Conditions: Risk Reduction Mammographic Density Reduction
Keywords: Breast cancer Prevention Tamoxifen
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 1440
Healthy volunteers: yes

Brief summary:
KARISMA2 is a randomized, double-blinded, six-armed placebo controlled study to identify a low dose of tamoxifen, with less side-effects and a density reduction non-inferior to the standard dose of 20 mg.

Detailed description:
This is a dose determination study aiming to identify the optimal tamoxifen dose for reducing the risk of breast cancer. The change in mammographic density is a very good marker of therapy response. Investigators will test if 1 mg, 2.5 mg, 5 mg and 10 mg reduce the mammographic density to the same extent as 20 mg.

1440 healthy women 40-74 yrs were included when adhering the national Swedish mammography screening program between 2016- 2019. Women were randomized and treated daily for 6 months. Mammograms were taken at baseline and at end of treatment and side effects were measured throughout the study trough schedueled questionnaires and spontaneous AE-reporting.

Arms:
arm 1: None arm 2: None arm 3: None arm 4: None arm 5: None arm 6: None

Interventions: intervention 1: Tamoxifen Oral Tablet intervention 2: Placebo Oral Tablet
Intervention descriptions: intervention 1: Randomised dose of tamoxifen 1 pill/day for 180 days intervention 2: Randomised dose of tamoxifen 1 pill/day for 180 days

Protocol document:
Prot_000.pdf: 
Clinical Study Protocol: Karisma II 
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Protocol code No: KARISMAII 
Version 2.1 
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Karisma II: A randomized, double blinded, six-armed placebo controlled study 
to investigate optimal dose of tamoxifen with the most favourable side effect 
spectra and with mammographic density reduction non-inferior to that of 20 
mg tamoxifen. 
 
 
 
 
 
 
 
Substance: 
 
 
 
 
Tamoxifen 
 
EudraCT:  
 
Coordinating Investigator, Principal 
Investigator and Sponsor: 
2016-000882-22  
 
 
Per Hall, MD, Professor 
Karolinska Institutet 
Institution for Medical Epidemiology and Biostatistics 
171 77 Stockholm 
Tel: +46733960590 
Email: per.hall@ki.se 
 
 
 
 
 
 
 


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PROTOCOL REVISION HISTORY 
 
 
 
Version 
Date 
Revised by 
Description of change  
Refers to 
chapter/page 
1.0 
2016-04-21 
NA 
Initial version. 
NA 
2.0 
2016-06-29 
NA 
Revised after feedback from MPA. For 
details see reply to MPA dated 1 July 2016 
NA 
2.1 
2017-01-02 
 
Revised after inclusion in Lund 20161114-
1202 and before recruitment in Sthlm 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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PROTOCOL SYNOPSIS 
 
Study Title 
Karisma II: A randomized, double blinded, six armed, placebo controlled study to investigate optimal dose of 
tamoxifen with the most favourable side effect spectra and with mammographic density reduction non-inferior to 
that of 20 mg tamoxifen.  
Study code KARISMA II 
EudraCT No: 2016-000882-22 
Coordinating Investigator 
Per Hall 
Study centre(s) – 
Karma Study Centre / Breast and Mammography screening unit, Södersjukhuset, Stockholm, Sweden 
Karma Study Centre / Unilabs mammography unit / Klinska prövningsenheten, Skånes Universitetssjukhus i 
Lund, Lund, Sweden 
Study period 
Estimated date of first subject enrolled, November 2016 
Estimated date of last subject completed, December 2017 
Phase of development 
Phase II 
Objectives and endpoints 
Primary objective: Identify the minimal dose of tamoxifen non-inferior in its ability to reduce mammographic 
density compared to 20 mg of tamoxifen 
Primary endpoint: Change in mammographic density and levels of side effects after 6 months. In particular, we 
will test for noninferiority in the proportion of women in the intervention arms (placebo, 1 mg, 2.5 mg, 5 mg, 10 
mg) who have a density reduction as great as or greater (after 6 months) than the median density reduction in the 
20 mg arm 
Secondary objectives: Assess the drop-out level and the level of side effects in the intervention arms compared to 
the 20 mg arm. 
Secondary endpoints: Drop-out rate. We will test for differences in the proportion of drop-outs after 6 months in 
the intervention arms compared to the 20 mg arm. 
Tertiary objectives: Relate levels of tamoxifen metabolites, proteins, lipids and hormones in blood and changes 
in breast tissue to different tamoxifen doses. Measure genetic polymorphism in germline DNA and relate it to the 
other tertiary objectives. Measure mammographic density and levels of side effects 6 months after tamoxifen 
cessation in relation to treatment arm 
Tertiary endpoints: Levels of tamoxifen metabolites, proteins, lipids and hormones in blood. Breast tissue 
changes. Genetic polymorphism in germline DNA. Mammographic density and levels of side effects 6 months 
after tamoxifen cessation 
Study design 
Randomized, double blinded, six-armed, placebo controlled clinical trial 
Number of subjects planned 
1,440 
Diagnosis and main eligibility criteria 
Healthy women participating in mammographic screening with a a measurable mammographic density, i.e. ≥4.5 
% density (volumetric) measured by Volpara. 
 
Investigational Medical Product 
Investigational product – Tamoxifen 


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Dosage – 20 mg, 10 mg, 5mg, 2.5 mg, 1 mg and 0 mg (placebo) 
Oral administration, one tablet once daily 
Duration of treatment 
6 months 
Duration of subjects involvement in the study 
6 months for the SÖS-cohort (1400 particip), 12 months for the Lund cohort (40 paticip) 
Efficacy assessments 
Mammographic density after 6 months 
Plasma levels of tamoxifen metabolites, proteins, lipids and hormones in blood 
Genetic polymorphism measured in germline DNA 
Safety assessments 
Monitoring and recording of all AEs and SAEs  
Statistical methods 
In two recent studies using change in mammographic density as a proxy for therapy response to daily intake of 
20 mg tamoxifen, exactly 48% of the women responded i.e. had a significant reduction in mammographic 
density. This implies that approximately 50% of the women experience the beneficial effect of tamoxifen with a 
daily dose of 20 mg. We therefore define the response threshold to be the median decrease in absolute 
mammographic density of women in the 20 mg tamoxifen arm (this definition ensures that the same proportion 
of women exceeds the threshold as observed in previous studies). Further, we define a responder to be a woman 
whose mammographic absolute decrease between baseline and 6 months exceeds the response threshold. 
The primary efficacy endpoint is the proportion of responders. We will test for non-inferiority with respect to the 
primary efficacy endpoint for groups of women treated with placebo, 1, 2.5, 5, and 10 mg compared to the group 
of women treated with 20 mg tamoxifen. The non-inferiority margin is defined to be 16.7 percentage points; that 
is, we define non-inferiority to mean that the fraction of responders is not less than one third of the treated 
individuals. 
The null hypothesis is thus that the proportion of responders in women treated with placebo, 1, 2.5, 5, and 10 mg 
is 16.7 percentage points less than those women treated with 20 mg tamoxifen (i.e. the fraction of responders is 
less than one third; 50%-16.7%=33.3%).  
Since little is known about the threshold when absolute mammographic density is used, we will perform 
sensitivity analyses where the threshold is varied ±10 percentage points. 


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Table of Contents 
Protocol Revision History ................................................................................................... 2 
PROTOCOL SYNOPSIS ......................................................................................................... 3 
1 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ................................................. 8 
2 
ETHICS ........................................................................................................................ 9 
 
Ethical and Regulatory review ......................................................................................... 9 
 
Ethical conduct of the study ............................................................................................ 9 
 
Subject information and consent .................................................................................... 9 
 
Subject data protection ................................................................................................... 9 
3 
ADMINISTRATIVE STRUCTURE .................................................................................... 9 
4 
INTRODUCTION ........................................................................................................ 10 
 
STUDY RATIONALE ........................................................................................................ 11 
5 
Risk-Benefit Assesment ............................................................................................ 13 
 
Benefits ........................................................................................................................ 13 
 
Risks of side effects ....................................................................................................... 13 
 
Study population .......................................................................................................... 14 
 
Study length and study period ...................................................................................... 14 
 
Risk-benefit of frequent mammograms ......................................................................... 15 
6 
STUDY OBJECTIVES and endpoints ............................................................................ 15 
 
Primary objective .......................................................................................................... 15 
 
Primary endpoint .......................................................................................................... 15 
 
Secondary objectives .................................................................................................... 15 
 
Secondary endpoint ...................................................................................................... 15 
 
Teritary objectives ........................................................................................................ 16 
 
Teritary endpoints ........................................................................................................ 16 
7 
INVESTIGATIONAL PLAN ........................................................................................... 17 
 
Overall study design ..................................................................................................... 17 
7.1.1 
Invitation ......................................................................................................................... 17 
7.1.2 
Pre-screening of eligibility of participation ..................................................................... 17 
7.1.3 
Baseline (Month 0) .......................................................................................................... 18 
7.1.4 
1 month after baseline (+2/-1 weeks) ............................................................................. 18 
7.1.5 
3 months after baseline (+/- 2 weeks) ............................................................................ 19 
7.1.6 
6 months after baseline (+/- 2 weeks), End of treatment all participants, end of study 
SöS-cohort .................................................................................................................................... 19 
7.1.7 
12 months after baseline (+/- 2 weeks), End of study Lund cohort ................................ 19 
7.1.8 
In between regular visits at the Karma Study Centre ..................................................... 19 
7.1.9 
Reminders ....................................................................................................................... 19 
 
Dropouts ....................................................................................................................... 20 
 
The Karma Study Centre ............................................................................................... 20 
 
Choice of tamoxifen as risk-reducing medication .......................................................... 20 
 
Choice of tamoxifen doses ............................................................................................ 21 
 
Design of follow-up scheme .......................................................................................... 21 
 
Selection of study population ....................................................................................... 21 
 
Inclusion criteria ........................................................................................................... 22 
 
Exclusion criteria ........................................................................................................... 22 


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Criteria for withdrawal .................................................................................................. 22 
 
Treatments ................................................................................................................... 23 
7.11.1 
Identity of Investigational Medicinal Product (IMP) ................................................... 23 
7.11.2 
Packaging, labelling and storage of IMP ..................................................................... 23 
7.11.3 
Doses and treatment regimens .................................................................................. 24 
7.11.4 
Investigational Medicinal Product accountability ....................................................... 24 
7.11.5 
Method of assigning participants to treatment groups .............................................. 24 
7.11.6 
Blinding ....................................................................................................................... 25 
7.11.7 
Prior and concomitant therapy ................................................................................... 25 
7.11.8 
Treatment compliance ................................................................................................ 25 
8 
Safety Assessments .................................................................................................. 25 
 
Participant Card ............................................................................................................ 25 
 
Birth control for women in fertile age ........................................................................... 26 
9 
Adverse Events ......................................................................................................... 26 
 
Definitions .................................................................................................................... 26 
9.1.1 
Adverse Event ................................................................................................................. 26 
9.1.2 
Serious Adverse Event ..................................................................................................... 26 
9.1.3 
Suspected Unexpected Serious Adverse Reaction (SUSAR) ............................................ 27 
 
Reporting of SUSARs ..................................................................................................... 27 
 
Eliciting and recording of AEs ........................................................................................ 27 
 
AEs of interest .............................................................................................................. 27 
 
Reporting of SAEs ......................................................................................................... 28 
9.5.1 
Follow-up of unresolved AEs ........................................................................................... 29 
9.5.2 
Procedures in case of pregnancy .................................................................................... 29 
10 
Data Quality Assurance ......................................................................................... 29 
 
Electronic Case Report Forms (eCRFs) ........................................................................... 29 
 
Monitoring .................................................................................................................... 29 
 
Source data ................................................................................................................... 30 
 
Audit and inspections ................................................................................................... 30 
 
Changes in the approved Study Protocol ....................................................................... 30 
 
Statistical methods and determination of sample size ................................................... 30 
10.6.1 
Demographics and baseline data ................................................................................ 30 
10.6.2 
Analysis of the primary endpoint (effectiveness ) ...................................................... 30 
10.6.3 
Sample size ................................................................................................................. 31 
10.6.4 
Analysis of the secondary outcome ............................................................................ 31 
10.6.5 
Analysis of tertiary outcomes ..................................................................................... 31 
10.6.6 
Examination of subgroups .......................................................................................... 31 
10.6.7 
Interim analysis ........................................................................................................... 32 
 
Data Management ........................................................................................................ 32 
11 
Study Management ............................................................................................... 32 
 
Study time table - End of study ..................................................................................... 32 
 
Insurance/indemnity .................................................................................................... 32 
 
Study agreements ......................................................................................................... 33 
 
Criteria for termination of the study ............................................................................. 33 
 
Reporting of results and publication policy ................................................................... 33 
 
Record retention ........................................................................................................... 33 
 
Disclosure and confidentiality ....................................................................................... 33 
12 
EMERGENCY PROCEDURES .................................................................................... 33 
 
Emergency contacts ...................................................................................................... 33 
 
Emergency unblinding for safety reason ....................................................................... 34 


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Procedures in case of medical emergency ..................................................................... 34 
 
Procedure in case of overdose ...................................................................................... 34 
13 
REFERENCES .......................................................................................................... 35 
14 
APPENDIxES .......................................................................................................... 36 
 
Signature pages ............................................................................................................ 36 
 
Questionnaire ............................................................................................................... 36 
 
Schedule of events ........................................................................................................ 36 
 
Additional translational protocol: Biopsy Karisma II ...................................................... 36 
 
Participant Card ............................................................................................................ 36 
 


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1 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
Abbreviation or term 
Explanation 
 
AE 
ATC 
Adverse event 
Anatomical Therapeutic Chemical 
BMI 
CIOMS 
CIOMS- 
form 
CMO 
Body mass index 
Council for International Organizations of Medical Sciences 
 
Suspect Adverse Reaction Report Form 
Contract Manufacturer Organization (in Pharma industry) 
CRF 
CRO 
CSA 
eCRF 
DCIC 
DDP 
DMP 
FAP 
Case report form 
Contract Research Organisation 
Clinical Study Agreement 
Electronic Case report form 
Ductal Carcinoma In Situ 
Data Display Plan 
Data Management Plan 
Full Analysis Population 
GCP 
ICF 
Good Clinical Practice 
Informed Consent Forms 
ICH 
International conference on harmonisation 
IEC 
Independent ethics committee 
IMP 
IMPD 
MedDRA 
PP 
Investigational medicinal product 
Investigational Medicinal Product Dossier 
Medical Dictionary for Regulatory Activities 
Protocol Population 
SAE 
SAP 
SP 
Serious adverse event 
Statistical Analysis Plan 
Safety Population 
SUSAR 
QC 
QP 
Suspected Unexpected Serious Adverse Event 
Quality Control 
Qualified person. Medicinal product can not be released for sale or 
supply prior to certification by a QP that the batch is in accordance 
with the relevant requirements 
 
 


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2 
ETHICS 
 
Ethical and Regulatory review 
Necessary approvals of the Study Protocol, the Subject Information and Informed Consent 
Form must be obtained before enrolment of any subject into the study. Furthermore, it is the 
responsibility of the Sponsor to keep the applicable Independent Ethics Committee (IEC) 
informed of any Suspected Unexpected Serious Adverse Reactions (SUSARs) and any 
substantial amendments to the protocol during the study period. The written approval from the 
IEC, including a study identification and the date of approval, will be filed with the Karma 
Group at the Karolinska Institutet and at the study site(s) together with a list of the IEC 
members, their titles or occupation, and their institutional affiliations.  
 
Ethical conduct of the study 
The study will be performed in accordance with the recommendations guiding physicians in 
biomedical research involving human subjects that were adopted in 1964 by the 18th World 
Medical Assembly, in Helsinki, Finland, with later revisions. 
 
Subject information and consent 
It is the responsibility of the Investigator to give each subject, adequate verbal and written 
information regarding the objectives and the procedures of the study as well as any risks or 
inconvenience involved before including the subject in the study. The subject should be 
informed that by signing the Informed Consent Form (ICF) she authorises monitor(s), 
auditor(s), the IEC and the Regulatory Authorities to have direct access to the subject’s medical 
records for verification of clinical study procedures. The women must be informed about the 
right to withdraw from the study at any time and that participation is on a voluntarily basis. The 
subject should be allowed sufficient time for consideration of the proposal.  
It is the responsibility of the Investigator to obtain signed informed consent from all subjects 
before including them in the study. The ICF must be signed and dated before any study-specific 
procedures are performed, including screening procedures. In case of possible future 
audits/inspections, the Investigator must file the signed ICF. 
The final version of the subject information and ICF is submitted to the IEC and concerned 
Regulatory Authorities and must not be changed without permission from Sponsor and the local 
IEC. 
 
Subject data protection 
The Investigator must file a subject identification list that includes sufficient information to link 
records, i.e. the electronic Case Report Form (eCRF) and clinical records. This list should be 
preserved for possible future inspections/audits.  
The subjects will be informed that the data will be stored and analysed by computer, that 
Swedish and local regulations for the handling of computerised data will be followed and that 
identification of individual subject data will only be possible for the Investigator.  
Representatives of the Karma Group including a Contract Research Organisation (CRO), and/or 
regulatory authorities will inform the subjects about the possibility of inspections/audits of 
relevant parts of the clinical records. Authorisation to direct access to the subject’s clinical 
records, as described above, is given by signing the ICF. 
3 
ADMINISTRATIVE STRUCTURE 
Qualified investigators under the sponsorship of the Karma Group will conduct the study. The 
name and contact details of the responsible persons at Karma Group and the CRO will be 
listed in the Investigator Site File provided at the site.  


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Role 
Name 
Contact 
Site 
Sponsor/Principal 
Investigator/ Coordinating 
Investigator/Responsible 
medical officer 
Per Hall 
073-396 05 90 
per.hall@ki.se 
Karolinska Institutet 
/Södersjukhuset /Karma Study 
Centre 
Medical doctor/Principal 
Investigator Lund 
Signe Borgquist 
0704-452558 
signe.borgquist@med.lu.se 
Skånes Universitetssjukhus 
Lund, Klinisk 
prövningsenhet/Karma Study 
Centre 
Medical 
doctor/Investigator 
Södersjukhuset 
Magnus Bäcklund 
Magnus.Backlund@ki.se 
Karolinska 
Universitetssjukhuset/KI/Karma 
Study Centre 
Head of radiology 
Södersjukhuset/ 
Responsible study 
specific mammography 
Karin Leifland 
karin.horstmann-
leifland@sodersjukhuset.se 
Department of Radiology, 
Södersjukhuset 
Head of Radiology 
Unilabs Skåne/ 
Responsible study 
specific mammography 
Catharina Behmer 
+46 (0)731-55 02 55 
catharina.behmer@unilabs.com 
Mammography Unit, Skånes 
Universitetssjukhus Lund 
Clinical Trial Manager 
Mattias Hammarström 
073- 7121476 
mattias.hammarstrom@ki.se 
Karolinska Institutet /Karma 
Study Centre 
Research nurses 
Ann-Sofie Andersson 
Agneta Lönn 
070-750 2110/08-524 82339 
karmastudien@ki.se 
Karma Study Centre 
MD, Gynaecological 
consultant 
Kenny Rodriguez-
Wallberg 
kenny.rodriguez-
wallberg@ki.se 
Karolinska 
Universitetssjukhuset / 
Karolinska Institutet  
Medical 
doctor/Radiologist 
Kristina Lång 
kristina.lang@med.lu.se 
Lunds Universitetssjukhus / 
Unilabs mammography 
QP-release 
ClinStorage AB 
 
Ola Camber 
 
Stockholm, Solna 
Monitor 
KTA, Karolinska Trial 
Alliance 
Lotta Mazouch@KTA.se 
Stockholm/Lund 
Holder of blinding-code 
(In case of emergency and 
blinding needs to be 
broken) 
Apoteket Lund AB 
Weekdays 08:00 – 16:00: 
 046-177139 
All other times: 046-171000, 
require jour pharmacist 
 
Stockholm/Lund 
SUSAR reporting 
KTA, Karolinska Trial 
Alliance 
Lotta Masousch 070-0011845 
Stockholm/Lund 
 
4 
INTRODUCTION 
The number of women diagnosed with breast cancer is increasing all over the world but little is 
done to lower these figures. The primary preventive measures that are available ranges from 
increased physical activity to prophylactic mastectomy. Several randomised controlled trials 
have shown a 40-60% decreased risk of breast cancer after anti-hormonal therapy (e.g. 
tamoxifen, raloxifen, aromatase inhibitors; [1]). Tamoxifen and aromatase inhibitors are 
normally used to treat breast cancer patients to reduce the risk of recurrence of the disease.  
Despite the remarkable risk reduction in preventive randomised controlled trials, primary 
preventive strategies are scarcely part of clinical routine [1]. There are several possible reasons 


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for the reluctance, the major one being that the side effects of these therapies are not trivial. 
Serious side effects could only be acceptable for those that benefit from therapy, that is, there 
is a need of identifying women at high risk. Also, there is not a consensus on dose needed in 
the preventive setting. So far only full therapeutic dose has been tested and no attempts have 
been made to determine if lower doses prevent women from being diagnosed with breast cancer. 
Karisma is an investigator-initiated study of primary prevention of breast cancer incidence. 
Karisma will be performed in three steps: 
Karisma I, Pilot study  
A previous application to Läkemedelsverket regarding the Karisma I study was accepted and 
the first pilot phase of Karisma was initiated in March 2015 and ended nine months later.  
The exploratory pilot study including 42 women who were randomised to either 10 or 20 mg 
of tamoxifen. The aims of the pilot study were to identify the time to mammographic density 
change, difference of density change between 10 mg and 20 mg tamoxifen and piloting 
procedures.  
The results from the pilot study show that density decreases within 6 months by about 50% for 
one quarter of the participants, about 20% for another quarter, and does not change for the 
remaining half of the participants.  
As expected, there were more reported side effects in those groups with most density decrease. 
We noted no difference in the density reduction nor side effect spectra when we compared 10 
and 20 mg tamoxifen. Approximately 25% of participants discontinued treatment due to 
adverse events and there was no dose dependant difference in discontinuation. 
Karisma II (which this application refers to) 
This is a dose determination study aiming to identify the optimal tamoxifen dose for reducing 
the risk of breast cancer. Instead of following a very large number of women, for many years, 
treated with different doses of tamoxifen and see if they develop breast cancer, we will include 
1,440 healthy women participating in the mammographic screening program at two 
Mammography units (Unilabs mammography, Lund and Södersjukhuset Breast Centre, 
Stockholm) and measure their change in mammographic density. The change in 
mammographic density is a very good marker of therapy response. We will test if 1 mg, 2.5 
mg, 5 mg and 10 mg reduce the mammographic density to the same extent as 20 mg. In 
summary Karisma II will be a randomized, double-blinded, six-armed placebo controlled study 
to identify the dose of tamoxifen with the most favourable side effect spectra and with a density 
reduction non-inferior to 20 mg tamoxifen. 
The Karisma II study will be run in accordance with the ICH GCP guidelines, the principals of 
which have their origins in the Helsinki 2000 Declaration.  
Karisma III 
When the optimal tamoxifen dose has been identified in the Karisma II study, we intend to test 
this dose in women at high risk of breast cancer and study long-term effect of breast cancer 
incidence. In this phase we plan to include 8,000 women. A separate application will be sent 
for Karisma III.  
 
STUDY RATIONALE 
For more than 40 years 20 mg of tamoxifen has been used in breast cancer patients to reduce 
the risk of recurrence. It has also been shown that 20 mg of tamoxifen reduces the risk of breast 
cancer in perfectly healthy women but that the treatment comes with side effects. [4] The side 
effects are probably one reason tamoxifen is not used in the preventive setting despite the 
substantial risk reducing effect. The aim of the Karisma II study is to identify the lowest 


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tamoxifen dose that could be used for primary preventive purposes, with a hypothesis that a 
lower dose will reduce the side effects.  
Mammographic density is the white part of the mammogram and consist of glandular and 
connective tissue. The black, non-radiolucent part is fat. The more glandular and more 
connective tissues, the whiter the breast, and thus, the higher the risk of breast cancer [2]. 
Factors that influence risk of breast cancer are known to influence density. Hormone 
replacement therapy increases density and risk of breast cancer, while tamoxifen has the 
opposite effect [3]. The molecular basis for the tamoxifen-induced changes in breast density is 
sparsely understood. Consequently, a secondary aim in the Karisma trial is to investigate 
changes in tissue markers by comparing breast tissue biopsies sampled prior to and after six 
months of treatment with tamoxifen or placebo. 
Mammographic density has been assessed in a nested case-control study within the IBIS-1 
study, a randomised prevention study of tamoxifen vs. placebo. The density decrease, over the 
first 18 months, in 123 women diagnosed with breast cancer, was compared to 942 women 
without breast cancer [4]. Forty-eight per cent of the women treated with tamoxifen experienced 
a 10% or greater reduction in breast density and had a 63% reduction in breast cancer risk 
compared to women in the placebo group. In contrast, those who took tamoxifen but 
experienced less than a 10% reduction in breast density had no risk reduction.  
Within the Karma group a parallel study has been conducted including breast cancer patients 
who where treated with adjuvant tamoxifen therapy [5]. Forty-eight per cent of the women 
experienced a decrease in density of > 20 % over a 12 month period and had a reduced breast 
cancer recurrence of 50%. These findings are fully supported by a very recent finding by a US 
group [6].  
The scientific “golden standard” for measuring mammographic density is a software supported 
method called Cumulus. In this semi-automated approach the breast contour is outlined and a 
human reader decides on what part of the breast is dense. The software then calculates the dense 
area. The obvious weaknesses of Cumulus are that it is time consuming, labor intensive and 
highly reader dependent. 
In clinical practice mammographic density is measured using BI-RADS. BI-RADS categorizes 
breast into four categories: entirely fatty, scattered areas of fibroglandular density, and 
extremely dense breast. The BI-RADS measurement is done by the radiologist by looking at 
the mammogram. BI-RADS measurements are very crude and highly reader dependent. 
Volpara is a FDA approved full automatic method for volumetric density measurement that the 
Karma project group in collaboration with the Mammography units at Södersjukhuset and 
Unilabs Lund has implemented for clinical use. The Volpara method has also been validated in 
the Karma cohort. We will use Volpara as a tool for assessment of eligibility (measurable 
density), since its implemented and the measurement can be done instantly.  
In the former mentioned Karma adjuvant tamoxifen therapy study we used a newly developed 
fully automated density measurement, the Stratus method, which mimics how Cumulus 
measures mammographic density but as automated tool is not reader dependent. In the Karisma 
II study we will use both Volpara and Stratus to analyse the mammographic changes.  
The results from the Karisma I study showed that the effect of tamoxifen on mammographic 
density can be determined after 6 months of treatment and indicates that the effect is equal for 
the doses 10 mg and 20 mg. Currently there are no studies including longitudinal measurement 
of mammographic density on healthy women using tamoxifen. By adding a lower dose in the 
Karisma I study, 10 mg, we have got an indication lower doses will be sufficient for reducing 
density. In Karisma II we will include six arms with 200 women in each arm; 20 mg, 10 mg, 5 


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mg, 2.5 mg, 1 mg and a control arm with placebo. The Subjects and Investigators will be 
blinded.  
Side effects and quality of life will be measured as a function of tamoxifen dose. In Karisma II 
we will use same questionnaires as in Karisma I to evaluate general symptoms (Memorial 
Symptom Assesment Scale), specific symtoms of tamoxifen (FACT-ES), compliance of 
medicine intake (Morisky scale) and health literacy (Health Literacy Scale). The questionnaires 
are validated instruments and include scoring system to evaluate the symptom burden of the 
study participants. (References to questionnaire instruments are to be found in appendix 15.2 
Questionnaires).  
The Karma project forms the basis for the Karisma study. The Karma project is lead by the 
same group that heads Karisma. The aim of Karma is to reduce incidence and mortality in breast 
cancer by a preventive approach. During 2010-2013 the Karma Study Centres at Södersjukhuset 
and at Skånes Universitetssjukhus, Lund were used to create the Karma Cohort witch is a 
prospective observational study including 70,877 women. Women invited for screening 
mammography were invited to Karma Study Centre to sign an informed consent form, donate 
blood, measure blood pressure and answer a comprehensive questionnaire on lifestyle factors. 
The women also allowed us to access their medical information in registers and records, and to 
store their mammograms. In Karisma II we plan to use the same infrastructure as for the 
recruitment to the Karma Cohort. 
 
5 
RISK-BENEFIT ASSESMENT 
 
Benefits 
There is no obvious advantage to participate in the study on an individual level. The benefits 
must be seen in a larger perspective, i.e. the possibility to develop a preventive measure against 
breast cancer. 
Every hour one Swedish woman is diagnosed with breast cancer and one in nine women will 
develop breast cancer during their lifetime. Tamoxifen has proven to reduce the incidence in 
breast cancer but treatment comes with side effects. If a reduced dose of tamoxifen could be 
proven to reduce the incidence in breast cancer and at the same time have less side effect it 
could potentially be used as a preventive measure. Such a finding has the potential to 
significantly reduce breast cancer incidence. We believe that a preventive approach with 
tamoxifen could lead to a reduction in the incidence of breast cancer by at least 10%. This 
corresponds to saving ≈ 800 women per year from getting the disease in Sweden alone. 
Previous preventive tamoxifen studies (there are four) have exposed healthy women to 
tamoxifen for at least three years. In the tamoxifen study by Jack Cuzick published in 2011 it 
was found that women who experienced a decrease in mammographic density had a reduced 
risk of breast cancer [4]. It is therefore possible that the reduction in mammographic density 
per see, and not the length of treatment, is what brings the reduction in breast cancer risk. In 
short, it is possible that participants in the study experiencing a reduction in mammographic 
density will have a reduced risk of breast cancer. 
 
Risks of side effects 
Tamoxifen at a dose of 20 mg has side effects that can be divided into common side effects, 
that are more or less harmless and reversible, and rarer serious side effects. 
The common side effects are menopause symptoms; sweating, hot flashes and insomnia. These 
side effects disappear shortly after stopping treatment. Women who during the study are 
bothered by these side effects will obviously be able to cancel participation. 


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The serious and uncommon side effects include cancer of the uterus. This is extremely rare and 
perishing of gynecological disorders. A gynecologist is involved in the study and the 
participants will be encouraged to contact the Karma Study Centre if they experience problems. 
Another serious and uncommon side effect is blood clots. It's the same kind of problems that 
can arise when using oral contraceptives. It should be emphasized that the rare side effects only 
have been observed for tamoxifen use in doses of 20-40 mg for at least 2 years. The spectra of 
side effects at lower doses and shorter treatment times are unknown.  
By applying strict exclusions criterias, such as a history of trombo- embolic disease, 
hypertension or use of hormonal contraceptive pills we will minimize the risks we expose to 
the participants. 
Participants will report side effects after 1, 3, and 6 months (and the Lund cohort will also report 
after 12 months). They will also report side effects whenever they occur and there is also an 
alarm system when adverse events, that need to be followed up, are reported in the web 
questionnaire form or in the application called Karmapp installed in their smartphone or tablets. 
Participants will be informed to contact the Karma Study Centre immediately if they suspect 
side effects. The contact channels are Karmapp, phone or email and they will be manned during 
long-weekends and holidays seasons on a jour basis.  
 
Study population 
The study will include healthy women that participate in the mammographic screening program 
at Södersjukhuset, Stockholm and at Skånes Universitetssjukhus, Lund, and have a negative 
screening mammogram. We will not include breast cancer patients as they receive a variety of 
drugs that might influence mammographic density. We are excluding women with no 
measurable density. This is because measurable density at study start is a prerequisite for being 
able to see reduction of mammographic density over time. We are not selecting women based 
on risk of breast cancer since we currently lack a reliable risk prediction model for Swedish 
standards. 
 
Study length and study period 
There are three studies [4,5,6] that used mammographic density change during one year as a 
proxy for tamoxifen effect. Two projects studied the association between mammographic 
density change in patients and prognosis and one study looked at density change among 
perfectly healthy woman and risk of breast cancer. All three studies came to the same result, 
approximately half of the treated participants respond to the therapy by a significant reduction 
in mammographic density after one year. Those that respond with a decreased mammographic 
density had a lower risk of breast cancer recurrence and a lower risk of breast cancer. 
The main reason we conducted the Karisma I study was to investigate time to density change. 
Radiologists tell us that the time to density change is most probably much shorter than one year, 
probably only a couple of months. However, the issue of time to density change during 
tamoxifen therapy has never been addressed. 
The results from Karisma I show that, regardless of dose, density decreases within 6 months by 
about 50% for one quarter of the participants, about 20% for another quarter and not at all for 
the other participants.  This result implicates that tamoxifen intake during 6 months gives a 
measurable density change of responders; therefore, the treatment period in Karisma II are set 
to 6 months.  
From a clinical point of view and for coming study (Karisma III) we will investigate the state 
of density change and decline of side effects 6 months after tamoxifen cessation in relation to 
doses. This will be done exploratory and only for the Lund-cohort. 


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The study period will be at least 14 months since we plan to run Karisma II at two sites 
successively: 
 
Site 
Karma Study Centre Lund 
Karma Study Centre Stockholm/SöS 
Number of 
participants 
Approximately 40 
Approximately 1400 
First 
enrolment 
November 2016 
February 2017 
End of 
treatment 
May 2017 
December 2017 
End of follow 
up 
November 2017 
December 2017 
 
 
Risk-benefit of frequent mammograms 
Eligible study participants will only be women that have negative screening mammograms 
before entering the study, i.e. no abnormalities have been identified. The participants will have 
extra mammograms taken at 6 (all participants) and 12 (only Lund cohort) months after study 
entry. In order to reduce radiation exposure, the mammograms consist of 2 images instead of 
4, which are standard at a screening examination. The images will be used for density 
measurement but radiologist will also screen them for abnormalities as in clinical practise. It 
could be that some few cancers are diagnosed at these extra visits, which should be seen as 
something that benefits the study participants.  
If a suspected alteration is seen, women will receive the normal routine work-up for a suspected 
breast cancer, that is, additional ultrasound examination and a possible fine needle biopsy. 
Surgical interventions are only performed if a cancer is detected.   
 
6 
STUDY OBJECTIVES AND ENDPOINTS 
 
Primary objective 
Identify the minimal dose of tamoxifen that is non-inferior in its ability to reduce 
mammographic density compared to 20 mg  tamoxifen. 
 
Primary endpoint 
Change in mammographic density and levels of side effects after 6 months. In particular, we 
will test for noninferiority in the proportion of women in the intervention arms (placebo, 1 
mg, 2.5 mg, 5 mg, 10 mg) who have a density reduction as great as or greater (after 6 months) 
than the median density reduction in the 20 mg arm 
 
 
Secondary objectives 
Assess the drop-out level and the level of side effects in the intervention arms compared to the 
20 mg arm. 
 
Secondary endpoint 
Drop-out rate. We will test for differences in the proportion of drop-outs after 6 months in the 
intervention arms compared to the 20 mg arm. 
 


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Teritary objectives  
Relate levels of tamoxifen metabolites, proteins, lipids and hormones in blood and changes in 
breast tissue to tamoxifen doses. Measure genetic polymorphism in germline DNA and relate 
it to the other teritary objectives. Measure mammographic density and levels of side effects 6 
months after tamoxifen cessation in relation to tamoxifen dose. 
 
Teritary endpoints  
Levels of tamoxifen metabolites, proteins, lipids and hormones in blood. Breast tissue 
changes. Genetic polymorphism in germline DNA. Mammographic density and levels of side 
effects 6 months after tamoxifen cessation  
 


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7 
INVESTIGATIONAL PLAN 
 
Overall study design  
 
 
7.1.1 Invitation  
Eligible women will be identified from women invited to mammography screening in 
Stockholm/Lund according to following: 
1) A comprehensive study information about Karisma II will be sent to all women invited to 
mammographic screening at the collaborating sites a few days after screening invitation. 
2) In connection with screening examination, interested women are invited to visit the Karma 
Study Centre (located within the screening facilities) to declare their interest of participation 
and to get further information about the study. 
7.1.2 Pre-screening of eligibility of participation 
At Karma Study Centre: 
1) A Karma staff checks that the woman has read and understood the study information. 
2) Volpara density measurement data are retrieved from the performed mammographic 
examination in order to see that the women meet inclusion criteria for density (≥4.5 % 
volumetric density) 


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3) Blood pressure is measured to ensure eligibility to participation. 
 If it at this stage turns out that the woman doesn’t meet inclusion criteria or meet any of the 
exclusion criteria, she will have an explanation from investigator why she can’t participate. 
A recording of non-participation is done in the Study file screening-list. 
7.1.3 Baseline (Month 0) 
Meeting with Investigator 
• The investigator will explain the nature and aim of the study, its procedures, possible 
side effects, requirements and restrictions.  
• The woman will sign the ICF before any further study related procedures are 
performed. 
• A woman fulfilling the inclusion and exclusion criteria will be randomised to one of 
the six treatment arms; tamoxifen 20 mg, 10 mg, 5 mg, 2.5 mg, 1 mg or placebo. All 
treatments will be on a daily basis for 6 months  
• The woman will receive a sufficient amount of IMP in order to take one tablet daily 
during 6 months  
• Screening form, randomization number and other applicable information will be filled 
in by investigator in the eCRF. 
• The participants will be asked if they allow to have two biopsies of the normal breast 
tissue taken at month 0 and 6. This is optional and complies with a separate study 
protocol (Protocol: Biopsy Karisma II) 
Procedures at baseline 
After meeting with investigator when ICF is signed and randomization is performed, the 
following will be obtained: 
• In total 24 ml blood will be collected. The blood will be analysed for tamoxifen 
metabolites, plasma hormones, plasma proteins, metabolic markers. In addition, DNA 
will be extracted and plasma aliquots will be biobanked for later analyses. 
• The participants will be asked to fill out two web-based questionnaires;  
o The Karma questionnaire including lifestyle factors that influence risk of breast 
cancer such as age at first birth, breast-feeding, etc., demographic, medical 
history, quality-of-life, and concomitant medication.  
o Karisma II baseline questionnaire including general symptoms and tamoxifen 
related symptoms 
• Pregnancy test (urine) will be offered women of childbearing age for safety 
• Measurement of weight, length and waist 
• Instructions are given on how to use and report events by the Karma-app  
• Biopsy of the breast. This is optional and complies with an additional translational 
study protocol (Protocol: Biopsy Karisma II) 
7.1.4 1 month after baseline (+2/-1 weeks) 
• Karisma II follow up questionnaire including general symptoms, tamoxifen related 
symptoms, compliance and health care contact connected to possible side effects 


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7.1.5 3 months after baseline (+/- 2 weeks) 
• Karisma II follow up questionnaire including general symptoms, tamoxifen related 
symptoms, compliance and health care contact connected to possible side effects 
7.1.6 6 months after baseline (+/- 2 weeks), End of treatment all participants, end of study 
SöS-cohort 
Visit at Karma Study Centre: 
• Karisma II follow up questionnaire including general symptoms, tamoxifen related 
symptoms, compliance and health care contact connected to possible side effects 
• In total 24 ml blood will be collected. Planned analyses are tamoxifen metabolites, 
plasma hormones, plasma proteins, metabolic markers. 
• Mammogram (one image per breast only for density measurement purposes) 
• Measurement of weight, length and waist 
• A 2nd biopsy will be performed if participation at baseline 
End of study SöS cohort. The women will be informed that the study period now has ended 
and she will be called to mammography within the national screening program.  
 
7.1.7 12 months after baseline (+/- 2 weeks), End of study Lund cohort 
Visit at Karma Study Centre: 
• Karisma II follow up questionnaire including general symptoms, tamoxifen related 
symptoms, compliance and health care contact connected to possible side effects 
• Mammogram (one image per breast only for density measurement purposes) 
• Measurement of weight, length and waist 
• Meeting with Investigator (Group meeting) 
The women will be informed that the study period now has ended and she will be called to 
mammography within the national screening program.  
7.1.8 In between regular visits at the Karma Study Centre 
In addition to the visits at the Karma Study Centre at baseline and 12 months, all participants 
will have access to web-based questionnaires at 1 and 3 months post baseline. The 
questionnaires will cover adverse events, change in concomitant medication and compliance. 
The participants can at any time in between regular visit at the Karma Study Center contact the 
Karma Study Centre with questions and issues regarding adverse events, compliance and 
concomitant medications. The Karma Study Centre will give participants fast access to medical 
doctors in case of need. 
7.1.9 Reminders 
Women will receive e-mail/sms reminders regarding their upcoming appointment and/or web-
questionnaire to complete. 


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Women not showing up at regular visits at the Karma Study Centre or fail to submit follow-up 
questionnaires will be contacted to ensure compliance and to ensure that no adverse events have 
occurred. 
 
Dropouts  
Participation in the Karisma II study is voluntary and the study participant may end her 
participation at any time. There may however be a scientific value to continue investigating the 
effects of tamoxifen after the participant discontinues her treatment to tamoxifen. Women that 
drop out after 1 week or more, will therefore be asked to still attend the planed follow-up 
questionnaires, blood sampling,  exit-mammogram and biopsy if applicable. This is however 
not a mandatory request and the study participant is free to decline. The time limit of exit exams 
is set to 2 weeks after stop of medication. 
 
The Karma Study Centre 
Women participating in the Karisma II study will conduct all study specific activities, except 
the mammography, at the Karma Study Centre. The Karma Study Centre is located adjacent to 
the Breast and Mammography Unit at both Skånes Universitetssjukhus Lund and at 
Södersjukhuset. They were established in 2011 and many of the women invited to the Karisma 
II study are also part of the Karma Cohort and have thus visited the Karma Study Centre 
previously.  
 
Choice of tamoxifen as risk-reducing medication 
Tamoxifen is a selective estrogen receptor modulator (SERM), which means that it affects the 
way estrogen influences the breast tissue. Breast cancer development is closely linked to the 
exposure of the female breast tissue to female sex hormones, among them estrogen. The 
hormones increase the rate by which cells divide, proliferate, and thereby increase the risk of a 
deleterious mutation that could lead to cancer. Tamoxifen blocks the estrogen receptor and 
thereby estrogens effect on breast tissue. 
The underlying mechanism driving tamoxifen to reduce breast density is not currently known, 
but rather one of the key scientific questions we struggle with right now. We do not know why 
more than 20% of the fibroglandular tissue “disappears” in approximately half of the woman 
treated with tamoxifen. It is not likely that the cells go into apoptosis but it could be that the 
reduced cell proliferation makes cells less radiolucent and therefore not seen on an x 
ray/mammogram.  
Tamoxifen has been used in the treatment of breast cancer for more than 40 years and is 
generally well tolerated. There are several reasons why we chose to use tamoxifen in the 
preventive setting: 
• The recently published American Association of Clinical Oncology (ASCO) clinical 
practice guideline on primary prevention of breast cancer, tamoxifen is one out of three 
suggested compounds and the only one suggested to be used in both pre- and post 
menopausal women [1]. 
• Tamoxifen is off patent and cheap (the cost is 10-20 that of aromatase inhibitors), thus 
is well suited from a health-economics perspective. 
• Alternative drugs, e.g. aromatase inhibitors, have side effects such as severe joint pain 
that influence adherence to therapy.  
• Aromatase inhibitors are not able to block the high production of estrogen seen in 
premenopausal women and are never used for younger women in the clinical setting. 
Thus aromatase inhibitors are not used for younger, premenopausal women. A woman 
identified with a very high risk of breast cancer at the age of 40 years should have the 


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possibility to start risk reducing medication and not have to wait until she enters 
menopause. 
 
 
 
Choice of tamoxifen doses 
In the pilot study we used 10 and 20 mg of tamoxifen. We choose the clinically accepted of 20 
mg that has so far also been used in the preventive setting. By adding a dose of 10 mg we got 
an indication of both density response and severity of side effects.  
There are currently no data supporting 10 mg of tamoxifen to have fewer and/or lower 
prevalence of side effects than 20 mg. This coincidence with what we saw in the pilot study but 
numbers were to small to make any significant conclusions. By adding 5 mg, 2.5 mg and 1 mg 
we believe that we will see a lower prevalence of side effects. The hypothesis that there is a 
linear dose – side effect relationship in tamoxifen therapy comes from the clinical experience 
in the early days of tamoxifen use. Initially the preferred dose was 40 mg/day but in the mid 
1980s doses were lowered to 20 mg mainly because of the severe side effects of 40 mg. If we 
believe in a linear dose – side effects relationship, even lower doses should further reduce side 
effects. 
 
Design of follow-up scheme 
Experience from clinical practice using tamoxifen in the adjuvant setting and from the set up in 
the pilot study, indicates that side effects from tamoxifen typically occur within the first 3 
months after initiation of treatment. The Karisma II study will therefore have frequent follow-
ups during the first 3 months post baseline, including a follow up questionnaire after 1 and 3 
month. Participants will also be instructed to contact Karma Study Center to report AE/side-
effects whenever they occur. 
 
Selection of study population 
Eligible women will be identified from the Mammographic screening cohort, i.e. women age 
40-74 years invited for screening at Södersjukhuset and University hospital in Lund during the 
inclusion period. Within this group of women, approximately 1/3 will be part of the Karma 
Cohort. The Karma Cohort is a prospective observational study including 70,877 women 
recruited in Stockholm and Region Skåne during 2010-2013. Women were recruited in to the 
Karma Cohort at screening centers in Lund, Landskrona, Helsingborg and at Södersjukhuset. 
Approximately 35 000 women were recruited from the Mammography department at 
Södersjukhuset and 10 000 from Mammography department in Lund. The only exclusion 
criteria were not being able to read and/or understand the informed consent form written in 
Swedish. For each participant we have gathered information on lifestyle factors, previous 
disorders, reproductive history, etc., through a web-based questionnaire. All participants have 
donated blood and allowed us to store mammograms. All Karma participants have signed an 
informed consent form allowing us to contact them to ask for participation in future studies.  
Only including Karma participants in the study will slow down inclusion. We will therefore 
invite all women attending screening at Södersjukhuset and University hospital in Lund during 
the study period. 
 
The rationale to exclude women with the lowest density is based on experience from our 
previous study of mammographic density reduction in patients on tamoxifen. In that study 
we had to exclude women with the lowest density since density change was not possible to 
measure in women with very low density. The cut off for inclusion is set to 4.5% density or 
more, measured by Volpara. The cut-off value is set to be sure that we will capture a change.  


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Inclusion criteria 
To participate, the women must meet the following inclusion criteria: 
• Attending the national mammography screening program, i.e. aged 40-74 and has 
performed a mammogram maximum 3 months prior to study inclusion 
• Having a measurable mammographic density, i.e. ≥4.5 % density (volumetric) 
measured by Volpara 
• Informed consent must be signed before any study specific assessments have been 
performed 
 
Exclusion criteria 
To participate, the women must not meet any of the following exclusion criteria: 
• Being pregnant or planning to become pregnant during the study period 
• Any previous or current diagnosis of breast cancer (including carcinoma in situ) 
• Mammographic BI-RADS code 3 or above at baseline mammography, or at a diagnostic 
mammography during time of treatment (the first 6 months of the study) 
• Any previous diagnosis of cancer with the exception of non-melanoma skin cancer and 
in situ cancer of the cervix 
• Currently using oral oestrogen and progesterone based hormone replacement therapy  
• Current use of hormone contraceptive with hormones, e.g. hormonal contraceptive pills, 
or progesterone implants. Hormonal intrauterine devices are accepted. 
• A history of thrombo-embolic disease such as embolies, deep vein thrombosis, stroke, 
TIA or cardiac arrest. 
• Known APC (Activated protein C )- resistance, an inherited hemostatic disorder 
• A history of major surgery of the breast, e.g. reduction or enlargement, which might 
affect density measurements 
• A history of immobilization, e.g. using wheelchair 
• Known uncontrolled diabetes  
• Hypertension at baseline, defined as systolic pressure higher than 140 mm Hg and 
diastolic higher than 90 mm Hg 
• Use of drugs that interfere with CYP2D6 expression such as Seroxat (paroxetine), 
Fontex (fluoxetin) and Zyban / Voxra (bupropion) 
• Use of Waran (warfarin) 
• Non-medical approved drugs against hot-flashes including phytooestrogen 
• Not able to understand study information and/or informed consent 
 Criteria for withdrawal 
The Subject should be excluded from the study if, in the opinion of the Investigator, it is 
medically necessary, or if it is the expressed wish of the subject. Women are free to discontinue 
their participation in the study at any time. 
Irrespective of the reason for withdrawal and whenever possible, the subject should be 
encouraged to return for a clinical visit at the time of or soon after discontinuation and complete 


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all evaluations which may be necessary to assure that the subject is free from any untoward 
effects and to perform appropriate follow-up for any continuing medical event. Relevant 
laboratory test samples should be obtained and all relevant assessments should be completed, 
preferably according to the scheme for the final assessment. The eCRF should be completed as 
far as possible. 
A withdrawn subject is not allowed to re-enter into the study. The criteria for withdrawal are 
the following:  
• A woman develops breast cancer, including ductal carcinoma in situ (DCIS). 
• Subject wishes to discontinue 
• Unacceptable adverse event/side effect 
• Non-compliance with the study protocol 
• Subject refuses to cooperate 
• Other medical reasons 
• Pregnancy 
 
 Treatments 
7.11.1 Identity of Investigational Medicinal Product (IMP) 
 
Tamoxifen 1, 2.5, 5, 10 , 20 mg and matching placebo are white to off white, flat, beveled 
edge uncoated tablets, plain on both sides. 
 
All investigational product will be provided as a tablet for oral administration and provided in 
blisters. 
All investigational product (tamoxifen and matching placebo) will be manufactured according 
to Good Manufacturing Practice (GMP). 
7.11.2 Packaging, labelling and storage of IMP  
The flow of the IMP: 
 
1) Producer (Khandelwal Laboratories PVT Ltd) 
2) Relabeling of CMO in Sweden (Galenica AB) 
3) Release (Clinstorage AB) 
4) Storage (Sjukhusapoteket Lund and Sjukhusapoteket Karolinska) 
5) Distribution to Karma Study Center 
At IMP producer (India): 
The producer of the IMP produces the six doses of tamoxifen in different batches. Every dose 
will be produced during one series i.e. one batch. The tablets are placed in blisters (primary 
package) then 18 blisters will be put in a box (secondary package). All boxes of the same dose 
are placed in one of six tertiary packages (a tertiary package per dose). The tertiary packages 
are than sealed. All the (primary, secondary and tertiary) packaging is labelled with a batch 


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number in accordance with GMP. Labelling on blister is an adhesive label that can be 
removed. The six tertiary packages are sent to a CMO in Sweden (Galencia) where the 
primary and secondary packaging will be relabelled according to a list of randomization. 
 
At CMO (Sweden): 
Blister is labeled with: 
Study: KarismaII 
Sponsor: Karolinska Institutet 
Investigator: Per Hall 
Randomization No: [1,2, ..., 1200] 
Batch No: xxx 
 
Secondary packaging is labelled with complete information including Randomization No.  
QP of release in Sweden is ClinStorage AB (QP name Ola Camber).  When released the IMP 
will be stored at Sjukhusapoteket Lund respectively Karolinska sjukhusapoteket Solna.  
 
The investigational product (tamoxifen and matching placebo) should be stored in the 
containers in which they are received from the Sponsor’s supplier, preferably at room 
temperature, but no specific storage conditions are needed. 
7.11.3 Doses and treatment regimens 
Participants will be randomised into: 
• Tamoxifen 20 mg 
• Tamoxifen 10 mg 
• Tamoxifen 5 mg 
• Tamoxifen 2.5 mg 
• Tamoxifen 1 mg 
• Placebo 0 mg 
All treatments will be on a daily basis (1 tablet daily) for 6 months. 
7.11.4 Investigational Medicinal Product accountability 
The IMP will be dispensed to the study participant at baseline. The number of tablets given 
out will be recorded in both CRF and in the Product Accountability log in the Study file. We 
will ask the participant to bring back the container at next visit (after 6 months or by 
withdrawal/dropout) in order to count remaining tablets. Any unused tamoxifen (i.e. 
remaining tablets) should be saved for drug accountability and sent for destruction. 
7.11.5 Method of assigning participants to treatment groups 
Randomisation will be performed by a CMO (Galenica AB). The participants will be 
randomized in blocks in where the 6 arms are distributed evenly. The study participant 
emergency code envelopes are kept by a 24/7 service provider (Apoteket AB in Lund) during 
the whole study. Researchers do not have access to the randomisation list. This means that the 
study is triple blinded, i.e. neither study participant, Karma Study Centre, nor researchers 
know which medication product was used for each study participant. 


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7.11.6 Blinding 
The study is double-blinded, placebo controlled randomised study and neither the investigators 
nor the participants will know who is on active treatment and at what doses (See 7.11.5 for 
method of assigning participants to treatment groups). 
The randomization code (emergency envelopes) are held by Apoteket in Lund. The treatment 
code can only be broken if an investigator considers there is a valid medical or safety reason to 
break the blinding. The Investigator must have a reason to break the blinding, for example when 
the studied drug is likely to have a significant effect on the clinical management of the study 
participant. When the code is broken for an individual subject, a notification has to be given in 
the eCRF. 
The study is also blinded for the person responsible for Mammographic Density assessments. 
7.11.7 Prior and concomitant therapy 
Other therapy that is considered necessary for the subject’s welfare may be given at the 
discretion of the Investigator. 
• Women using drugs that interfere with CYP2D6 expression such as Seroxat 
(paroxetine), Fontex (fluoxetin), Zyban / Voxra (bupropion) are not allowed during 
the study or used at study enrolment. 
• Oestrogen and progesterone based hormone replacement therapy (oral), hormone 
contraceptive with hormones, e.g. hormonal contraceptive pills, or progesterone 
implants are not allowed during the study or used at study enrolment. 
• Waran (warfarin) is not allowed during the study or used at study enrolment 
• Non-medical approved drugs against hot-flushes including phytoestrogen are not 
allowed during the study or used at study enrolment. 
 
All concomitant therapy must be recorded in the eCRF. No other drug under investigation may 
be used concomitantly with the study medication. Medication will be coded according to 
WHO’s Anatomical Therapeutic Chemical (ATC) classification. 
7.11.8 Treatment compliance 
Compliance will be assessed throughout the study by i) calculating the number of tablets 
remaining at the final visit at the Karma Study Centre and comparing with the expected usage, 
ii) by web-questionnaires targeting adherence to therapy. The participant is defined as a 
complier if more than 80% of the tablets are taken. 
 
8 
SAFETY ASSESSMENTS 
Safety assessment will consist of monitoring and recording Adverse Events (AE) and Serious 
Adverse Events (SAE). Women in fertile age will be asked to use high efficient birth control 
and pregnancy tests will be done if the women approve. 
 
Participant Card  
Participants will get a wallet sized Participant card containing essential information about the 
study and contact information to Karma Study Center (see appendix). 


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Birth control for women in fertile age 
Premenopausal participants will be asked if they use any of the highly effective birth control 
methods mentioned below: 
1. Hormonal contraception inhibiting ovulation. 
a. Women on using exogenous female sex hormones will be excluded from the 
study since hormones affects mammographic density. 
2. Intrauterine devices 
a. Fully acceptable 
3. Intrauterine hormone releasing systems 
a. Fully acceptable since we do not believe that the low systemic hormone levels 
affects mammographic density. They do not influence the risk of breast cancer. 
4. Bilateral tubal exclusion 
a. Fully acceptable 
5. Vasectomised partner 
a. Fully accepted  
6. Sexual abstinence 
a. In most circumstances not an accepted method since the study continues for 6 
months. 
Methods #2-5 will be considered acceptable and in some few circumstances #6, depending on 
the wish of the participant.  
 
9 
ADVERSE EVENTS 
 
Definitions 
9.1.1 Adverse Event 
Any untoward medical occurrence in a subject, who has received IMP, will be registered. The 
occurrence does not necessary have to have a causal relationship with the IMP. An AE can 
therefore be any unfavourable and unintended sign (e.g. an abnormal laboratory finding), 
symptom, or disease temporally associated with the use of an IMP, weather or not considered 
related to the IMP. 
9.1.2 Serious Adverse Event 
An SAE is any untoward medical occurrence or event, at any dose, that: 
• Results in death 
• Is life threatening 
• Requires hospitalisation or prolongation of existing hospitalisation 
• Results in persistent or significant disability/incapacity 
• Is a congenital anomaly/birth defects. 
Or 


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• Other important medical event (example of such events are convulsions that do not 
result in hospitalisation) 
Medical and scientific judgment should be exercised in deciding whether expedited reporting 
is appropriate in other situations, such as important medical events that may not be immediately 
life-threatening or result in death or hospitalisation but may jeopardise the subject’s health or 
may require intervention to prevent one of the other outcomes listed in the definitions above. 
These AEs should also usually be considered as serious. 
9.1.3 Suspected Unexpected Serious Adverse Reaction (SUSAR) 
A SUSAR is a suspected unexpected serious adverse reaction, potentially causally related to 
the investigational medicinal product, and will be reported to the appropriate IEC and 
Regulatory Authorities. A Serious Adverse Reaction is expected if it is mentioned in the 
Summary of Product Characteristics (SPC) for Tamoxifen Mylan. 
 
Reporting of SUSARs 
Only SAEs that are both unexpected and assessed as related to IMP(s), i.e. SUSARs, are subject 
to expedited reporting by the Sponsor to the appropriate IEC and Regulatory Authorities, as per 
local requirements. 
The sponsor must report all SUSARs that resulted in death or was life threatening to the 
authority and to the IEC within 7 days, and if necessary complete the follow up report within 
the following 8 days. Other SUSAR should be reported within 15 days.  
 
Eliciting and recording of AEs 
The method for collecting and recording AEs will be through the web-based questionnaire each 
study participant has to fill in at a regular basis during the study period. Additionally, the 
participants can at any time report AEs to Karma Staff by contacting Karma Study Centre. All 
AEs, serious and non-serious, will be recorded in the eCRFs. If no AE has occurred during the 
study period, this should also be recorded. 
 
AEs of interest 
In the questionnaire (Appendix 14.2) are AE:s of interest listed.  
In the questionnaire it’s also possible to address not stated adverse events by writing free text. 
These answers are continuously monitored by Karma staff in order to take immediate action if 
there are any signs of adverse events of interest or possible serious adverse event. Potential 
serious adverse events are signs of thromboembolism or gynaecological bleedings. 
An alarm system in the e-CRF will alert personnel at the Karma Study Centre when an AE of 
interest is recorded so evaluation and follow up can be performed throughout the study period.  
All AEs of interest will be coded according to Medical Dictionary for Regulatory Activities - 
MedDRA. The Investigator will perform the coding. 
The following evaluations are to be done by the Investigator in connection with the AE of 
interest: 
• Description of the AE. If possible the investigator uses the diagnosis for description of 
the event. If no diagnosis is decided, each symptom will be described.  
• Seriousness 
- Yes or No, see definition of a SAE for seriousness, as of above. 


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• Intensity (if the intensity changes during the period of the AE the maximum intensity 
should be recorded) 
- Mild - symptoms which are easily tolerated (acceptable) 
- Moderate - symptoms which interfere with usual activity (disturbing) 
- Severe - symptoms which give incapacity to do usual activity (unacceptable) 
• Duration of the AE (start - end) 
• Action taken due to the AE 
- None 
- IMP stopped 
- Additional details on action taken, including diagnostic procedures, will be recorded for 
any women with vaginal bleeding or discharge. Any women who report abnormal vaginal 
bleeding or discharge or pelvic pain or pressure, should be promptly investigated. It is not 
mandatory that the study code is broken in such cases, but this may be done if requested 
by the treating physician, necessary for further treatment. 
• Causality rating with investigational medicinal product 
- Not related:  Indicates that the AE is definitely not related to the study drug. 
- Unlikely: The adverse onset and the medicinal product administration is such that the 
medicinal product is not likely to have any reasonable association to the AE. 
- Possible: The subject´s clinical status or the medicinal product could have produced the 
AE. 
- Probable: The AE follows a reasonable temporal sequence from the time of 
administration of the medicinal product, abates upon discontinuation of the medicinal 
product and cannot be reasonably explained by the known characteristics of the subject´s 
clinical status.  
- Not assessable: There is not sufficient information to assess causality between the 
medicinal product and the suspected reaction. 
• Outcome of the AE 
- Recovered/Resolved 
- Recovering/Resolving 
- Not recovered/Not resolved 
- Recovered/Resolved with sequelae 
- Fatal 
- Unknown 
 
Reporting of SAEs 
It is important to distinguish between serious and severe adverse events. Severity is a measure 
of intensity whereas seriousness is defined by the criteria in the previous sections. An adverse 
event of severe intensity need not necessarily be considered serious. For example nausea, which 
persists for several hours, may be considered severe nausea but not a serious adverse event. On 
the other hand, a stroke resulting only in limited degree in disability may be considered a mild 
stroke but would be considered a serious adverse event. 


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All SAEs must be reported by the Investigator using phone or fax within 24 hours from the 
Investigator's knowledge of the event to the Sponsor regardless of the time that may have 
elapsed from the time the event occurred to when the Investigator first learns of it. The initial 
report should contain as a minimum the following information: 
• Subject identification 
• Treatment specification 
• Diagnosis or symptoms  
• Name of the original reporter 
A SAE report form must also be completed, signed by the Investigator and submitted to the 
sponsor no later than five working days after the initial information was received. Apart from 
the information above, this follow-up report should also contain the following information: 
• Assessment of intensity 
• Assessment of causality 
9.5.1 Follow-up of unresolved AEs 
If a subject is withdrawn due to an AE, or if an AE persists at the end of the study treatment 
period, this should be followed up until the condition has ceased or until the subject is under 
professional medical care and a potential causality between IMP and the AE has been 
penetrated. An outcome assessment should be performed when an AE persists. 
9.5.2 Procedures in case of pregnancy 
In case of pregnancy or suspicion of possible pregnancy, the study treatment must be stopped 
immediately, and the subject discontinued from participation in the study. Pregnancy itself is 
not regarded as an AE unless there is a suspicion that the IMP may have interfered with the 
effectiveness of the contraceptive medication. However, the outcome of all pregnancies (e.g. 
spontaneous miscarriage, elective termination, normal birth or congenital abnormality) must be 
followed up and documented even after the subject was discontinued from the study. 
All events of congenital abnormalities/birth defects are SAEs. Spontaneous miscarriages should 
also be reported and handled as SAEs. All outcomes of pregnancy must be reported to Karma 
Study Centre. 
 
10 DATA QUALITY ASSURANCE 
 Electronic Case Report Forms (eCRFs) 
The site personnel will enter the information required for the study in the eCRF. Data will also 
be imported from web questionnaire, medical records and laboratory findings.  
 Monitoring  
The Karisma II study will be monitored according to ICH-GCP guidelines, Regulatory 
requirements and the study specific Monitoring plan. The Karisma II study will be monitored 
by an independent Monitor according to ICH GCP Guidelines. Study conductance, source data 
and adherence to the study protocol will be monitored. The Investigator's duty is to inform all 
personnel concerned about the trial and, if necessary, educate and train all persons participating 
in the study. This should be checked by the monitor.  


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 Source data 
Source data will be handled according to regulatory requirements. A source data location list 
will be kept at the investigational site for definition of source data.  
 Audit and inspections 
The study site may be subject to quality assurance audit by the Sponsor or someone appointed 
for this task by the Sponsor. A Regulatory Authority may request to make an inspection of the 
study site. The procedures of such a visit would be similar to those of a monitoring visit, and 
data already checked by the Monitor may be checked again. The Investigator is required to 
inform Karisma II study immediately of an inspection requested by a Regulatory Authority. 
The investigator and other relevant personnel must be available during the audit/inspection and 
must devote sufficient time. 
 Changes in the approved Study Protocol 
Study procedures must not be changed without the mutual agreement of the Investigator and 
Sponsor. 
Any substantial change to the approved Final Study Protocol will be documented in a written 
and numbered Protocol Amendment. Any proposed substantial change to the Final Study 
Protocol must be discussed with and approved by Sponsor/Investigator before submitted to IEC 
and Regulatory Authority for approval, according to applicable national regulations.  
 Statistical methods and determination of sample size  
All analyses will be performed after the study is completed and the database is released. All 
statistics, including tables, figures and listings, will be performed using SAS®, Version 9.3, and 
R, Version 3.x.  
The statistical analysis are described in detail in the Statistical Analysis Plan (SAP) including 
a Data Display Plan (DDP), which will be in a final draft version prior to study start and 
finalised and approved before database lock.  
10.6.1 Demographics and baseline data 
All data will be presented using descriptive statistics. Results will be presented in total, by 
treatment group, age and as menopausal status. Continuous variables will be summarised using 
number of women, mean, standard deviation, median, minimum and maximum. Categorical 
variables will be summarised using the number and percentage of women. 
10.6.2 Analysis of the primary endpoint (effectiveness ) 
The primary analysis will be performed on an intention to treat (ITT) basis.  
The results of both papers [4] and [5] showed that a threshold value in mammographic density 
decrease needs to be exceeded in order for a woman to experience the beneficial effect of 
tamoxifen. This threshold value was different in [4] compared to [5]. A 10% or greater reduction 
was needed in [4] whereas a 20% reduction was needed in [5], a difference most likely 
explained by different methods of measuring mammographic density. However, the proportion 
of women exceeding this threshold was exactly 48% in both studies, implying that 
approximately 50% of the women experience the beneficial effect of tamoxifen irrespective of 
the way mammographic density is measured.  
In the Karma II study, we will use area based mammographic density, which was used in both 
[4] or [5]. With support of the results in [4] and [5], we define the response threshold to be the 
median decrease in mammographic density of women in the 20 mg tamoxifen arm (this 
definition ensures that the same proportion of women exceeds the threshold as observed in 


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previous studies). Further, we define a responder to be a woman whose mammographic density 
decrease between baseline and 6 months exceeds the response threshold. 
Compliance of study drug intake is defined as at least 80% of the prescribed numbers of tablets 
should be used during time of treatment (i.e. 6 months). 
The primary efficacy endpoint is the proportion of responders (women with a reduction of 
mammographic density, from baseline to 6 months). We will test for non-inferiority with 
respect to the primary efficacy endpoint for groups of women treated with placebo, 1, 2.5, 5, 
and 10 mg compared to the group of women treated with 20 mg tamoxifen. The non-inferiority 
margin is defined to be 16.7 percentage points; that is, we define non-inferiority to mean that 
the fraction of responders is not less than one third of the treated individuals (50% minus 16.7% 
= 33.3%). 
The null hypothesis is thus that the proportion of responders in women treated with placebo, 1, 
2.5, 5, and 10 mg is 16.7 percentage points less than those women treated with 20 mg tamoxifen 
(i.e. the fraction of responders is less than one third).  
We will also perform sensitivity analyses where the threshold is varied ±10 percentage points. 
10.6.3 Sample size  
With 168 patients in each treatment group, the lower limit of the observed one-sided 98.5% 
confidence limit for the difference will be expected to exceed the non-inferiority margin 16.7 
percentage points with a 80% power, assuming that the expected difference in responders 
between two arms is 0 and the proportion responders in the 20 mg tamoxifen group is 50%. 
With an estimated 30% drop-out rate, 240 patients in each group need to be randomized. The 
computations are based on the following formula: 
n = f(α, β) × [πs × (100 − πs) + πe × (100 − πe)] / (πs − πe − d)2 
where πs and πe are the true percent 'success' in the standard (20mg) and experimental treatment 
group respectively, and 
f(α, β) = [Φ-1(α) + Φ-1(β)]2 
Φ-1 is the cumulative distribution function of a standardised normal deviate. 
10.6.4 Analysis of the secondary outcome 
We will assess the proportion of drop-outs at six months and test for differences in the 
proportion of drop-outs in the intervention arms compared with the 20 mg arm.  We will use 
Fisher’s exact test with an p-value of less than 0.05 (corrected for multiple testing, see SAP 
Section 6.7) as a threshold to declare statistical significance. 
10.6.5 Analysis of tertiary outcomes 
Tertiary outcomes will be analysed with statistical methods appropriate for the specific 
outcome. For example, the association of plasma concentrations of endoxifen with responder 
status will be analysed with logistic regression both within treatment groups and among 
treatments groups (treatment group will be handeled as a random effect in the latter case). Full 
details of the analysis of tertiary outcomes will be in the statistical analysis plan (SAP) prior to 
database release. 
10.6.6 Examination of subgroups 
We will perform subgroup analyses, where the subgroups are CYP2D6 polymorphism status 


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(excellent, intermediate, poor metaboliser). CYP2D6 polymorphism status will be handled as 
a categorical fixed effect variable. 
10.6.7 Interim analysis 
We will three months into the study perform an interim analysis on overall dropout level. If 
the proportion of drop out at three months, we will – at best – have 73% power for our 
primary analysis (see SAP Section 6.6). In this case, we will increase the number of women 
the study needs to accrue according to:  
ntot =168×6/(1−d),  
where d is the overall proportion of drop outs at three months.  
 
 Data Management 
A quality control (QC) of data will be performed to ensure that data entry and verification have 
been performed correctly in accordance to pre-defined instructions. The QC will be performed 
as described in the Data Management Plan (DMP). The QC will be performed before data is 
declared clean. 
 
11 STUDY MANAGEMENT 
 Study time table - End of study 
The study is planned in two steps geographically (see 5.4). The first inclusion phase will start 
in November 2016 in Lund. Last participant in Lund will end medication in May 2017 and will 
do the final examinations in November 2017. In the overlapping second phase we will start 
inclusion at Södersjukhuset, Stockholm, by November 2016 and end the study by May 2018. 
 Insurance/indemnity 
All participants are covered from accidents in connection with the trial by the Patient Injury 
Act insurance (Patientförsäkringslagen) and from injuries caused by study drug by the Swedish 
Pharmaceutical Drug Insurance.  
The Sponsor agrees to indemnify (legal and financial coverage) and hold the Investigator free 
of harm from any claim, whether based on legal principles or on generally accepted liability 
standards within the pharmaceutical industry, made against him by reason of personal injury, 
including death, to any person arising out of or connected with the performance of the study to 
the extent that the injury is not caused by: 
1) Failure by the Investigator to adhere to the terms of the Protocol; 
2) Failure by the Investigator to comply with any applicable governmental regulations; 
3) Malpractice, negligence or wilful malfeasance by the Investigator. 
The Investigator agrees to notify the Karisma study personnel whenever he/she becomes aware 
of a claim or action, and to co-operate with and to authorise Karisma study personnel to carry 
out sole management of such claim or action. 
The Sponsor’s responsibility is covered by LIF Läkemedelsförsäkring via an association 
agreement with Kammarkollegiet. The insurance covers Karisma II liability under law and 


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generally accepted liability standards within the pharmaceutical industry towards any third 
parties, including participating women, as Sponsor of the study. 
 Study agreements 
The Principal Investigator at the investigational site must comply with all the terms, conditions, 
and obligations of the Clinical Study Agreement (CTA) for this study.  
 Criteria for termination of the study 
The Karisma II study personnel reserves the right to discontinue the study at any time, but 
intends only to exercise this right for valid scientific or administrative reasons. 
After such a decision, the Investigator must contact all participating women within 2 weeks and 
inform the women as well as perform relevant assessments, preferably according to the scheme 
for the final assessments. All delivered and unused study products and other study materials 
must be returned and all eCRFs completed as far as possible. 
 Reporting of results and publication policy 
A clinical study report, in compliance with ICH E3; Guideline for Industry, will be prepared 
by the Karisma study personnel , describing the conduct of the study, the statistical analysis 
performed and the obtained results. 
The data generated by this study are considered confidential information and the property of 
the Karisma Study. Said confidential information may be published only in collaboration with 
the Karisma study leadership. The Karisma study leadership reserves the right to review and 
comment on the proposed publication prior to being submitted and/or published. 
 Record retention 
The Investigator must arrange for retention at the investigational site of a list of the women and 
their identifying code, subject files and other study documents. The archiving period must be 
adapted to regulations in force but should not be shorter than ten years after the termination of 
the study and the presentation of the final report.   
It is the responsibility of the Karisma study personnel to inform the Investigator/institution as 
to when these documents no longer need to be retained 
 Disclosure and confidentiality 
All unpublished information concerning the test product and research carried out within the 
study, including patent applications, manufacturing processes, basic scientific data, etc., is 
considered confidential and the sole property of Karisma. Disclosure to third parties must be 
limited to those undertaking legitimate peer reviews of the scientific and ethical aspects of the 
study and to those participating, including the recipients of drugs, so that customary medical 
care and informed consent can be achieved. 
 
12 EMERGENCY PROCEDURES 
 Emergency contacts 
In case of a medical emergency, contact Karisma study personnel: 
• Per Hall, Responsible Medical Officer, Coordinating and Principal Investigator 
Mobile +46733960590, email: per.hall@ki.se 
• Signe Borgqusit, Principal Investigator, Lund 
Mobile  +46704452558, email: Signe.Borgquist@med.lu.se 


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• Mattias Hammarström, Clinical Trial Manager 
Mobile +46737121476, email: Mattias.Hammarstrom@ki.se 
 Emergency unblinding for safety reason 
The randomization code (emergency envelopes) are held by Apoteket in Lund. In case of a 
rare emergency where, in the opinion of the Investigator, discontinuation of the study 
treatment is not sufficient and the study treatment must be unblinded in order to evaluate 
further course of action, the Investigator should contact Apoteket Lund (Phone numbers and 
routines are to be found in the Study Site File) to initiate patient unblinding. In the event the 
blind is broken, the sponsor should be informed as soon as possible. The date, time, and 
reason for unblinding must be documented in the appropriate section of the eCRF, and in the 
source document.  
Information about the treatment assignment must be restricted to designated study site staff who 
are providing immediate care to the patient. Any documentation of the treatment assignment 
must be maintained separately (i.e. in a secured file). The information must not be included in 
the patient’s source files to ensure the treatment assignment will remain blinded to the monitor 
and other study personnel not involved with the patient’s immediate care. 
 Procedures in case of medical emergency 
The Investigator is responsible for ensuring that there are procedures and expertise available to 
cope with medical emergencies during the study. The study is performed at the hospitals 
Södersjukhuset and Skånes Universitetssjukhus Lund, and any acute medical illness should be 
dealt with at the hospital. 
 Procedure in case of overdose 
Overdosing tamoxifen may on theoretical grounds increase the pharmacological side effects 
of the drug. Any study participant with a suspected or confirmed overdose of tamoxifen 
should contact the Swedish Poison Information Center.  
 
 


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13 REFERENCES 
1. 
Visvanathan K, Hurley P, Bantug E, Brown P, Col NF, Cuzick J, Davidson NE, Decensi A, Fabian 
C, Ford L, Garber J, Katapodi M, Kramer B, Morrow M, Parker B, Runowicz C, Vogel VG 3rd, 
Wade JL, Lippman SM. Use of pharmacologic interventions for breast cancer risk reduction: 
American Society of Clinical Oncology clinical practice guideline. J Clin Oncol. 2013 Aug 
10;31(23):2942-62.  
2. 
Boyd NF, Guo H, Martin LJ, Sun L, Stone J, Fishell E, Jong RA, Hislop G, Chiarelli A, Minkin S, 
Yaffe MJ. Mammographic density and the risk and detection of breast cancer. N Engl J Med. 2007 
Jan 18;356(3):227-36. 
3. 
Brand JS, Czene K, Eriksson L, Trinh T, Bhoo-Pathy N, Hall P, Celebioglu F.  Influence of lifestyle 
factors on mammographic density in postmenopausal women. PLoS One. 2013 Dec 
9;8(12):e81876. doi: 10.1371/journal.pone.0081876. eCollection 2013. 
4. 
Cuzick J, Warwick J, Pinney E, Duffy SW, Cawthorn S, Howell A, Forbes JF, Warren RM. 
Tamoxifen-induced reduction in mammographic density and breast cancer risk reduction: a nested 
case-control study. J Natl Cancer Inst. 2011 May 4;103(9):744-52. doi: 10.1093/jnci/djr079. Epub 
2011 Apr 11. 
5. 
Li J, Humphreys K, Eriksson L, Edgren G, Czene K, Hall P. J Clin Oncol. 2013 Jun 
20;31(18):2249-56. 
6. 
Nyante SJ, Sherman ME, Pfeiffer RM, Berrington de Gonzalez A, Brinton LA, Aiello Bowles EJ, 
Hoover RN, Glass A, Gierach GL. Prognostic significance of mammographic density change after 
initiation of tamoxifen for ER-positive breast cancer. J Natl Cancer Inst. 2015 Feb 6;107(3) 
7. 
Li J, Czene K, Brauch H, Schroth W, Saladores P, Li Y, Humphreys K, Hall P. Association of 
CYP2D6 metabolizer status with mammographic density change in response to tamoxifen 
treatment. Breast Cancer Res. 2013 Oct 3;15(5):R93. [Epub ahead of print] 
8. 
Early Breast Cancer Trialists’ Collaborative Group: Tamoxifen for early breast cancer: an overview 
of the randomised trials. Lancet 1998, 351:1451-1467. 
9. 
Maltoni C, Minardi F, Belpoggi F, Pinto C, Lenzi A, Filippini F: Experimental results on the 
chemopreventive and side eff ects of tamoxifen using a human-equivalent animal model. In The 
Scientifi c Bases of Cancer Chemoprevention. Edited by Maltoni C, Soff ritti M, Davis W. Elsevier 
Science BV; 1996:197-207. 
10. Decensi A, Robertson C, Viale G, Pigatto F, Johansson H, Kisanga ER, Veronesi P, Torrisi R, 
Cazzaniga M, Mora S, Sandri MT, Pelosi G, Luini A, Goldhirsch A, Lien EA, Veronesi U: A 
randomized trial of low-dose tamoxifen on breast cancer proliferation and blood estrogenic 
biomarkers. J Natl Cancer Inst 2003, 95:779-790. 
11. Guerrieri-Gonzaga A, Robertson C, Bonanni B, Serrano D, Cazzaniga M, Mora S, Gulisano M, 
Johansson H, Intra M, Latronico A, Franchi D, Pelosi G, Johnson K, Decensi A: Preliminary results 
on safety and activity of a randomized, double-blind, 2 x 2 trial of low-dose tamoxifen and 
fenretinide for breast cancer prevention in premenopausal subjects. J Clin Oncol 2006, 24:129-135. 
12. Decensi A, Robertson C, Guerrieri-Gonzaga A, Serrano D, Cazzaniga M, Mora S, Gulisano M, 
Johansson H, Galimberti V, Cassano E, Moroni SM, Formelli F, Lien EA, Pelosi G, Johnson KA, 
Bonanni B: Randomized double-blind 2 x 2 trial of low-dose tamoxifen and fenretinide for breast 
cancer prevention in highrisk premenopausal subjects. J Clin Oncol 2009, 27:3749-3756. 
 


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14 APPENDIXES 
 Signature pages 
 Questionnaire 
 Schedule of events 
 Additional translational protocol: Biopsy Karisma II 
 Participant Card 
 
 
 
 
 
 


Clinical Study Protocol: Karisma II 
 
Appendix 14.1; Signature pages 
 
 
Sponsor, Coordinating Investigator and Principal Investigator 
 
Study title: 
Karisma II: A randomized, double blinded, six-armed placebo controlled 
study to investigate optimal dose of tamoxifen with the most favourable side 
effect spectra and with mammographic density reduction non-inferior to 
that of 20 mg tamoxifen. 
 
 
By signing this page I acknowledge that I have read and understood the study protocol 
“Karisma II: A randomized, double blinded, six-armed placebo controlled study to investigate 
optimal dose of tamoxifen with the most favourable side effect spectra and with 
mammographic density reduction non-inferior to that of 20 mg tamoxifen.”. 
 
I also agree by signing this page that I will conduct the study in accordance with the 
procedures specified in the protocol, the ethical principles in the latest version of the 
Declaration of Helsinki, ICH Good Clinical Practice and applicable regulatory requirements. 
 
 
Sponsor, Coordinating Investigator and Principal Investigator, Södersjukhuset 
 
 
Per Hall 
 
 
 
2017-01-12 
Name 
 
Signature 
 
Date 
 
 
 
 


Clinical Study Protocol: Karisma II 
 
Principal Investigator 
 
Study title: 
Karisma II: A randomized, double blinded, six-armed placebo controlled 
study to investigate optimal dose of tamoxifen with the most favourable side 
effect spectra and with mammographic density reduction non-inferior to 
that of 20 mg tamoxifen. 
 
 
By signing this page I acknowledge that I have read and understood the study protocol 
“Karisma II: A randomized, double blinded, six-armed placebo controlled study to investigate 
optimal dose of tamoxifen with the most favourable side effect spectra and with 
mammographic density reduction non-inferior to that of 20 mg tamoxifen.”. 
 
I also agree by signing this page that I will conduct the study in accordance with the 
procedures specified in the protocol, the ethical principles in the latest version of the 
Declaration of Helsinki, ICH Good Clinical Practice and applicable regulatory requirements. 
 
 
Principal Investigator, Lund 
 
 
 
 
Signe Borgquist 
 
 
 
 
 
2017-01-12 
name 
 
Signature 
 
Date 
 
 
 
 


Clinical Study Protocol: Karisma II 
 
Appendix 14.2; Questionnaires 
 
Questionnaires sent to MPA and IRB are in Swedish and 
therefore removed from Clinical Trial version of protocol 
 
 
 


Clinical Study Protocol: Karisma II 
 
 
 
Appendix 14.3 
 
 


Clinical Study Protocol: Karisma II 
 
 
Appendix 15.4 
 
 
 Additional translational study Protocol: Biopsy Karisma II 
 
1. Objective 
Relate changes in breast tissue to different tamoxifen doses and placebo 
 
2. Endpoint 
Breast tissue changes between baseline and 6 months of tamoxifen treatment 
 
3. Rational  
The molecular basis for the tamoxifen-induced changes in breast density is sparsely understood. 
Consequently, a secondary aim in the Karisma trial is to investigate changes in tissue markers 
by comparing breast tissue biopsies sampled prior to and after six months of treatment with 
different doses of tamoxifen or placebo. 
Mammographic density is the white part of the mammogram and consist of glandular and 
connective tissue. The black, non-radiolucent part is fat. The more glandular and connective 
tissues, the whiter the breast, and thus; the higher the risk of breast cancer [1]. Factors that 
influence risk of breast cancer are known to influence density. Hormone replacement therapy 
increases density and risk of breast cancer, while tamoxifen has the opposite effect [2].  
 
4. Design  
Donation of breast biopsies is optional and not a criteria to participate in Karisma II. All 
Karisma II participants will be invited to donate a biopsy upon study entry. Based on the 
recruitment frequency of a previous study with breast biopsies conducted within the Karma 
project, we anticipate that approximately 10% of the Karisma II participants will donate 
biopsies. Breast biopsies will be collected at study baseline and at month 6 (end of treatment). 
 
5. Investigational plan 
5.1. Procedure at baseline 
5.1.1. Women have to meet inclusion and not fulfil exclusion criteria of the Karisma II 
study. Women are informed about the study and accompanying procedures by the 
investigator. If consenting women are invited for breast biopsy.  
5.1.2. Only participants with a negative mammogram, defined as BI-RADS score 1 or 
2, at baseline are excluded from the study. 
5.1.3. The responsible mammography physician performs an ultrasound-led breast 
biopsy, guided by the mammogram, in the densest part of the left breast under local 
anaesthesia. 
5.1.4. Four core needle biopsies will be extracted from one single incision site. Two 
cores will be formalin-fixed and paraffin embedded (FFPE) and two cores will be 
freshly frozen. All four biopsies will be biobanked. 
5.1.4.1. 
Biopsy #1 is the “clinical” FFPE biopsy  
5.1.4.2. 
Biopsy #2 is the “research” FFPE biopsy 


Clinical Study Protocol: Karisma II 
 
5.1.4.2.1. 
The biopsies for FFPE will be sent to Histocenter, Gothenburg, for 
preparation. HE-stained slides will be sent to the Department of 
Pathology, Skåne University Hospital, Lund or the South General 
Hospital, Stockholm, for clinical evaluation. In case of pathological 
histopathology, participants will be referred for routine clinical work up 
by the responsible Karisma physician. Diagnostic HE-stained slides will 
be stored at the relevant Department of Pathology, whereas remaining 
biopsies will be stored at the Karolinska Institutet Biobank. 
5.1.4.2.2. 
Biopsy #3 and #4 are the fresh frozen biopsies for research. Biopsies 
will be stored at the Karolinska Institutet Biobank. 
5.1.5. For Biopsy procedure details see SOP Biopsy Karisma II. 
 
5.2. Procedure at month 6 (end of treatments) 
Procedure according to 5.1.3 to 5.1.4.3. 
The procedure at 6 month will follow the schedule of Karisma II and the mammography 
will not be part of the ordinary screening program.  
 
6. Possible side effects  
Breast biopsy is a routine procedure frequently used in clinical practice. The physicians 
performing the biopsies are all experienced medical professionals. Furthermore, the Karisma II 
participants are healthy women. The risk of side effects is thus relatively low. The most 
commonly associated side effects are tenderness at the incision site, contusion and bleeding. 
Clinical routines apply to deal with these issues.  
 
 
7. References  
13. Boyd NF, Guo H, Martin LJ, Sun L, Stone J, Fishell E, Jong RA, Hislop G, Chiarelli A, Minkin S, 
Yaffe MJ. Mammographic density and the risk and detection of breast cancer. N Engl J Med. 2007 
Jan 18;356(3):227-36. 
14. Brand JS, Czene K, Eriksson L, Trinh T, Bhoo-Pathy N, Hall P, Celebioglu F.  Influence of lifestyle 
factors on mammographic density in postmenopausal women. PLoS One. 2013 Dec 
9;8(12):e81876. doi: 10.1371/journal.pone.0081876. eCollection 2013. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Clinical Study Protocol: Karisma II 
 
Appendix 15.5 
 
 
Participant Card (in Swedish, therefore removed from Clinical Trial version) 
 
 
 

SAP_001.pdf: 
KARISMA II
Statistical Analysis Plan
October 25, 2016
Approvals
Author
Martin Eklund, statistician
Approved by
Per Hall, PI
Kamila Czene, co-PI
Revision history
Version
Date
Reason for change
0.3
October 17, 2016
First draft
0.4
October 25, 2016
Split of primary analysis
1


Contents
1
Preface
3
2
Abbreviations
3
3
Background
3
4
Design
4
4.1
Study population . . . . . . . . . . . . . . . . . . . . . . . . .
5
4.2
Study period
. . . . . . . . . . . . . . . . . . . . . . . . . . .
6
5
Endpoints
6
5.1
Primary objective . . . . . . . . . . . . . . . . . . . . . . . . .
6
5.2
Primary endpoint . . . . . . . . . . . . . . . . . . . . . . . . .
6
5.3
Secondary objectives . . . . . . . . . . . . . . . . . . . . . . .
6
5.4
Secondary endpoint . . . . . . . . . . . . . . . . . . . . . . . .
6
5.5
Tertiary objectives . . . . . . . . . . . . . . . . . . . . . . . .
7
5.6
Tertiary endpoints . . . . . . . . . . . . . . . . . . . . . . . .
7
6
Statistical methods
7
6.1
Populations . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7
6.2
Demographics and baseline data
. . . . . . . . . . . . . . . .
8
6.3
Primary analysis: Proportion of responders (eﬃcacy) . . . . .
8
6.4
Secondary analysis: Side eﬀects (safety) . . . . . . . . . . . .
9
6.5
Tertiary analyses . . . . . . . . . . . . . . . . . . . . . . . . .
9
6.5.1
Sensitivity analyses of the primary endpoint . . . . . .
9
6.5.2
Sensitivity analyses of the primary and secondary end-
points simultaneously
. . . . . . . . . . . . . . . . . .
10
6.5.3
Tertiary endpoints . . . . . . . . . . . . . . . . . . . .
10
6.6
Sample size . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11
6.7
Adjustment for multiplicity, confounders, and heterogeneity .
12
6.8
Interim analysis . . . . . . . . . . . . . . . . . . . . . . . . . .
13
6.9
Handling of missing data . . . . . . . . . . . . . . . . . . . . .
13
2


1
Preface
This Statistical Analysis Plan (SAP) describes the planned analyses for
Karisma II: A randomized, double blinded, six-armed placebo controlled study
to investigate optimal dose of tamoxifen with the most favourable side eﬀect
spectra and with mammographic density reduction non-inferior to that of 20
mg tamoxifen, EudraCT number 2016-000882-22.
The planned analyses identiﬁed in this SAP will be included in future
manuscripts. Exploratory analyses not necessarily identiﬁed in this SAP
may be performed to support planned analyses. Any post-hoc exploratory
or unplanned analyses not speciﬁed in this SAP will be identiﬁed as such in
manuscripts for publication.
This SAP was written by a statistician and investigators who were
blinded to treatment allocation and treatment-related study results and will
remain so until the central database is locked and the ﬁnal data are ex-
tracted for analysis. To ensure blinding, treatment allocations are stored in
a separate location accessible only by an un-blinded statistician.
2
Abbreviations
Abbreviation
Explanation
SAP
Statistical analysis plan
ITT
Intention to treat
PP
Per protocol
MP
Multiple imputation
3
Background
For more than 40 years, 20 mg of tamoxifen has been used in breast cancer
patients to reduce the risk of recurrence. It has also been shown that 20 mg
of tamoxifen reduces the risk of breast cancer (by 40-60%) in healthy women
but that the treatment comes with side eﬀects. The side eﬀects are probably
one reason to that tamoxifen is not used in the preventive setting despite
the substantial risk reducing eﬀect. The aim of the Karisma II study is to
identify the lowest tamoxifen dose that could be used for primary preventive
purposes, with a hypothesis that a lower dose will reduce the side eﬀects.
Mammographic density is the white part of the mammogram and consist
of glandular and connective tissue. The black, non-radiolucent part is fat.
3


The more glandular and more connective tissues, the whiter the breast,
and the higher the risk of breast cancer [1]. Factors that inﬂuence risk of
breast cancer are known to inﬂuence density. Hormone replacement therapy
increases density and risk of breast cancer, while tamoxifen has the opposite
eﬀect.
The molecular basis for the tamoxifen-induced changes in breast
density is sparsely understood.
There are three studies that used mammographic density change during
one year as a proxy for tamoxifen eﬀect [2–4]. Two projects studied the
association between mammographic density change in patients and progno-
sis and one study looked at density change among perfectly healthy woman
and risk of breast cancer. All three studies came to the same result, ap-
proximately half of the treated participants respond to the therapy by a
signiﬁcant reduction in mammographic density after one year. Those that
respond with a decreased mammographic density had a lower risk of breast
cancer recurrence and a lower risk of breast cancer.
The conclusion is that change in mammographic density is a very good
marker of Tamoxifen therapy response.
4
Design
This is a dose determination study aiming to identify the optimal tamoxifen
dose for reducing the risk of breast cancer. Instead of following a very large
number of women, for many years, treated with diﬀerent doses of tamoxifen
and see if they develop breast cancer, we will include 1440 healthy women
participating in the mammographic screening program at two Mammogra-
phy units (Unilabs mammography, Lund and S¨odersjukhuset Breast Centre,
Stockholm) and measure their change in mammographic density. We will
test if 1 mg, 2.5 mg, 5 mg and 10 mg reduce the mammographic density to
the same extent as 20 mg. In summary Karisma II will be a randomized,
double-blinded, six-armed placebo controlled study to identify the dose of
tamoxifen with the most favourable side eﬀect spectra and with a density
reduction non-inferior to 20 mg tamoxifen.
Secondary aims of the Karisma II trial are to investigate changes in
tissue markers by comparing breast tissue biopsies sampled prior to and
after six months of treatment with tamoxifen or placebo, as well as relate
levels of tamoxifen metabolites, proteins, lipids and hormones in blood and
changes in breast tissue to tamoxifen doses. Moreover, we will study genetic
polymorphism in germline DNA and their association to the other endpoints.
4


Figure 1: Overall study design.
4.1
Study population
The study will include healthy women that participate in the mammographic
screening program at S¨odersjukhuset, Stockholm and at Sk˚anes Univer-
sitetssjukhus, Lund, and have a negative screening mammogram. We will
not include breast cancer patients as they receive a variety of drugs that
might inﬂuence mammographic density. We are excluding women with no
measurable density (< XXX *** according to the Stratus breast density
measurement method *** this is 4.5% or more by Volpara according to the
protocol (p. 17), is this still the way it should be done?). This is because
measurable density at study start is a prerequisite for being able to see re-
duction of mammographic density over time. We are not selecting women
based on risk of breast cancer since we currently lack a reliable risk predic-
tion model for Swedish standards.
5


4.2
Study period
Karisma I was a proof of concept study preceding Karisma II. The main
reason we conducted the Karisma I study was to investigate time to density
change. Radiologists tell us that the time to density change is most probably
much shorter than one year, probably only a couple of months. However,
the issue of time to density change during tamoxifen therapy has never been
addressed.
The results from Karisma I show that, regardless of dose, density de-
creases within 6 months by about 50% for one quarter of the participants,
about 20% for another quarter and not at all for the other participants. This
result implicates that tamoxifen intake during 6 months gives a measurable
density change of responders; therefore, the treatment period in Karisma II
are set to 6 months. From a clinical point of view and for coming study
(Karisma III) we will investigate the state of density change and decline of
side eﬀects 6 months after tamoxifen cessation in relation to doses.
5
Endpoints
5.1
Primary objective
Identify the minimal dose of tamoxifen that is non-inferior in its ability to
reduce mammographic density compared with 20 mg tamoxifen.
5.2
Primary endpoint
Change in mammographic density. In particular, we will test for noninferi-
ority in the proportion of women in the intervention arms (placebo, 1 mg,
2.5 mg, 5 mg, 10 mg) who have a density reduction as great as or greater
(after 6 months) than the median density reduction in the 20 mg arm (see
Section 6.3).
5.3
Secondary objectives
Assess the drop-out level and the level of side eﬀects in the intervention arms
compared to the 20 mg arm.
5.4
Secondary endpoint
Drop-out rate. We will test for diﬀerences in the proportion of drop-outs
after 6 months in the intervention arms compared to the 20 mg arm.
6


5.5
Tertiary objectives
Relate levels of tamoxifen metabolites, proteins, lipids and hormones in
blood and changes in breast tissue [*** according to what measure] to ta-
moxifen doses. Measure genetic polymorphism in germline DNA and relate
it to the other secondary objectives. Measure mammographic density and
levels of side eﬀects 6 months after tamoxifen cessation in relation to tamox-
ifen dose. Study associations between dose of tamoxifen, polymorphisms in
CYP2D6 and blood levels of endoxifen. Quantify the level of side eﬀects
(according to the FACT-ES instrument) and compliance of medicine intake
(Morisky scale).
5.6
Tertiary endpoints
Levels of tamoxifen metabolites (e.g.
endoxifen), proteins [*** which?],
lipids [*** which?]
and hormones [*** which?]
in blood.
Breast tissue
changes. Genetic polymorphism in germline DNA, in particular CUP2D6.
Mammographic density and levels of side eﬀects 6 months after tamoxifen
cessation. Side eﬀects (according to the FACT-ES instrument) and compli-
ance of medicine intake (Morisky scale).
6
Statistical methods
All analyses will be performed after the study is completed and the database
is released. All statistics, including tables, ﬁgures and listings, will be per-
formed using SAS, Version 9.3, or R, Version 3.x.
6.1
Populations
The intention to treat (ITT) population consists of all women who were
randomized and had eligible measure at baseline and at least one eligible
measure post baseline. The Per Protocol (PP) Population consists of all
women in the ITT population who remained in the study for at least 6
months (the time period during which Tamoxifen is administered) in the
study and who had no signiﬁcant protocol violations (compliance of study
drug intake is deﬁned as at least 80% of the prescribed numbers of tablets
should be used during time of treatment). The analyses will be preformed
on both ITT and PP, however the primary analyses will be carried out on
an ITT basis. ITT analysis will likely bias the results toward equivalence for
the primary eﬃcacy endpoint compared to if all participants completed the
7


study (assuming that non-compliance is dose dependant). The per-protocol
analysis, on the other hand, excludes data from patients with major protocol
violations and may bias the results in either direction. We will therefore
analyse the primary endpoint using both intention-to-treat and per-protocol
approaches.
Since we expect around 30% dropout rate, we will also create a dataset
where missing data is imputed; the multiple imputation (MP) population
(see Section 6.9 for details).
6.2
Demographics and baseline data
All data will be presented using descriptive statistics. Results will be pre-
sented in total, by treatment group, age and menopausal status. Continuous
variables will be summarized using number of women, mean, standard de-
viation, median, minimum and maximum.
Categorical variables will be
summarized using the number and percentage of women.
6.3
Primary analysis: Proportion of responders (eﬃcacy)
The results of both papers [2] and [3] showed that a threshold value in
mammographic density decrease needs to be exceeded in order for a woman
to experience the beneﬁcial eﬀect of tamoxifen. This threshold value was
diﬀerent in [2] compared to [3]. A 10% or greater reduction was needed
in [2] whereas a 20% reduction was needed in [3], a diﬀerence most likely
explained by diﬀerent methods of measuring mammographic density. How-
ever, the proportion of women exceeding this threshold was exactly 48% in
both studies, implying that approximately 50% of the women experience the
beneﬁcial eﬀect of tamoxifen irrespective of the way mammographic density
is measured.
In the Karma II study, we will use the Stratus method for measuring
breast density, which was not used in either [2] or [3].
With support of
the results in [2] and [3], we deﬁne the response threshold to be the me-
dian decrease in mammographic density of women in the 20 mg tamoxifen
arm (this deﬁnition ensures that the same proportion of women exceeds the
threshold as observed in previous studies). Further, we deﬁne a responder
to be a woman whose mammographic density decrease between baseline and
6 months exceeds the response threshold.
The primary eﬃcacy endpoint is the proportion of responders (women
with a reduction of mammographic density, from baseline to 6 months). We
will test for non-inferiority with respect to the primary eﬃcacy endpoint for
8


groups of women treated with placebo, 1, 2.5, 5, and 10 mg compared to
the group of women treated with 20 mg tamoxifen. We will use Fisher’s
exact test with an p-value of less than 0.05 (corrected for multiple testing,
see Section 6.7) as a threshold to declare statistical signiﬁcance. The non-
inferiority margin is deﬁned to be 16.7 percentage points; that is, we deﬁne
non-inferiority to mean that the fraction of responders is not less than one
third of the treated individuals (50% minus 16.7% = 33.3%).
The null
hypothesis is thus that the proportion of responders in women treated with
placebo, 1, 2.5, 5, and 10 mg is 16.7 percentage points less than those women
treated with 20 mg tamoxifen (i.e. the fraction of responders is less than one
third). For rejection of the null hypothesis, the lower end of the conﬁdence
interval needs to by higher than -16.7%. For this to hold, the point estimate
of an intervention arm can not be more than 5 percentage points lower than
the percent responders in the 20 mg arm.
6.4
Secondary analysis: Side eﬀects (safety)
We will assess the proportion of drop-outs at six months and test for dif-
ferences in the proportion of drop-outs in the intervention arms compared
with the 20 mg arm. We will use Fisher’s exact test with an p-value of less
than 0.05 (corrected for multiple testing, see Section 6.7) as a threshold to
declare statistical signiﬁcance.
6.5
Tertiary analyses
We will perform two types of tertiary analyses: (i) sensitivity analysis on
the primary and secondary endpoints using diﬀerent methods than used in
the primary and secondary analyses in order to assess sensitivity to choice
of analyses pathway, assumptions, and missing data; and (ii) on tertiary
endpoints.
6.5.1
Sensitivity analyses of the primary endpoint
1. We will perform sensitivity analyses to the primary analyses where the
threshold for being deﬁned as a responder is varied ±10 percentage
points, to assess whether the overall conclusion of the trial is sensitive
to the within study deﬁnition of what a responder is. Similarly, we
will perform a tipping point analysis, where the the threshold for being
deﬁned as a responder is changed until the result of the trial changes
(either from a nonsigniﬁcant result to a signiﬁcant result or vice versa).
9


2. Using absolute change in density instead of relative change to deﬁne
a responder.
3. We will analyze data from all arms in a regression model (as opposed
to one arm against another arm).
I.e., responder status (a binary
variable) will be used as the response variable in a logistic regression
model, where dose allocation is the independent variable.
4. We will impute missing data (as described in Section 6.9) and re-
perform the primary analysis using imputed datasets (in a multiple
imputation protocol).
5. We will analyse the results using the recorded continuous endpoint
(density reduction) instead of discretising the reduction into either
responder or non-responder. the We will use both relative and absolute
density reduction is endpoints in this analysis.
6.5.2
Sensitivity analyses of the primary and secondary end-
points simultaneously
We will perform an analysis where the distance to the diagonal line in a plot
of proportion responders vs proportion drop-outs by study arm is quantiﬁed
(see Figure 2 for a schematic mock ﬁgure). Putatively, the arm with the
greatest distance to the diagonal line gives the best tradeoﬀbetween eﬃcacy
and drop-out level. Conﬁdence intervals of the distance will be computed
using the bootstrap.
6.5.3
Tertiary endpoints
Tertiary outcomes will be analysed with statistical methods appropriate
for the speciﬁc outcome. A large number of tertiary endpoints are being
recorded within the trial, e.g.
levels of tamoxifen metabolites, proteins,
lipids and hormones in blood, breast tissue changes, genetic polymorphism
in germline DNA, mammographic density and levels of side eﬀects 6 months
after tamoxifen cessation. Since this the ﬁrst study of its type, the asso-
ciations of nearly every possible combination of primary, secondary, and
tertiary endpoints are of interest. We therefore expect a number of tertiary
analyses to be performed. In the list below, we brieﬂy specify some obvious
examples of such analyses:
1. The association of dose of tamoxifen to blood concentration of ta-
moxifen metabolites (e.g. endoxifen) by CYP2D6 polymorphism sta-
tus (a woman can be classiﬁed as an excellent, intermediate, or poor
10


Proportion responders 
Proportion drop-outs 
Placebo 
1 mg 
2.5 mg 
5 mg 
10 mg 
20 mg 
Δ1mg 
Δ2.5mg 
Δ5mg 
Δ10mg 
0 
0 
0.5 
0.35 
Figure 2: Schematic picture of what a plot of proportion responders vs
proportion drop-outs by study arm may look like. The arm with the greatest
distance to the diagonal line gives the best tradeoﬀbetween eﬃcacy and
drop-out level.
metaboliser of tamoxifen based on polymorphism patterns in CYP2D6
[5]) will be analysed in a linear regression model with tamoxifen dose,
CYP2D6 polymorphism status, and age as independent variables.
2. The association of plasma concentrations of endoxifen with responder
status will be analysed with logistic regression both within treatment
groups and among treatments groups (treatment group will be handled
as a random eﬀect in the latter case).
6.6
Sample size
Since we have ﬁve intervention arms and since we do the primary analy-
sis on a ”per arm basis”, we have ﬁve chances to show noninferiority of
the primary endpoint and we therefore need to correct for multiple testing.
Standard methods for correcting for multiple testing (according to the meth-
ods by Bonferroni or Holm) are too conservative in this context, since they
11


rely on an assumption of independence between the tests whereas the tests
here are clearly correlated. We will instead use the resampling based step-
down method suggested by Romano, Shaikh, and Wolf [6], which seamlessly
handles correlated tests (see also Section 6.7). The correlation structure of
the tests is unknown and we therefore assume that the multiplicativity leads
to that the α decreases from 0.05 to 0.03 in order to declare a signiﬁcant
results (correction by Bonferroni would lead to an α of 0.01).
With 168 participants in each study arm, the lower limit of the observed
one-sided 98.5% conﬁdence limit for the diﬀerence will be expected to ex-
ceed the non-inferiority margin 16.7 percentage points with a 80% power,
assuming that the expected diﬀerence in responders between two arms is 0
and the proportion responders in the 20 mg tamoxifen group is 50%. With
an estimated 30% drop-out rate, 240 patients in each group need to be
randomized.
The trial is powered for the primary analysis. However, we here also
brieﬂy investigate the power for showing both noninferiority with respect to
the primary endpoint and superiority with respect to the secondary end-
point. With 240 participants randomized to each study arm, we have 79%
power to show both noninferiority with respect to the primary endpoint and
superiority with respect to the secondary endpoint assuming that the pro-
portion of drop outs is reduced by two thirds (from 30% to 10%) and 67%
power if the proportion of drop outs is reduced by 50% (from 30% to 15%).
(For these calculations, we use the same assumptions about the correlation
of the tests as for the analysis of primary endpoint.)
The computations are based on the following formula:
n = f(α, β)πs(100 −πs) + πe(100 −πe)
(πs −πe −d)2
,
where πs and πe are the true percent ’success’ in the standard (20mg) and
experimental treatment group respectively, and
f(α, β) =

Φ−1(α) + Φ−1(β)
2 ,
and Φ−1 is the cumulative distribution function of a standardised normal
distribution.
6.7
Adjustment for multiplicity, confounders, and hetero-
geneity
Since we have ﬁve treatment arms and therefore ﬁve comparisons, we will
need to adjust for multiple testing.
The treatment arms are correlated,
12


since they are deﬁned by diﬀerent doses of the same drug. Thus, standard
adjustment for multiple testing using the methods by Bonferroni or Holm are
too conservative. Instead, we will use the bootstrap based method suggested
by Romano, Shaikh, and Wolf [6], which seamlessly handles correlated tests.
The correlation structure of the tests is unknown and we therefore assume
that the multiplicativity leads to that the α decreases from 0.05 to 0.03 in
order to declare a signiﬁcant results (correction by Bonferroni would lead to
an α of 0.01).
We will perform subgroup analyses, where the subgroups are CYP2D6
polymorphism status (excellent, intermediate, poor metaboliser [5]). CYP2D6
polymorphism status will be handled as a categorical ﬁxed eﬀect variable.
Other subgroups will likely be studied in non-prespeciﬁed analyses.
6.8
Interim analysis
We will three months into the study perform an interim analysis on overall
dropout level. If the proportion of drop out at three months, we will – at best
– have 73% power for our primary analysis (under the same assumptions as
described in Section 6.6, apart from the assumption about the proportion
of drop outs). In this case, we will increase the number of women the study
needs to accrue according to:
ntot = 168 × 6/(1 −d′),
where d′ is the overall proportion of drop outs at three months.
6.9
Handling of missing data
The primary analysis will be performed on an ITT basis using the last-
observation-carried-forward method.
This analysis makes sense from an
eﬀectiveness (de facto) point of view, however it will likely bias the results
towards equivalence for the primary eﬃcacy endpoint (assuming that non-
compliance is dose dependant) from an eﬃcacy (de jure) point of view (i.e.
compared to if no women dropped out from the study). We will therefore
perform sensitivity analyses (Section 6.5) where we impute mammographic
density 6 months into the study for women who dropped out. Speciﬁcally,
we will use a multiple imputation protocol where mammographic density at
6 months is imputed using a regression model with density at the time points
recorded within the study, age, HRT status, CYP2D6 polymorphisms, and
endoxifen levels (at the available time points). The exact choice of multiple
imputation model will be done using the study data after database lock using
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cross-validation. We will also perform tipping point analyses with respect
to the missing data by exploring how extreme the imputation of the missing
data will need to be to change the overall conclusion of the trial (see also
Section 6.5.1).
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doi: 10.1007/s11749-008-0126-6. URL http://dx.doi.org/10.1007/
s11749-008-0126-6.
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