................................................................28 
8.3.2 Medications susceptible to interfere with outcome measure ........................................29 
8.4 
Prior and Concomitant Therapy ..........................................................................29 
8.5 
Packaging, Labelling and Supply........................................................................30 
8.6 
Blinding ..............................................................................................................30 
8.7 
Storage Conditions .............................................................................................31 
8.8 
Study Drug Accountability ..................................................................................31 
8.9 
Return or Destruction of Study Drug ...................................................................31 
9. 
STUDY PROCEDURES ..............................................................................................32 
9.1 
Blood samples for CTC/NK assessment .............................................................32 
9.2 
Study drug administration ...................................................................................32 
9.3 
Pregnancy test ...................................................................................................32 
10. 
SAFETY ......................................................................................................................33 
10.1 Safety Variables .................................................................................................33 
10.2  Definition of (Serious) Adverse Events ...............................................................33 
10.3 Recording of (Serious) Adverse Events ..............................................................34 
10.4 Assessment of Causatility (Serious) Adverse Events .........................................35 
10.5 Reporting of Serious Adverse Events .................................................................35 
10.6 Follow up of (Serious) Adverse Events ...............................................................37 
11. 
DATA QUALITY ASSURANCE ...................................................................................38 
11.1 Routine Monitoring .............................................................................................38 
11.2 Audits and Inspections .......................................................................................38 
11.3 Specification of Source Documents ....................................................................38 
12. 
STATISTICS ...............................................................................................................40 
12.1 Statistical and Analytical Plans ...........................................................................40 
12.2 Null and Alternative Hypotheses.........................................................................40 
12.3 Planned Analyses ..............................................................................................40 
12.3.1 
Primary Analyses ................................................................................................41 
12.3.2 
Secondary Analyses ...........................................................................................41 
12.3.3 
Interim Analyses .................................................................................................41 
12.4 Handling of Missing Data ...................................................................................41 
12.5 Determination of Sample Size ............................................................................42 
13. 
DATA HANDLING AND RECORD KEEPING ..............................................................43 
14. 
CONFIDENTIALITY ....................................................................................................44 
15. 
INSURANCE ...............................................................................................................45 
16. 
STUDY REGISTRATION ............................................................................................46 
17. 
PUBLICATION POLICY ..............................................................................................47 
18. 
FINANCIAL DISCLOSURE .........................................................................................48 
19. 
SIGNATURES .............................................................................................................49 
20. 
REFERENCES ............................................................................................................51 

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1.  LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
 
 
AE
Adverse Event 
ASA
American Society of Anesthesiologists
BIS
Bispectral index
COX
Cyclo-Oxygenase
CRF
Case Report Form 
CTC
Circulating Tumor Cells
eCRF
Electronic Case Report Form 
ECS
Extra Capsular Spreading
EpCAM
Epithelial Cell Adhesion Molecule 
GCP
Good Clinical Practice 
HIV
Human Immunodeficiency Virus
HR
Hazard Ratio 
IB
Investigator’s Brochure
ICH
International Conference on Harmonization
ISF
Investigator Site File 
ITT
Intention to treat
KISIM
Electronic patient chart at the UHZ
MAC
Minimal Alveolar Concentration
NK
Natural Killer
PACU
Post Anesthesia Care Unit
PI
Principal Investigator 
PONV
Postoperative nausea and vomiting
RT-PCR
Reverse Transcription Polymerase Chain Reaction
SAE
Serious Adverse Event 
SDV
Source Data Verification 
SOP
Standard Operating Procedure
SUSAR
Suspected Unexpected Serious Adverse Reaction 
TCI
Target-Controlled Infusion
Th1/Th2
T-helper cell 1 and 2
TMF
Trial Master File 
TNF
Tumor Necrosis Factor
TNM stage
Classification of malignant tumors (Tumor; Node; Metastasis)
UHZ
University Hospital of Zürich
VAS
Visual Analog Scale

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2.  INVESTIGATORS AND STUDY ADMINISTRATIVE STRUCTURE  
 
 
Sponsor-Investigator 
(Principal Investigator): 
 
Name: Prof. Dr.med. Beatrice Beck 
Schimmer 
Address: Institute of Anesthesiology, 
University Hospital Zurich,  
Raemistrasse 100, CH-8091 Zürich 
Email: beatrice.beck@usz.ch 
Phone: +41 44 255 20 32 
Fax: +41 44 255 44 09 
Local Principal Investigator 
Hirslanden 
Name: Prof. Dr.med. Manfred Seeberger 
Institute of Physicians for Anesthesia and 
Intensive Care 
Klinik Hirslanden 
Wittelikerstr. 40 
CH-8032 Zurich 
Email: manfred.seeberger@hirslanden.ch 
Phone: +41 44 387 38 80 
Fax: +41 44 387 38 85 
Study Site(s): 
Name: University Hospital Zurich 
Address: Rämistrasse 100, CH-8091 
Zurich 
 
Name: Klinik Hirslanden 
Address: Witellikerstr. 40, CH-8032 
Zurich 
Co-Investigator: 
UniversitätsSpital Zürich 
 
Name: Dr. med. Frédérique Hovaguimian  
Address: Institute of Anesthesiology, 
University Hospital Zurich, 
Rämistrasse 100, CH-8091 Zurich 
Email: frederique.hovaguimian@usz.ch 
Phone: +41 44 255 11 11 
Fax: +41 44 255 44 09 
Co- Investigator 
Hirslanden 
Name: med. pract. Urs Rölli  
Institute of Physicians for Anesthesia and 
Intensive Care 
Klinik Hirslanden 
Wittelikerstr. 40 
CH-8032 Zurich 
Email: urs.roelli@hirslanden.ch 
Phone: +41 44 387 21 11 
Fax: +41 44 387 38 85 

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3. ETHICS 
3.1 
Independent Ethics Committee (IEC) and Regulatory Authorities 
Before this study will be conducted, the protocol, the proposed subject information and 
consent form as well as other study-specific documents will be submitted to a properly 
constituted Independent Ethics Committee (IEC) in agreement with local legal 
requirements, for formal approval. Any amendment to the protocol must be approved 
by these institutions. 
The decision of the IEC concerning the conduct of the study will be made in writing to 
the Sponsor-Investigator before commencement of this study. 
 
3.2 
Ethical Conduct of the Study 
The study will be carried out in accordance with principles enunciated in the current 
version of the Declaration of Helsinki, the guidelines of Good Clinical Practice (GCP) 
issued by ICH, and Swiss regulatory authority’s requirements.  
IEC and regulatory authority will receive annual safety and interim reports and be 
informed about study stop/ end in agreement with local requirements. 
 
3.3 
Subject Information and Informed Consent 
The investigator must explain to each subject the nature of the study, its purpose, the 
procedures involved, the expected duration, the potential risks and benefits and any 
discomfort it may entail. Each subject must be informed that the participation in the 
study is voluntary and that he/she may withdraw from the study at any time and that 
withdrawal of consent will not affect his/her subsequent medical treatment.  
The subject must be informed that his/her medical records may be examined by 
authorized individuals other than their treating physician. 
All subjects for this study will be provided a subject information sheet and a consent 
form describing this study and providing sufficient information for subjects to make an 
informed decision about their participation in this study.   
The subject information sheet and the consent form will be submitted with the protocol 
for review and approval for the study by the IEC. The formal consent of a subject, using 
the approved consent form, must be obtained before that subject is submitted to any 
study procedure.   
The subject should read and consider the statement before signing and dating the 
informed consent form, and should be given a copy of the signed document. The 
consent form must also be signed and dated by the investigator (or his designee) and 
it will be retained as part of the study records. 

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4. INTRODUCTION 
This document is a protocol for a human research study. This study is to be conducted 
according to international standards of GCP (International Conference on 
Harmonization guidelines), the current version of the Declaration of Helsinki, and 
applicable national regulatory authority’s requirements. 
 
4.1 
Background and Rationale 
Breast cancer is the most prevalent tumor affecting adult women in worldwide 
countries.1 Each year, 1.38 million of new cases are diagnosed, accounting for an 
incidence rate of 89.9 per 100’000 women in Western Europe.2 Despite surgical 
treatment in more than 90% of newly diagnosed breast cancer patients,3 3.5 to 6.3% 
will face tumor recurrence due to occult metastases.4 Developing methods for early 
detection of metastatic disease has thus become a priority in cancer research.  
 
It has been claimed that peripheral blood testing for circulating tumor cells (CTC) may 
represent a promising tool to identify early relapse and monitor therapy response 
among cancer patients.5,6 In a meta-analysis pooling data from more than 6’800 
subjects, patients with CTC levels below 5 cells per 7.5ml blood were found to have 
lower risk of disease recurrence and lower mortality rates compared to patients with 
levels ≥5 CTC/7.5ml blood.7 These findings were reported for both early-stage and 
metastatic breast cancer patients, regardless of the method used for CTC detection 
(CellSearch® assay versus reverse-transcriptase polymerase chain reaction (RT-
PCR) approach). These results are also consistent with newly published trials, which 
reported reduced progression-free survival and decreased overall survival with CTC 
levels as low as 1 cell per 7.5ml of blood.8-10  
 
Whilst there is sound evidence that the presence of CTC correlates with poor 
prognosis, uncertainty remains as to the factors triggering CTC release in peripheral 
blood.11 Increasing concern about the perioperative period has emerged, since it 
seems to create conditions promoting metastatic spreading.12 Tumor manipulation 
during surgery, for instance, has been claimed to participate in CTC spreading, but in 
4 trials, there was no significant change in CTC levels before and after surgical 
treatment.13-16 Other authors suggested that pharmacological properties of anesthetic 
drugs may promote tumor recurrence: (1) either indirectly through 
immunosuppressive mechanisms, such as decreasing the cytotoxic activity of natural 
killer (NK) cells (which have a key role in tumor cells destruction and limitation of 
tumor growth); (2) or by interfering directly with tumor cells biology.17,18  
 
Although these effects have been well documented in a large amount of pre-clinical 
studies, their relevance at the clinical level is still matter of debate. Several factors 
may account for this lack of consensus: First, the outcome of tumor cells (elimination 
versus further spreading) seems to result from complex immune interactions that 
have – to date – not been fully elucidated.18 This delicate interplay between immune 
and tumor cells might be disrupted by several factors intervening during the 
perioperative phase, such as surgical stress, pain, hypothermia or blood 
transfusions.12  A further complicating aspect in onco-anesthesia research is related 
to the very experimental nature of in vitro studies, which usually target specific 

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immune or tumor cells, thereby failing to recreate the complex micro-environment 
seen in vivo. Finally, clinical data remain scarce, conflicting and mostly driven by 
retrospective analyses, which are prone to important methodological limitations. 
Several authors already pointed out this manifest gap of knowledge and stressed the 
need for large, well-designed, randomized controlled trials addressing specifically the 
effects of anesthetics on tumor recurrence.19,20  
 
One of the main issues in cancer research resides in the extensive amount of time 
needed to ascertain tumor recurrence, particularly when considering modestly 
aggressive tumors such as breast cancer. To overcome this limitation, the use of 
surrogate markers (i.e. markers predictive of the clinical endpoint) might be of 
interesting value, especially if they are lying early in the causal pathway. In this 
respect, CTC levels may represent an ideal surrogate to explore the effect of 
anesthesia on tumor spreading since they are predictive of tumor relapse, have been 
described in a variety of oncologic settings,21 are objectively measured and easily 
obtained through simple blood test.  
 
Therefore, the aim of this study is to evaluate if findings from pre-clinical onco-
anesthetic research are consistent and applicable in a clinical trial setting. To achieve 
this goal, we will use CTC as a surrogate marker for tumor spreading and evaluate 
the impact of 2 different anesthetic regimens on CTC levels among patients 
undergoing surgery for primary breast cancer. More specifically, we will compare the 
effect of inhalational anesthesia (i.e. sevoflurane) with conventional intravenous 
anesthesia (i.e. propofol) on perioperative CTC levels measured at 4 different time 
points.    
4.2 
Pre-clinical data and evidence from clinical research 
We performed 2 systematic searches for relevant reports in the Medline/Ovid 
database without language or date restriction (last search: November 2014), using 
both MeSH terms and keywords combined with Boolean operators as follows:  
> Pre-clinical data: “exp anesthesia, general”, “exp anesthesia, intravenous”, 
"anesthesia.tw", "anesthetics.tw", "inhalational.tw", "volatile.tw", "intravenous 
anesthesia.tw", "propofol.tw", "cell line, tumor", "neoplasms, experimental", "killer 
cells, natural". We identified 433 reports, of which 356 were excluded after title and 
abstract screening. Another 47 records were discarded since they were not 
investigating currently used anesthetics (i.e. isoflurane, sevoflurane or propofol) or 
were comparing regional with general anesthesia. Thus, we considered 30 studies 
reporting the effect of propofol and volatile anesthetics on tumor cells (20 studies) 22-
41 and on NK cells (10 studies) 42-51. 
> Clinical research: “exp anesthesia, general”, “exp anesthesia, intravenous”, 
"anesthesia.tw", "anesthetics.tw", "inhalational.tw", "volatile.tw", "intravenous 
anesthesia.tw", "propofol.tw", "neoplastic cells, circulating", "neoplasm recurrence, 
local", "circulating tumor cells.tw", "CTC.tw", "perioperative.tw". We identified 1586 
records, of which most (1564) were excluded after title and abstract screening. From 
the remaining 22, 52-73 we discarded 19 reports that were not comparing sevoflurane 
with propofol. Thus, only 3 clinical studies were eventually considered. 57,60,71 
 
 
 
 

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4.2.1 Effects of anesthetics on tumor cells 
 
Table 1 outlines the main results of pre-clinical studies reporting on tumor cells lines 
exposed to a variety of anesthetics. Although propofol seems to have a protective 
effect against tumor growth, migration and invasiveness, the heterogeneity in 
designs, cell lines, interventions and outcomes makes these findings difficult to 
interpret. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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4.2.2 Effect of propofol and sevoflurane – clinical evidence 
 
As outlined previously, clinical data remain scarce, conflicting and mostly driven by 
retrospective analyses. We retrieved only 3 studies comparing the effects of 
sevoflurane versus propofol on tumor recurrence (Table 2).57,60,71 The most recent 
study was a large retrospective register review including data from 2838 patients 
undergoing surgery for breast or colorectal carcinoma.57 After adjustment for multiple 
confounders, there was no difference between groups in terms of survival. In breast 
cancer patients receiving sevoflurane anesthesia, a beneficial trend towards better 
survival was identified, although not statistically significant (HR propofol group: 1.33 
(0.91–1.94)). These findings were consistent with a small randomized controlled trial 
that was not able to show any difference in metastasis rates, overall mortality and 
cancer-related mortality in patients receiving either propofol or sevoflurane 
anesthesia.71 Finally, a retrospective chart review of moderate size yielded conflicting 
results, but several aspects limit the validity of these results.60 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
4.2.3 Secondary Outcomes: 
4.2.3.1 
NK cells and tumor spreading 
 
Under physiologic circumstances, tumor cells are believed to be caught and 
destroyed through cell-mediated immune responses involving NK cells.74,75 As 
mentioned previously, these complex immune interactions are usually impaired 
during the perioperative period: surgical stress, for instance, has been suggested to 
induce a generalized inflammatory state that is thought to suppress NK cells activity 
and therefore enhance tumor spreading.76,77 Further weakening of the immune 
response is believed to occur through drug-related effects of anesthetics on NK cells 
(table 3).The vast majority of these studies were either not controlled or not 
appropriately controlled, yielding results that are difficult to compare. 
 
 

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4.2.3.2 
CTC and extracapsular spread in lymph nodes  
 
While lymph node status has been regarded as the most important prognostic factor 
for disease-free and overall survival in breast cancer patients,78 a substantial amount 
of node-positive patients will surprisingly remain free of systemic relapse.9 By 
contrast, some patients expected to have a favorable outcome will experience the 
opposite scenario, suggesting that other factors play a role in the metastatic cascade. 
 

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Recent findings suggests that extracapsular spread (ECS) of tumor cells observed in 
axillary lymph node contents might correlate with poorer prognosis in breast cancer 
patients.79 According to the yearly statistics of our hospital, axillary node dissection is 
realized in more than 30% of women undergoing surgery for primary breast cancer.  
The correlation between CTC positivity and the presence of ECS in the 
histopathological findings of axillary lymph node contents will be assessed in these 
patients.    
 
4.3 
Investigational Product and Dose Rationale 
Sevoflurane is a halogenated general inhalation anesthetic drug. It is chemically 
stable and is neither flammable nor explosive in clinical concentrations. It belongs to 
the group of halogenated ethers, and its structural formula is:   
 
 
 
 
 
fluoromethyl 2,2,2,-trifluoro-1-(trifluoromethyl) ethyl 
ether 
 
 
 
Sevoflurane is an inhalational anesthetic agent for induction and maintenance of 
general anesthesia. The minimum alveolar concentration (MAC) of sevoflurane in 
oxygen for a 40-year-old adult is 2.1%. Because of its little solubility in blood and 
tissues its effects are of rapid onset. Recovery of consciousness is usually achieved 
within 8 to 10 minutes after discontinuation of application. The use of sevoflurane as 
a general anesthetic is well established, and considered as a safe, routinely 
prescribed, anesthetic agent.  
Propofol is an intravenous hypnotic agent, poorly soluble in water, thus dissolved in a 
fat emulsion vehicle that contains soya bean oil, triglycerides, egg lecithin, glycerol, 
sodium oleate and water.  
 
 
 
 2,6-diisopropylphenol 
 
 
 
Its hypnotic effect occurs rapidly (less than 60 seconds), but the effect duration is 
short, due to rapid redistribution and metabolism. It is a well-established drug, which 
has been successfully used for general anesthesia or sedation since more than 20 
years. 

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Since the emergence of short acting intravenous anesthetics in the late 80’s, an 
impressive amount of literature comparing volatile with intravenous anesthesia has 
been produced, focusing on pharmacological effects, patient safety, quality process, 
patient satisfaction, or organ protection. No consensus regarding the superiority of 
one technique has been reached so far, mainly because complications related to 
anesthesia are more the result of multiple factors (surgery, patient specific health 
problems) rather than due the drug itself. Both sevoflurane and propofol contribute 
equally to the following systemic effects observed during general anesthesia: 
- Cardiovascular: hypotension (fall in systemic resistance), bradycardia. 
- Respiratory: reduced minute ventilation, reduced respiratory threshold to hypoxia, 
increased tolerance to hypercapnia. 
- Central nervous system: autoregulation maintained, decrease in cerebral blood flow 
and oxygen consumption.      
If both drugs produce similar physiologic effects, they differ however regarding 
specific side effects:   
- in susceptible patients, Sevoflurane can trigger malignant hyperthermia, a 
hypermetabolic state due to abnormal calcium release in skeletal muscle 
(pharmacogenetic disease, autosomal dominant).  
- Sevoflurane and other volatile anesthetics are suggested to confer organ protection, 
a property known as “volatile preconditioning”  
- Propofol is known to decrease the incidence of postoperative nausea and vomiting 
in susceptible individuals  
- In soya- or egg-allergic patients, the administration of propofol can induce 
anaphylactic reactions  
The choice between the different regimens of anesthesia maintenance (volatile vs. 
intravenous) is mostly dictated by habits and local hospital practices. 
 
4.4 
Risk/Benefits and Ethical Considerations 
 
By means of this trial, we might be able to identify potential targets playing a crucial 
role in the metastatic cascade. Being able to find the best (or the least harmful) 
anesthesia regimen for cancer patients is of primary relevance, and raises important 
ethical concerns in terms of beneficence and non-maleficence.  
 
 
 
 
 
 
 
 
 
 
 

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5. STUDY OBJECTIVES AND OUTCOMES 
We will conduct a prospective, randomized, double blinded, controlled study over a 
52-month period to investigate whether commonly used anesthetics (volatile versus 
intravenous) affect the level of circulating tumor cells in patients undergoing surgery 
for primary breast cancer.  
 
Hence, the primary objective is to compare the effects of 2 anesthetic regimens 
(sevoflurane versus propofol) on perioperative CTC levels measured at 4 different 
time points. Blood levels (i.e. number) of CTC will be determined using the 
CellSearch approach, as described below.  
 
Two secondary objectives will be determined: 
 
A) To determine if anesthesia interferes with NK cells by measuring NK cells number 
and activity at 4 different time points; 
 
B) To establish if the level of CTC correlates with the presence of ECS in the 
histopathological findings of axillary lymph nodes (exclusively in patients undergoing 
breast surgery with axillary dissection). 
 
5.1 
Primary Variable: circulating tumor cells (CTC) 
 
Blood CTC levels (i.e. number of CTC per 7.5ml of blood) will be measured before 
and after the administration of anesthetics using the CellSearch system, as 
described elsewhere:9,80,81 15ml sample of peripheral blood (necessary amount for 2 
CellSearch tubes) will be collected at 4 pre-defined time points and screened for 
CTC within 96 hours of blood collection.  
 
5.2 
Secondary Variables 
5.2.1 Secondary variable A: NK cells (number and activity) 
 
Assessment of NK cells will be performed at 4 pre-definite time points, concomitant to 
CTC determination, using 2x8ml of peripheral venous blood collected in sterile tubes 
containing sodium heparin. The number of NK cells will be assessed using flow 
cytometry, whilst the NK cells activity will be determined in vitro by a direct, cell-based 
cytotoxicity assay against the K562 (human chronic myelogenous leukaemia) cell line 
with a fluorescent read-out, as previously described.82,83  
 
5.2.2 Secondary variable B: extracapsular tumor spread (ECS) 
 
After study completion, pathology reports from patients undergoing surgery with 
axillary lymph node dissection will be reviewed for extracapsular tumor spread with 
the aim to establish if blood CTC levels correlates with the presence of ECS.  
 

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5.2.3 Other study variables 
 
Demographics and perioperative data will be collected as presented in table 4. 
Since opioids have been suggested to inhibit the immune function,84 perioperative 
analgesia will be administered following a standardized protocol and will be 
documented thoroughly. Restriction and stratified randomization are expected to 
minimize the risk of unbalanced confounders (section12). However, since we will 
perform exploratory analyses whenever there is a need to control for residual 
confounders, the following variables will be collected: hypothermia, blood transfusion, 
administration of cyclooxygenase inhibitors, beta-blockers, glucocorticoids and date 
of last menstruation.85-87 
5.2.4 Safety variables 
 
Any adverse event (AE) or serious adverse event (SAE) as described below. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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6. STUDY DESIGN  
6.1 
General Design 
 
We will conduct a prospective, randomized, double blinded, controlled study over a 
period of 52 months. We will consider adult female patients undergoing surgery for 
primary breast cancer. Expecting a dropout rate of 10% over time, we will recruit a 
total of 232 patients (i.e. 116 in each study group). 
 
Patients will be screened for eligibility according to the inclusion and exclusion criteria 
on the day prior to surgery. Written informed consent will be subsequently obtained. 
A pregnancy test will be performed for all included patients.  
 
On the day of operation, the patients will be premedicated with midazolam per os 
(3.75mg to 7.5mg). Randomization will be performed according to a computer-
generated anesthesia-assignment (www.randomizer.at) once the patient will be 
installed for induction of anesthesia. By this means, concealment of random 
allocation will be ensured.  
After preoxygenation, induction of anesthesia will be administrated in both groups 
similarly, using fentanyl 2-3mcg/kg, Pentothal 4-6 mg/kg and rocuronium 0.6mg/kg 
intravenously. Maintenance of anesthesia will be conducted following the assignment 
group:  
1) in “Group Sevo”, anesthesia will be maintained with sevoflurane in 80% oxygen, 
adjusted to keep an adequate depth of anesthesia (minimal alveolar concentration 
(MAC) between 0.8 and 1.2), attested by bispectral (BIS) index values between 40 
and 60.  
2) in “Group Propofol” anesthesia will be maintained with a target-controlled infusion 
(TCI) device providing an intravenous propofol dose adjusted to keep an adequate 
depth of anesthesia (TCI values: start after induction with 2.0mcg/ml, then ranging 
from 3.0mcg/ml to 5.0mcg/ml), attested by BIS index values between 40 and 60. 
Patients will be ventilated with 80% oxygen in air.  
Patients requiring a rapid sequence induction will receive rocuronium 0.9mg/kg 
instead of 0.6mg/kg. Per- and postoperative analgesia will be provided following a 
standardized protocol: fentanyl 2mcg/kg (total dose ranging from 5-10mcg/kg) will be 
used during surgery; surgical wound infiltration with local anesthetics will not be 
allowed; morphine intravenous 0.02-0.04mg/kg for a VAS <4 will be used in the 
PACU, whilst subcutaneous morphine (0.1-0.2mg/kg will be prescribed as second 
line analgesic once patients will be discharged from PACU. Basic postoperative 
analgesia will be provided using paracetamol and non-steroidal anti-inflammatory 
drugs. Postoperative nausea and vomiting (PONV) prophylaxis will be prescribed 
following a strict protocol (no glucocorticoids).  
 
 
 
 
 
 

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6.2 
Primary Study Endpoint: blood CTC levels 
 
The primary endpoint, blood CTC levels, will be assessed at 4 pre-defined time 
points using the CellSearch approach: two 7.5ml samples of blood will be collected 
pre-operatively (visit 2, before induction of anesthesia), at the end of surgery (visit 3, 
before extubation) and two times postoperatively (visit 4, 48h postoperative, and visit 
5, 72h after surgery, before hospital discharge). The number of CTC per 7.5ml of 
peripheral blood will be determined by the CellSearch system within 96 hours of 
blood collection. The analysis will account for the effect of repeated measurements 
over time (within-subject factor) and the effect of anesthesia regimen (between-
subject factor) as described under section 12.    
 
The CellSearch® approach 
 
The CellSearch® assay contains a ferro fluid-based capture reagent and 
immunofluorescent reagents. The ferrofluid reagent consists of particles with a 
magnetic core surrounded by a polymeric layer coated with antibodies targeting the 
epithelial cell adhesion molecule (EpCAM) antigen for capturing CTC. After 
immunomagnetic capture and enrichment, fluorescent reagents (anti-CK-
phycoerythrin, DAPI, and anti-CD45-allophycocyanin) are added for identification and 
enumeration of CTC. (www.veridex.com)  
 
6.3 
Secondary Endpoints 
6.3.1 Anesthesia, NK cells and tumor spreading 
The assessment of NK cells (mean number and activity) will be performed at 4 pre-
definite time points, concomitant to CTC determination. The analysis will account for 
the effect of repeated measurements over time (within-subject factor) and the effect 
of anesthesia regimen (between-subject factor) as described under section 12.    
6.3.2 CTC positivity and extracapsular tumor spreading 
 
Pathology reports from patients undergoing surgery with axillary lymph node 
dissection will be reviewed for extracapsular tumor spread with the aim to establish a 
correlation between CTC positivity and ECS.      
 
 
6.3.3 Safety Endpoints 
 
Adverse events (AE) or serious adverse events (SAE) will be actively sought and 
reported. For comprehensive description of AE/SAE, see paragraph 10 below.  
 
6.4 
Study procedures (fig.1) 
6.4.1 Visit 1 (preoperative day) 
• Standard pre-anesthesia visit: medical history, physical examination, 
demographics (as listed in Table 1) 

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• Check for eligibility according to the inclusion and exclusion criteria 
• Information of patient and written informed consent 
• Pregnancy test: if not pregnant, the patient will qualify for the study 
• Concomitant therapy* will be administered 
• Potential confounders will be documented   
 
6.4.2 Visit 2 (day of surgery – before induction of general anesthesia) 
• Vital signs will be recorded, concomitant therapy* will be administered 
• Patient randomization to “Group Sevo” (experimental) or to “Group Propofol” 
(control) 
• 2x7.5ml + 2x8ml of blood will be collected and sent for CTC/NK cells 
assessment (1° variable and 2° variable A) 
 
6.4.3 Visit 3 (day of surgery – end of surgery, before extubation) 
• Maintenance of anesthesia following the assignment group (sevoflurane or 
propofol) 
• Vital signs will be recorded, concomitant therapy* will be administered 
• 2x7.5ml + 2x8ml of blood will be collected and sent for CTC/NK cells 
assessment (1° variable and 2° variable A) 
• Histopathological findings (2° variable B): data related to this endpoint will be 
collected after completion of the study.  
• Perioperative data collection as listed in Table 3  
• AE or SAE will be recorded 
 
6.4.4 Visit 4 and 5 (48h and 72h postoperative) 
• Vital signs will be recorded, concomitant therapy* will be administered 
• 2x7.5ml + 2x8ml of blood will be collected and sent for CTC/NK cells 
assessment (1° variable and 2° variable A), concomitant to routine 
postoperative laboratory tests 
• Perioperative data collection as listed in Table 3  
• AE or SAE will be recorded 
 
 
 
 
 
 
 
* For medications susceptible to interfere with outcome measure, see paragraph 8.5 below 

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Fig.1 Trial profile. Each visit is highlighted in a different color. 

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7. SUBJECT SELECTION AND WITHDRAWAL 
Patients undergoing surgery for breast cancer will be enrolled at the University 
Hospital of Zürich (USZ) and at Klinik Hirslanden Zurich. The Department of 
Gynecology and Breast Cancer Center of the USZ perform about 150-200 operations 
per year for breast cancer. Based on USZ last year records, we expect to enroll 5-10 
patients per month; slightly lower enrollment rates are expected at Klinik Hirslanden, 
(3-8 patients/month).  
Meetings with the anesthesia team USZ and the investigators of Klinik Hirslanden  
will be organized periodical at least all 6 month,  to identify at an early stage potential 
enrolment or organizational issues. These meetings are also expected to act as a  
positive feedback and motivational tool.   
Patients fulfilling all of the following inclusion criteria will be enrolled in the study: 
 
7.1 
Inclusion Criteria 
• 18 to 85 years old 
• Female patients 
• Primary breast cancer without known remote extension  
• Scheduled for tumor resection or mastectomy, +/- axillary node dissection 
• ASA status I-III, as defined by the American Society of Anesthesiologists 
• Capable of giving informed consent 
 
7.2 
Exclusion Criteria 
 
The presence of any one of the following exclusion criteria will lead to exclusion of 
the subject: 
 
• 
Metastatic breast cancer 
• 
No primary surgery (recurrence, reconstruction) 
• 
Pre-operative chemotherapy or radiotherapy  
• 
 Significant immune impairment: auto-immune disease, HIV, other active    
cancer, age>85, ASA IV-V 
• 
Concomitant regional anesthesia (epidural catheter, paravertebral blockade, 
wound infiltration with local anesthetics) 
• 
Chronic opioids medication 
• 
Any systemic immunosuppressive therapy (glucocorticoids, cytostatics, 
antibodies, drugs acting on immunophilins, interferon, TNF bindings proteins, 
mycophenolate) 
• 
Known hypersensitivity or suspected allergy to propofol, soya or egg proteins 

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• 
Known hypersensitivity to volatile anesthetics (malignant hyperthermia) 
• 
Women who are pregnant or breast feeding  
• 
Inability to follow the procedures of the study, e.g. due to language problems, 
psychological disorders, dementia, etc. of the subject 
• 
Participation in another study with investigational drug within the 30 days 
preceding and during the present study 
Intraoperative radiotherapy is not an excluding criteria.  
7.3 
Subject Recruitment and Screening 
 
All consecutive patients admitted for breast cancer surgery will be assessed for study 
eligibility by the anesthesiology staff. The attending anesthetist will inform patients 
about the study orally and in writing. Patients willing to participate will subsequently 
provide written informed consent. A pregnancy test will be performed. 
 
Participation is on a strictly voluntary base. No compensation in any form will be 
granted to the subjects. 
 
 
7.4 
Early Withdrawal of Subjects 
 
Patients will be informed about the possibility to retract their participation to the study 
at any time. Investigators may withdraw a patient prior to expected completion of the 
study for safety reasons (for instance as the result of an adverse event (AE) with 
unacceptable consequence or risk for the patient). In both cases, for patient safety, a 
final medical examination will be performed. Notification of the discontinuation will be 
clearly documented on the patient’s case report form (CRF). 
Since data collection and analysis will be performed following an intention-to-treat 
model, all consecutive patients will be analyzed according to the initial randomization 
group, regardless of the study drug administered. These patients will not be 
withdrawn from analysis.    
7.4.1 Criteria for Early Withdrawal of Subjects 
 
Initially included patients meeting unexpectedly exclusions criteria during the course 
of the study (such as: incidental findings of metastatic cancer, other surgical 
procedure as initially planned, reoperation within the first 5 postoperative days, 
patients receiving chemo- or radio- or any immunotherapy during the study course), 
or patients unable to follow study procedures (for instance, patients transferred to 
another hospital) will be considered as drop-outs and will not appear in the study 
analysis.  
 
7.4.2 Data Collection and Follow-up for Withdrawn Subjects 
 

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Patients withdrawn because of an adverse event (AE) or patients unexpectedly 
meeting exclusion criteria will be followed during the study course. Data available 
through routine care will be recorded in the CRF unless the patient specifically 
refuses the investigators to access the medical record. 
 
7.5 
Early Termination of Study 
The Sponsor-Investigator may terminate the study prematurely according to certain 
circumstances: 
• 
ethical concerns, 
• 
insufficient patient recruitment, 
• 
when the safety or benefit of the subjects is doubtful or at risk, respectively, 
• 
alterations in accepted clinical practice that make the continuation of a clinical 
trial unwise,  
• 
reaching a positive or negative statistical end point earlier than anticipated.  
 
 
 

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8. TREATMENTS 
8.1 
Treatments to be Administered 
 
Sevoflurane group:  
Anesthesia will be maintained with sevoflurane (gaseous form via the endotracheal 
tube) in 80% oxygen, adjusted to keep an adequate depth of anesthesia (minimal 
alveolar concentration (MAC) between 0.8 and 1.2, corresponding to an age adjusted 
sevoflurane expiratory concentration ranging from 1.5% to 2.5%), attested by BIS 
index values between 40 and 60. Drug administration will be limited to the length of 
the surgical procedure. 
 
Propofol group: 
 
Anesthesia will be maintained with a TCI device providing an intravenous propofol 
dose adjusted to keep an adequate depth of anesthesia (TCI values: start after 
induction with 2.0mcg/ml, then ranging from 3.0mcg/ml to 5.0 mcg/ml), attested by 
BIS index values between 40 and 60. Patients will be ventilated with 80% oxygen in 
air. Drug administration will be limited to the length of the surgical procedure. 
 
Identity of Investigational Product(s) 
 
Sevoflurane (test group) 
Sevorane®, AbbVie, 250ml bottle  
 
Propofol (control group) 
Propofol-®Lipuro 1% (10mg/ml), B.Braun, 50ml glass bottle  
 
8.2 
Method of Assigning Subjects to Treatment Groups 
 
Fixed randomization based on a 1:1 allocation ratio will be performed. Balanced 
treatment allocation will be ensured using block sizes of 4. Patients will be stratified 
according to their ASA class during the randomization process, which will be 
performed using a web-based interactive allocation platform (www.randomizer.at). 
 
8.3 
Treatment Compliance 
8.3.1 Study drugs  
 
Because the attending anesthesiologist will provide the administration of study drugs, 
treatment compliance is not considered as a potential issue.  

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8.3.2 Medications susceptible to interfere with outcome measure  
8.3.2.1 
 Opioids  
 
Since opioids have been suggested to inhibit the immune function,84 per- and 
postoperative analgesia will be provided following a standardized protocol: 
 
• Fentanyl 2mcg/kg (total dose ranging from 5-10mcg/kg) will be used during 
surgery  
• Surgical wound infiltration with local anesthetics will not be allowed  
• Morphine intravenous 0.02-0.04mg/kg for a VAS <4 will be used in the PACU 
• Basic postoperative analgesia will be provided using paracetamol and non-
steroidal anti-inflammatory drugs 
• Subcutaneous morphine 0.1-0.2mg/kg will be prescribed as second line 
analgesic on the ward (reserve medication) 
 
With the aim to obtain high protocol compliance, postoperative analgesics in USZ will 
be prescribed in the patient electronic chart (KISIM) following a predefined schema 
saved in the KISIM database. The attending anesthesiologist has access to the 
database and will upload this schema to the electronic patient chart, regardless of the 
assignment group.    
For patients enrolled at Klinik Hirslanden, postoperative analgesics will be prescribed 
using a standardized paper form specifically designed for this study.  
8.3.2.2 
Glucocorticoids 
 
Glucocorticoids are hardly prescribed in the perioperative setting but can be used as 
rescue treatment for postoperative nausea and vomiting, in conjunction with other 
antiemetics. Thus, PONV medication will be administered following a protocol strictly 
avoiding the use of glucocorticoids.  
  
• Ondansetron amp 4mg intravenous 2x/day (reserve medication) 
• Metoclopramid amp 10mg intravenous 3x/day (reserve medication) 
• Meclozin supp 20mg intrarectal 1-2x/day (reserve medication) 
 
Postoperative PONV medication will be prescribed in the patient electronic chart 
(KISIM) following a predefined schema saved in the KISIM database. The attending 
anesthesiologist has access to the database and will upload this schema to the 
electronic patient chart, regardless of the assignment group. 
For patients enrolled at Klinik Hirslanden, postoperative PONV medication will be 
prescribed using a standardized paper form specifically designed for this study.     
 
8.4 
Prior and Concomitant Therapy 
 
Patients being on any systemic immunosuppressive therapy (glucocorticoids, 
cytostatics, antibodies, drugs acting on immunophilins, interferon, TNF bindings 
proteins, mycophenolate), as well as patients taking chronically opioids, planned for 
pre-operative chemo- or radiotherapy, or undergoing surgery with concomitant 

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regional anesthesia (epidural catheter, paravertebral blockade, wound infiltration with 
local anesthetics) will not be included, as these factors are known to interfere with the 
immune system.84,88,89  
Of note is that patients undergoing intraoperative radiotherapy with Intrabeam 
immediately after removal of the tumor are included. This is a new localized 
intraoperative “standard of care” therapy in breast cancer.  
 
Long-term medications, such as beta-blockers or COX-inhibitors, as well as 
perioperative blood transfusion or postoperative opioids, have also been suggested 
to affect the immune system.84,87 However, because the benefits of these treatments 
outweigh their presumable detrimental effect on the immunity, and because strict 
avoidance of opioids in the postoperative phase would lead to increased pain and 
distress, these treatments will not be interrupted nor avoided. Opioids will be 
administrated following a strict protocol mentioned previously (8.4.2.1). Adjustment 
for residual confounders will be performed at the analytical level.  
 
All concomitant and/or rescue treatment(s) have to be recorded in the eCRF, except 
for perioperative standard medication such as prophylactic antibiotics or heparin. 
 
8.5 
Packaging, Labelling and Supply 
Both propofol (Propofol-®Lipuro 1% (10mg/ml), B.Braun) and sevoflurane 
(Sevorane®, AbbVie) are anesthetics used on a daily base in our Anesthesiology 
department. These products are provided by the Kantonsapotheke and will be 
labeled as follows:  
 
 
 
 
 
 
 
 
 
 
 
 
Study products will be stored in a storage facility accessible exclusively to members of 
the study group. The study investigators will be in charge of the resupply. Regarding 
the volatile anesthesia, a vaporizer will be filled with sevoflurane and stored in the 
locker. This vaporizer will be used exclusively for study patients and will be stored back 
in the locker at the end of the anesthesia. 
8.6 
Blinding 
Adequate double-blinding will be performed: patients will not be told about their group 
assignments, the surgical team and nurses on the ward will not be informed during 
FOR CLINICAL TRIAL USE ONLY 
CTC_BREAST STUDY 
 
Sponsor: Beck Schimmer Beatrice  
Study ID:  
Propofol (B.Braun) OR sevoflurane (AbbVie)  
Charge:  
Expiry Date:  
Storage: at room temperature  
Patient ID:  

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the perioperative period about the anesthesia regimen provided, anesthesia records 
will be placed in sealed opaque envelopes at discharge from PACU and kept in a 
secure locker in the anesthesia office until the patient is discharged from the hospital. 
The pathologist responsible for histopathological assessment, the lab staff performing 
the CTC detection and the research fellow in charge for isolation and culture of NK 
cells (outcome assessors) will not have any access to the operation theatre or to the 
patient chart. The attending anesthesiologist responsible for randomization and 
providing the anesthesia and collecting the first two blood samples cannot per se be 
blinded but will not participate in any data collection or analysis. The study research 
team collecting the data will not have access to patient allocation until the final 
analysis.    
In situations requiring a breach in the blinding process, such as the occurrence of 
SAEs, an Emergency Code Break will be available to the investigator. This Code 
Break should be opened only in emergency situations when the identity of the 
investigational product must be known by the investigator in order to provide 
appropriate medical treatment. 
 
8.7 
Storage Conditions 
Both drugs will be stored in the operation theater, at room temperature, in a storage 
facility with a secured locker. 
 
8.8 
Study Drug Accountability 
 
Investigational product supplies will be kept in a secure, limited access storage area 
under the recommended storage conditions. 
  
The investigator will maintain accurate and adequate records including dates, lot 
number, doses given, individual usage, etc. 
 
 
8.9 
Return or Destruction of Study Drug 
 
At the completion of the study, there will be a final reconciliation of drug shipped, drug 
consumed, and drug remaining. This reconciliation will be logged on the drug 
accountability form, signed and dated. Any discrepancies noted will be investigated, 
resolved, and documented prior to return or destruction of unused study drug. Drug 
destroyed on site will be documented in the study files. 
 
 

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9. STUDY PROCEDURES 
9.1 
Blood samples for CTC/NK assessment 
 
Blood samples will be collected before induction of general anesthesia (visit 2), at the 
end of surgery, before extubation (visit 3), 48h (visit 4) and 72h (visit 5) 
postoperatively.  
The CTC detection procedure will be performed as described by the CellSearch 
system manufacturer: two 7.5ml samples (i.e. 15ml) of whole blood will be aseptically 
collected into the CellSave Preservative tubes and will be transported to the 
University Hospital Zurich; IFA D LAB 26, Sternwarstr. 14, 8091 Zurich  for 
subsequent analysis within 96 hours of blood collection.  
Assessment of NK cells will be performed at the same 4 pre-definite time points, 
concomitant to CTC determination, using 2x8ml of peripheral venous blood collected 
in sterile tubes containing sodium heparin. After subsequent transport to the ZKF, the 
number of NK cells will be performed using flow cytometry, while NK cells activity will 
be determined in vitro by a direct, cell-based cytotoxicity assay against the K562 
(human chronic myelogenous leukaemia) cell line with a fluorescent read-out as 
previously described.82,83  
 
Tubes for CTC/NK cells detection will be kept in plastic bags specifically labeled with 
the visit number. Four bags per patient will be available: 2 for the attending 
anesthetist (visit 2 and 3) and 2 for the attending nursing staff on the ward (visit 4 and 
5). The blood samples will be eliminated after subsequent analysis. 
 
  
 
9.2 
Study drug administration 
 
Sevoflurane or propofol will be administrated according to patient allocation. Because 
patients scheduled for breast cancer surgery will need a general anesthesia in any 
case, regardless of their participation to this study, both drugs will be provided by the 
UHZ.  
 
 
9.3 
Pregnancy test 
 
Patients of childbearing age scheduled for breast surgery are routinely screened for 
pregnancy one day prior to surgery. After obtaining written informed consent, the 
attending anesthetist will look for pregnancy test results in the KISIM database. 
Considered as a routine prescription at the UHZ, the test should not create additional 
costs. 
Patients of childbearing age at Klinik Hirslanden will be screened for pregnancy at 
enrollment. The UHZ study team will provide the pregnancy tests and will bear the 
associated costs.  
 
 

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10. SAFETY  
10.1 Safety Variables 
 
Serious adverse events occurring within the two study groups during the course of 
the study will be recorded. The attending anesthetist, the surgeon and the nursing 
staff on the ward will be instructed to report any (S)AE to the investigators of the 
study. A dedicated study nurse will retrospectively review the chart of patients after 
hospital discharge to identify any unreported (S)AE.  
 
10.2  Definition of (Serious) Adverse Events  
 
Adverse events 
Adverse events (AEs) are defined as any untoward medical occurrence in a patient 
or clinical investigation subject administered a pharmaceutical product and which 
does not necessarily have a causal relationship with this treatment. An AE can 
therefore be any unfavourable and unintended sign (including an abnormal laboratory 
finding), symptom, or disease temporally associated with the use of a medicinal study 
product, whether or not related to the medicinal study product. An AE may also 
consist of a new disease, an exacerbation of a pre-existing illness or condition, a 
recurrence of an intermittent illness or condition, a set of related signs or symptoms, 
or a single sign or symptom. 
 
AEs observed by the investigator and/or reported by the subject must referring to the 
HFG, not be reported in the eCRF - 
 
All AEs, qualifying as an SAE , observed by investigator must be reported in the 
eCRF.  For all SAE`s sufficient information will be pursued and/or obtained so as to 
permit an adequate determination of the outcome of the event  and an assessment of 
the casual relationship between the SAE and the investigational drug or study 
treatment(s). The causality assessment of the event to the study drug will be made 
by investigator  and Sponsor-investigator, based on criteria listed in the ICH 
guidelines 
    
 
Whenever available, the underlying disease or condition for which a therapeutic or 
diagnostic procedure is required should be reported as the AE term. Surgeries or 
other invasive procedures that had already been planned prior to the start of the 
study do not have to be documented as AEs. These planned procedures will be 
recorded in the eCRF by the investigator at the baseline visit. It is not important if the 
condition was known before enrolment, only if the procedure was planned before. 
 
Pregnancy per se does not classify as an AE. However, AEs related to a pregnancy 
have to be reported like any other AEs. Pregnancy should be confirmed by a reliable 
laboratory test. Pregnant subjects must be immediately withdrawn from the clinical 
study. All pregnancies occurring during the treatment phase of the study and within 
30 days after discontinuation of study medication have to be reported to the 

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Investigator-Sponsor within one working day of the investigational sites knowledge of 
the pregnancy on the Initial Pregnancy Report Form. The Sponsor-Investigator will 
contact the attendant physician by phone during pregnancy and after the estimated 
date of delivery to enquire about course and outcome of the pregnancy. Course of 
the pregnancy and health status of the new born child have to be documented on the 
Follow-Up Pregnancy Report Form.  
 
 
Serious adverse event 
An SAE is any untoward medical occurrence that at any dose results in  
 
• results in death,  
• is life-threatening,  
• requires subject hospitalization or prolongation of current hospitalization,  
• results in persistent or significant disability/incapacity, or  
• is a congenital anomaly/birth defect,  
• any important medical event and any event which, though not included in the 
above, may jeopardise the subject or may require intervention to prevent one of the 
outcomes listed above. 
 
Any other medically important condition that may be not immediately life-threatening 
or results in death or hospitalization but may jeopardize the subject or may require 
intervention to prevent one of the outcomes listed above should also usually (i.e. 
based on medical and scientific judgment) be considered serious. For example: 
intensive treatment at home for allergic bronchospasm; certain laboratory 
abnormalities (e.g. blood dyscrasias); convulsions that do not result in hospitalisation; 
development of drug dependency or drug abuse. 
 
 
10.3 Recording of (Serious) Adverse Events 
Clinical investigators and ultimately the protocol Principal Investigator (PI) have the 
primary responsibility for AE identification, documentation, grading, and assignment 
of attribution to the investigational agent/intervention. 
 
Clinical study subjects will be routinely questioned about AEs at study visits. The 
well-being of the subjects will be ascertained by neutral questioning ("How are 
you?"). The investigator is responsible for reporting all AEs occurring during the 
course of the study.  
 
All observed or volunteered adverse drug events (serious or non-serious) and 
abnormal test findings, regardless of treatment group or suspected causal 
relationship to the investigational drug or study treatment(s) will be recorded in the 
patient file and subsequently in the eCRF.   
 
 

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All SAEs, will be fully documented in the appropriate eCRF. For each SAE, the 
investigator will provide the onset, duration, intensity, treatment required, outcome 
and action taken with the investigational product.  
 
The intensity of SAEs will be assessed as being  
 
• mild (hardly noticeable, negligible impairment of well-being),  
• moderate (marked discomfort, but tolerable without immediate relief), or  
• severe (overwhelming discomfort, calling for immediate relief). 
 
The investigator will determine the relationship of the investigational drug to all AEs 
as defined on the AE page of the eCRF. 
 
10.4 Assessment of Causatility (Serious) Adverse Events 
 
 
The assessment by the investigator with regard to the study drug relation is done 
according to the following definitions: 
 
Relationship 
Description 
Definitely 
Temporal relationship 
Improvement after dechallenge* 
Recurrence after rechallenge 
(or other proof of drug cause) 
Probably 
Temporal relationship 
Improvement after dechallenge 
No other cause evident 
Possibly 
Temporal relationship 
Other cause possible 
Unlikely 
Any assessable reaction that does not fulfil the above 
conditions 
Not related 
Causal relationship can be ruled out 
*Improvement after dechallenge only taken into consideration, if applicable to reaction 
 
 
 
 
10.5 Reporting of Serious Adverse Events 
All SAEs must be reported immediately and within a maximum of 24 hours to the Sponsor-
Investigator of the study. The Sponsor-Investigator will re-evaluate the SAE and return the 
form to the site. 
 
 
The Sponsor-Investigator is responsible for SAE reporting to the IEC, according to the 
following details: 
 

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• Reporting to IEC any SAE which resulted in death: 
- without delay, and no later than 7 calendar days. 
• Reporting to IEC of fatal and life-threatening SAEs if evaluated as “drug related” 
(SADR):  
- without delay and no later than 7 calendar days following awareness that event  
   meets criteria for an SADR. 
• Reporting to IEC of fatal and life-threating SAEs if evaluated as “suspected”, 
“unexpected” and “drug related” (SUSAR) 
- without delay and no later than 7 calendar days following awareness that event  
   meets criteria for an SUSAR. 
- follow-up information regarding the SUSAR within further 8 calendar days.  
• Reporting to IEC of non-fatal and not life-threatening SAEs if evaluated as 
“suspected”, “unexpected” and “drug related” (SUSAR): 
- promptly and no later than 15 calendar days following awareness that event  
   meets criteria for a SUSAR. 
• All other SAEs will be summed up in the annual safety update report. The annual 
safety report contains information from all sites including information from all sites.  
The Sponsor-Investigator prepares it, and then submits it to the participating 
Investigators. The participating Investigators submit it to the local committees 
 
The Sponsor-Investigator is responsible for SAE reporting to Swissmedic according to 
the following details: 
 
• 
Compliance with the regulatory requirements of Swissmedic regarding prompt 
reporting of unexpected SAEs for which a causal relationship with the study drug 
or device cannot be ruled out.  
• 
Reporting to Swissmedic of fatal and life-threatening SAEs if evaluated as 
“suspected”, “unexpected” and “drug related” (SUSAR): 
- without delay and no later than 7 calendar days following awareness that 
event  
   meets criteria for a SUSAR; 
- follow-up information regarding the SUSAR within further 8 calendar days. 
• 
Reporting to Swissmedic of non-fatal and not life-threatening SAEs if evaluated 
as “suspected”, “unexpected” and “drug related” (SUSARs): 
- promptly and no later than 15 calendar days following awareness that event  
   meets criteria for a SUSAR. 
• 
Sending yearly safety reports, starting one year after the date of notification to 
Swissmedic. These reports should contain: 
 
- A concise critical summary of the safety profile of the drug studied as well as the 
  safety issues that have arisen; 
- A listing of all SUSARs that have occurred in Switzerland and at international 
   level (if applicable);  

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- Ideally all adverse drug reactions at international level. 
- The accompanying letter provided with the Annual Safety Report should 
contain a short summary of the status of the clinical trial in Switzerland 
(number of centers open/closed, number of patients recruited/recruitment 
closed, and number of SAR/SUSAR. 
 
 
An unexpected SAE refers to any AE, the nature or severity of which is not consistent 
with the applicable product information (see Appendix) 
 
 
10.6 
Follow up of (Serious) Adverse Events 
Subjects terminating the study (either regularly or prematurely) with 
• reported ongoing SAE, or 
• any ongoing SAEs of laboratory values or of vital signs being beyond the alert limit 
will return for a follow-up investigation. This visit will take place up to 30 days after 
terminating the treatment period. Follow-up information on the outcome will be 
recorded on the respective AE page in the eCRF. All other information has to be 
documented in the source documents. Source data has to be available upon request. 
In case of subjects lost to follow-up, efforts should be made and documented to contact 
the subject to encourage him/her to continue study participation as scheduled. In case 
of minor AEs a telephone call to the subject may be acceptable. 
All new SAE or pregnancies that the investigators will be notified of within 30 days after 
discontinuation of study medication have to be reported in appropriate report forms 
and in the eCRF if required.  
Follow-up investigations may also be necessary according to the investigator’s medical 
judgment even if the subject has no AE at the end of the study. However, information 
related to these investigations does not have to be documented in the eCRF but must 
be noted in the source documents. 
 

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11. DATA QUALITY ASSURANCE 
The Sponsor-Investigator is implementing and maintaining quality assurance and 
quality control systems with written SOPs and Working Instructions to ensure that 
trials are conducted and data are generated, documented (record), and reported in 
compliance with the protocol, GCP, and applicable regulatory requirement(s).  
Monitoring and Audits will be conducted during the course of the study for quality 
assurance purposes. 
 
11.1 Routine Monitoring 
Regular monitoring visits at the investigator’s site prior to the start and during the 
course of the study will help to follow up the progress of the clinical study, to assure 
utmost accuracy of the data and to detect possible errors at an early time point. The 
Sponsor-Investigator organizes professional independent monitoring for the study.  
All original data including all patient files, progress notes and copies of laboratory and 
medical test results must be available for monitoring. The monitor will review all or a 
part of the eCRFs and written informed consents. The accuracy of the data will be 
verified by reviewing the above referenced documents.  
 
11.2 Audits and Inspections 
A quality assurance audit/inspection of this study may be conducted by the regulatory 
authority or IEC, respectively. The quality assurance auditor/inspector will have 
access to all medical records, the investigator's study related files and 
correspondence, and the informed consent documentation that is relevant to this 
clinical study. 
The investigator will allow the persons being responsible for the audit or the 
inspection to have access to the source data/documents and to answer any 
questions arising. All involved parties will keep the patient data strictly confidential. 
 
11.3 Specification of Source Documents 
The following documents are considered source data, including but not limited to: 
• SAE worksheets 
• Nurse records, records of clinical coordinators, and  
• Medical records from other department(s), or other hospital(s), or discharge letters 
and correspondence with other departments/hospitals, if subject visited any during 
the study period and the post study period.  
 
Source data must be available at the site to document the existence of the study 
subjects and substantiate the integrity of study data collected. Source data must 

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include the original documents relating to the study, as well as the medical treatment 
and medical history of the subject. 
 
The following information (at least but not limited to) should be included in the source 
documents: 
• Demographic data (age, sex) 
• Inclusion and Exclusion Criteria details  
• Participation in study and signed and dated Informed Consent Forms  
• Visit dates  
• Medical history and physical examination details  
• Key efficacy and safety data (as specified in the protocol)  
• AEs and concomitant medication  
• Results of relevant examinations  
• Laboratory printouts  
• Dispensing and return of study drug details  
• Reason for premature discontinuation  
• Randomization number 
 
 
 
 
 
 
 

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12. STATISTICS 
12.1 Statistical and Analytical Plans 
 
This is a multicentric randomized controlled trial comparing the effect of 2 different 
anesthetic regimens (sevoflurane versus propofol) on perioperative levels of CTC in 
patients undergoing surgery for primary breast cancer.  
Using a strict 4 time-points schedule, the number of CTC in peripheral blood will be 
measured before and after exposition to general anesthesia (primary outcome). 
Using a mixed model analysis, the effect of two independent variables on the mean 
level of CTC will be explored: a within-subject time factor (i.e. variation of CTC levels 
over time) and a between-subject treatment factor (i.e. anesthesia regimen).    
 
Fixed randomization based on a 1:1 allocation ratio will be performed using 
minimization (www.randomizer.at).  Patients will be stratified according to their ASA 
class during the randomization process, which will be performed using a web-based 
interactive allocation platform (www.randomizer.at).   
 
Accordingly, the risk of allocation and selection bias will be negligible. Restriction and 
stratified randomization are likely to minimize the risk of unbalanced known and 
unknown confounders. Blinding of participants and outcome assessors (laboratory 
staff, pathologist) will prevent measurement/detection bias. 
 
12.2 Null and Alternative Hypotheses 
 
The null hypothesis: there is no difference in the mean CTC levels over time among 
patients exposed to volatile anesthesia compared to patients in the control group 
(propofol anesthesia). 
The alternative hypothesis: there is a difference in the mean CTC levels over time 
among patients exposed to volatile anesthesia compared to patients in the control 
group (propofol anesthesia). 
 
The primary objective of this study is to reject the null hypothesis H0.  
 
 
12.3 Planned Analyses 
All primary and secondary analyses will be performed following an intention-to-treat 
(ITT) model. This analysis will be subsequently compared to a per-protocol analysis 
to assess the impact of patients who were not treated as randomized. Results will be 
reported as OR, 95% confidence intervals and their associated p-values. A p-value 
<0.05 will be considered statistically significant. 
Data analyses will be performed after enrolment of the last patient and completion of 
the study. No interim analyses are planned.  
 

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12.3.1 Primary Analyses 
 
The primary endpoint, i.e. blood CTC levels, is a continuous variable. In our study, 2 
independent variables may interact with CTC levels: a time factor, which results from 
repeated measurements over time (i.e. within-subjects factor) and an intervention 
factor, which is the anesthesia regimen (between subjects factor). Thus, our primary 
analysis will be performed using a linear mixed model with random effects matrix to 
account for within-subjects correlated observations as indicated for longitudinal data 
analysis. 
 
12.3.2 Secondary Analyses 
Since most reports refer to CTC levels as a binary outcome (i.e. positive versus 
negative endpoint using a cut-off value of ≥5 CTC/7.5ml blood), data transformation 
from a continuous into a binary variable will be performed using the same cut-off 
value as described previously. This new data set will be analyzed using a logistic 
regression mixed model with random effects matrix. 
Provided our sample size is large enough, adequate randomization is likely to reduce 
imbalance in baseline characteristics between groups. However, further exploratory 
analyses will be performed whenever there is a need to control for residual 
confounding.  
 
12.3.3 Interim Analyses 
No interim analyses are planned. 
 
12.4 Handling of Missing Data 
 
Longitudinal data analyses are likely to suffer from error and bias due to missing or 
incomplete data. In the case of dropouts or incomplete dataset, it remains thus 
essential to collect a maximal amount of information to determine the nature of 
missing data (data missing completely at random (MCAR), data missing at random 
(MAR) or data missing not at random (MNAR)). In case of missing randomness we 
will consider using multiple imputation for analyses where complete data are needed 
(linear mixed models can deal with missing data without need for imputation), which 
is considered superior to a complete case analysis (assuming that all data are MCAR 
or MAR) or a last observation carried forward analysis.  
In our trial, missing data is not expected to exceed 10% for any variable. This trial will 
be conducted in a university hospital where clinical research is common practice 
(trained study nurses, research-orientated infrastructure and care, high standards of 
quality control). Considerable efforts will be made to ensure high protocol compliance 
(postoperative medication using a predefined schema provided by the electronic 
patient database; regular meetings with the anesthesia team to identify potential 
enrolment or organizational issues).  
 

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12.5 Determination of Sample Size 
 
Sample size calculation was performed using a method accounting for a longitudinal 
design with 2 groups, which allows for repeated measurements and subject attrition. 
Since most studies investigating perioperative CTC only provide positivity rates rather 
than numerical levels, we were not able to use these data to estimate our sample 
size. Therefore, we adopted a conservative approach and assumed that the 
expected effect size between groups would be small (0.3). This should ensure that 
our estimation is adequately powered to detect even a small difference between 
groups. Moreover, we estimated a within-subject correlation over time of 0.4. Thus, to 
detect a 30% difference between groups, with a power of 80% at a significance level 
of 5% (two-sided) and assuming a dropout rate of 10% over time, we would need 116 
patients in each group (i.e. a total of 232 patients).   
 
 

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13. DATA HANDLING AND RECORD KEEPING 
The study will strictly follow the protocol. If any changes become necessary, they 
must be laid down in an amendment to the protocol. All amendments of the protocol 
must be signed by the Sponsor-Investigator and submitted to IEC and Swissmedic.  
The investigators will use electronic case report forms (eCRF), one for each enrolled 
study participant, to be filled in with all relevant data pertaining to the subject during 
the study. All subjects who either entered the study or were considered not-eligible or 
were eligible but not enrolled into the study additionally have to be documented on a 
screening log. The investigator will document the participation of each study subject 
on the Enrollment Log.  
For data and query management, monitoring, reporting and coding an internet-based 
secure data base secuTrial® developed in agreement to the Good Clinical Practice 
(GCP) guidelines provided by the Clinical Trials Center (CTC) Zurich will be used for 
this study. It is the responsibility of the investigator to assure that all data in the 
course of the study will be entered completely and correctly in the respective data 
base. Corrections in the eCRF may only be done by the investigator or by other 
authorised persons. In case of corrections the original data entries will be archived in 
the system and can be made visible. For all data entries and corrections date, time of 
day and person who is performing the entries will be generated automatically. 
eCRFs must be kept current to reflect subject status at each phase during the course 
of study. Subjects must not to be identified in the eCRF by name. Appropriate coded 
identification (e.g. Subject Number) must be used.  
It must be assured that any authorised person, who may perform data entries and 
changes in the eCRF, can be identified. A list with signatures and initials of all 
authorised persons will be filed in the study site file and the trial master file, 
respectively. 
Documented medical histories and narrative statements relative to the subject's 
progress during the study will be maintained. These records will also include the 
following: originals or copies of laboratory and other medical test results must be kept 
on file with the individual subject's eCRF.  
The investigators assure to perform a complete and accurate documentation of the 
subject data in the eCRF. All data entered into the eCRF must also be available in 
the individual subject file either as print-outs or as notes taken by either the 
investigator or another responsible person assigned by the investigator.  
Essential documents must be retained for at least 10 years after the regular end or a 
premature termination of the respective study (VKlin Art. 25).  
Any patient files and source data must be archived for the longest possible period of 
time according to the feasibility of the investigational site, e.g. hospital, institution or 
private practice. 
 
 

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14. CONFIDENTIALITY 
The investigators are liable to treat the entire information related to the study and the 
compiled data strictly confidentially. Any passing-on of information to persons that are 
not directly involved in the study must be approved by the owner of the information.  
Data generation, transmission, archiving and analysis of personal data within this 
study, strictly follows the current Swiss legal requirements for data protection. 
Prerequisite is the voluntary approval of the subject given by signing the informed 
consent prior start of participation of the clinical trial.  
 
Individual subject medical information obtained as a result of this study is considered 
confidential and disclosure to third parties is prohibited. Subject confidentiality will be 
further ensured by utilising subject identification code numbers to correspond to 
treatment data in the computer files. 
 
Data generated as a result of this study are to be available for inspection on request 
by the monitors, by the IEC and the regulatory health authorities. 
 
 

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15. INSURANCE 
Insurance is covered by “Haftpflichtversicherung für den Kanton Zürich betreffend 
das UniversitätsSpital Zürich“ (Certificate n°1192 ; insurance Policy n°14.970.888). 
Any damage developed in relation to study participation is covered by this insurance. 
So as not to forfeit their insurance cover, the subjects themselves must strictly follow 
the instructions of the study personell. Subjects must not be involved in any other 
medical treatment without permission of the principal investigator (emergency 
excluded). Medical emergency treatment must be reported immediately to the 
investigator. The investigator must also be informed instantly, in the event of health 
problems or other damages during or after the course of study treatment. 
The investigator will allow delegates of the insurance company to have access to the 
source data/documents as necessary to clarify a case of damage related to study 
participation. All involved parties will keep the patient data strictly confidential. 
A copy of the insurance certificate will be placed in the Investigator’s Site File. 
 
 

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16. STUDY REGISTRATION 
The study will be registered in the local trial registry of the University Hospital Zürich 
(„Studienregister USZ“), the international ClinicalTrials.gov registry (clinicaltrials.gov) 
and SNTCP N° 0952 
 

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17. PUBLICATION POLICY 
After the statistical analysis of this trial the sponsor will make every endeavour to 
publish the data in a medical journal.  
The results of this trial will be published irrespective of its findings. Any form of 
publication bias including selective outcome reporting will be avoided. Third parties 
with financial or other support will not have any influence in the decision to publish 
the results of this trial. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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18. FINANCIAL DISCLOSURE 
This study will be supported by institutional funds (“Betriebsmittelkredit Beck-
Schimmer”).   

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19. SIGNATURES 
Sponsor-Investigator (Principal Investigator): 
 
This clinical trial protocol was subject to critical review and has been approved by the 
Sponsor-Investigator. The information herein is consistent with 
- the current risk/benefit evaluation of the investigational product(s) 
- the moral, ethical and scientific principles governing clinical research as set out in 
the  
  current version of the Declaration of Helsinki, Good Clinical Practice and the  
  respective SAMW guidelines. 
 
 
Prof. Dr. med. Beatrice Beck Schimmer  
Institute of Anesthesiology  
University Hospital Zurich  
Raemistrasse 100  
CH-8091 Zurich  
Switzerland 
 
 
 
 
Zürich, 23.03.2017 
 
Place/Date 
 
Signature 
 
 
 
 
Co-Investigator:  
Dr. med. Frédérique Hovaguimian  
Institute of Anesthesiology  
University Hospital Zurich  
Raemistrasse 100  
CH-8091 Zurich  
Switzerland 
 
 
 
 
Place/Date 
 
Signature 
 
 
 
 
 

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Local Principal Investigator at study site: 
 
I have read and understood this trial protocol and agree to conduct the trial as set out 
in this study protocol, the current version of the World Medical Association 
Declaration of Helsinki, ICH-GCP guidelines or ISO 14155 norm and the local legally 
applicable requirements. 
 
Site 
Klinik Hirslanden 
Principal 
investigator 
Prof. Dr. med. Manfred Seeberger 
 
 
 
 
 
 
 
 
 
Zürich, 23.03.2017 
Place/Date 
 
Signature 
 
 
 
 
Investigator :  
 
med. pract. Urs Rölli 
Institute of Anaesthesiology and Intensive Care  
Klinik Hirslanden 
Wittellikerstr. 40 
CH-8032 Zurich  
Switzerland 
 
 
 
 
 
Place/Date 
 
Signature 
 

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