Presurgical Treatment With Ribociclib and Letrozole in Patients With Locally Advanced Breast Cancer: the NEOLETRIB Study.
Phase: PHASE2
Conditions: Breast Cancer, HER2-negative Breast Cancer, ER-positive Breast Cancer, Locally Advanced Breast Cancer, Luminal A Breast Cancer, Luminal B Breast Cancer
Sponsor: University Hospital, Akershus

 
1
Presurgical Treatment with Ribociclib and 
Letrozole in Patients with Locally Advanced 
Breast Cancer: the NEOLETRIB study. 
 
 
Research protocol:  October 25th.  2020                                              
Version: 1.0 
 
 
Principal and corresponding investigator: 
 
 
Jürgen Geisler, MD, PhD, Senior Consultant Oncologist, Head, Translational 
Cancer  Research Group at Akershus University Hospital & Professor, Medical 
Oncology & Radiotherapy, Institute of Clinical Medicine, University of Oslo 
(Campus AHUS);  
E-mail: <juergen.geisler@medisin.uio.no>; Tel. (AHUS): (+47) 02900; 
Cell phone: (+47) 91 18 74 47; Fax: (+47) 67963820 
 
 
Complete list of involved investigators / co-workers  
(in alphabetical order; confirmed collaborators, if not marked otherwise): 
 
 
- 
Bahrami, Nazli; M.D., Breast- and Endocrine Surgery, Akershus 
University Hospital, Norway; 
 
- 
Bemanian, Vahid, Ph.D., Senior Scientist, Clinical Molecular Biology, 
Akershus University Hospital; 
 
- 
Buvarp, Unn-Cathrin; Research nurse, Department of Oncology, 
Akershus University Hospital; 
 
- 
Carroll, Jason; M.D., Ph.D., Professor and group leader, Cambridge 
Biomedical Campus, University of Cambridge, UK; 
 
- 
Chen, Shiuan; Ph.D., Professor, Head, Department of Cancer Biology, 
Beckman Research Institute of the City of Hope, National Cancer Center, 
Duarte, CA, USA; 
 
- 
Fallang, Lars-Egil, PhD., Medical Advisor, Novartis Oncology, Novartis 
Norge AS, Oslo, Norway; 
 
- 
Geisler, Stephanie; M.D., Senior consultant, medical oncology and section 
leader, Department of Oncology, Akershus University Hospital;  
 
- 
Geitung, Jonn Terje, M.D., Ph.D., Professor, Department of Radiology, 
Akershus University Hospital; 

 
2
 
- 
Goel, Shom, M.D, Ph.D., Professor and group leader, Peter MacCallum 
Cancer Centre, Melbourne, Australia; 
 
- 
Gravdehaug, Berit; M.D., Senior consultant in Breast- and Endocrine 
Surgery, Department of Surgery, Akershus University Hospital; 
 
- 
Holm, Barbro, Ph.D., Nordic Medical Disease Area Lead, Novartis 
Oncology, Novartis Sverige AB, Kista, Sweden; 
 
- 
Hurtado, Antoni, Ph.D., Prof., Department of Biomedical Sciences, 
University of Barcelona, Spain; 
 
- 
Jabeen, Shakila, Ph.D., Molecular Biology, (EPIGEN); Akershus University 
Hospital; 
 
- 
Jahnsen, Jørgen; M.D., Ph.D., Professor and Senior Consultant, 
Department of Gastroenterology, Akershus University Hospital, Norway; 
 
- 
Kiil, Erik, M.D., Consultant in Oncology, Department of Oncology, 
Akershus University Hospital; 
 
- 
Kristensen, Vessela; Ph.D., Oslo University Hospital, Ullevål Hospital, 
Molecular Cancer Biology; Professor, Institute of Medicine, University of 
Oslo; 
 
- 
Lehtiö, Janne; Ph.D., Prof., Science for Life Laboratory, Department of 
Oncology-Pathology, Karolinska Institute, Solna, Sweden; 
 
- 
Lindem, Ida Caroline, M.D., Department of Oncology, Akershus 
University Hospital; 
 
- 
Loeng, Marie; Research nurse, Department of Oncology, Akershus 
University Hospital; 
 
- 
Lyngra, Marianne; M.D., Senior Consultant, Department of Pathology, 
Akershus University Hospital; 
 
- 
Manouchehr, Seyedzadeh, M.D., Senior Consultant, Radiologist and 
Leader, Breast Diagnostic Center, Akershus University Hospital; 
 
- 
Moen, Aina Elisabeth Fossum; PhD, Senior researcher, Clinical 
Molecular Biology (EpiGen), Division of Medicine, Akershus University 
Hospital 
 
- 
Porojnicu, Alina Carmen; MD, Senior Consultant in Medical Oncology, 
Vestre Viken Hospital Trust, Drammen, Norway; 
 

 
3
- 
Reis, Joana; M.D., Department of Radiology, Akershus University 
Hospital; 
 
- 
Reitsma, Laurens Cornelus, M.D., Head, Department of Breast- and 
Endocrine Surgery, Akershus University Hospital; 
 
- 
Sahlberg, Kristine Kleivi, Ph.D.; Research Director, Vestre Viken 
Hospital Trust, Drammen Hospital, Norway; 
 
- 
Sauer, Torill; PhD, Senior Consultant, Department of Pathology, Akershus 
University Hospital; Prof. emerit., University of Oslo, Norway; 
 
- 
Seyedzadeh, Manouchehr; M.D.; Head, Breast Diagnostic Center, 
Akershus University Hospital.; 
 
- 
Selsås, Knut; M.D., Department of Breast. & Endocrine Surgery, Akershus 
University Hospital, Norway; 
 
- 
Skjerven, Helle Kristine; M.D., Senior Consultant, Breast and Endocrine 
Surgery, Vestre Viken Hospital Trust, Drammen, Norway; 
 
- 
Tahiri, Andliena; PhD., Postdoc, Molecular Biology (EPIGEN); Akershus 
University Hospital, Lørenskog, Norway; 
 
- 
Tekpli, Xavier; PhD; Oslo University Hospital, Ullevål University Hospital, 
Department of Cancer Research; Oslo, Norway; 
 
- 
Touma, Joel; MD, Breast and Endocrine Surgery, Akershus University 
Hospital, Norway; 
 
- 
Ødegård, Hilde Presterud; M.D., Senior Consultant in Oncology, 
Department of Oncology, Akershus University Hospital; 
 
 
 
 
 
 
 
 
 

 
4
SHORT SUMMARY 
 
 
Aim of the study:  
 
To study the direct and indirect anti-tumor effects of targeted cancer therapy 
with letrozole and ribociclib given in combination with special focus on the 
immune system in patients suffering from locally advanced, luminal-A/B breast 
cancer; 
 
Primary objective:  
1. To evaluate the intra-tumor immunological effects of individual patients 
treated with letrozole and ribociclib in the neoadjuvant setting  
a. Endpoints  
i. Evolution of the intra-tumor immune profile during 
neoadjuvant therapy of breast cancer [Time Frame: D1, D21, 
and at surgery] determined through IHC or immunoblotting, 
scRNAseq, and other relevant methods described in the 
following chapters. 
 
Secondary objectives: 
 
 
1. To study early and late mechanisms of adaptation and resistance to 
aromatase inhibitor (letrozole) in combination with ribociclib;  
b. Endpoints  
i. Evolution of the tumor cell profile during neoadjuvant therapy 
of breast cancer [ Time Frame: D1, D21, and at surgery ] 
determined through whole tumor sequencing, scRNAseq etc. 
 
2. To evaluate clinical and pathological response of targeted neoadjuvant 
therapy with letrozole and ribociclib  
a. Endpoints 
i. Determine the on treatment effects assessed by PEPI-0 and 
CCCA (Ki67<2,7%) at D 21 & at surgery. Change in Ki67 BL-

 
5
D 21 to surgery, as well as ROR-score, based on genomic 
analysis. [Time Frame: D1, D21, and at surgery] 
ii. Long term effects and outcome will be assessed at yearly 
intervals in line with clinical routine. 
 
3. To study the effect of ribociclib and letrozole treatment on the gut 
microbiome.  
a. Endpoints 
i. The longitudinal effect of neoadjuvant treatment will be 
determined through next generation amplicon sequencing 
on the V4 region of the fecal bacterial 16S rRNA gene by 
comparing baseline and follow up samples 
[Time Frame: D1, D21, D90 and at surgery (D180)] 
 
4. To search for surrogate parameters (tumor- or immune related markers 
etc.) allowing to estimate the anti-tumor effects in liquid biopsies before 
and during therapy;  
a. Endpoint: 
i. Assess serum cytokine levels, serum metabolite levels and 
circulating free tumor DNA (cftDNA) [Time Frame: D1, D21, 
D90 and at surgery (D180)] 
  
Exploratory objectives: 
 
1. To study the relationship between cancer treatment effect, the immune 
system and gut microbiota; 
 
2. To develop optimal methods to evaluate the tumor response by novel CT 
/ MRI techniques (tumor measurements); 
 
Study Design:  
Multicentre, single-arm, open-label, neoadjuvant study; 
 
Clinical Setting:  
Collaboration of two Norwegian Cancer Centers: 

 
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1. Department of Oncology, Akershus University Hospital, 
Norway); 
2. Department of Oncology, Drammen Hospital (Vestre 
Viken Health region, Norway); 
 
(additional breast cancer centers in Norway may be 
involved if patient recruitment is not reaching the 
expected pace); 
 
Patients:  
Patients diagnosed with (biopsy-confirmed) locally 
advanced (defined as either large T2, or T3/T4, or N2-3), 
ER-positive (>50% pos. cancer cells), HER-2 negative, 
luminal-A/B breast cancer suitable for neoadjuvant anti-
hormone therapy will be considered for this protocol; 
At least 100 patients should complete all study procedures. 
Thereafter, the protocol will be closed for further inclusion, 
but each patient included up to that time will be completed 
according to the protocol. The patients will be followed by 
the involved medical oncologists for up to 10 years after 
surgery or until relapse (whatever comes first). 
  
 
 

 
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Statistical considerations: 
Based on the results of the CORALLEEN trial 1 investigating 
neoadjuvant ribociclib treatment of luminal B patients, the 
spread between level of treatment response (24 weeks of 
treatment) based on MRI was 14% Complete Response 
(CR), 43% Partial Response (PR), 33% Stable Disease (SD) 
and 10% N/A. We propose a minimum sample size in either 
group to be at least >30 patients, thus an inclusion goal of 
100 patients in total would be sufficient for hypothesis 
generating results. However, it is important to mention that 
the purpose of this trial is to deliver basic research results 
and not primarily to evaluate the efficacy or comparisons of 
treatments. Based on the major endpoints and their 
anticipated data variation, the data will be sufficient to 
come to reliable results. Finally, statistical power is added 
as all patients will serve as their own controls based on 
sequential intra-patient data obtained over time (in general 
180 days all in all).  
This is aligned with advice given by gastroenterologists and 
on par with earlier studies that allowed for characterization 
of the tumor immune microenvironment and the gut 
microbiota 2,3 and gives sufficient statistical power to 
prioritize biomarkers for larger companion diagnostic 
studies in the future. 
 
Treatment: 
Suitable patients will receive neoadjuvant therapy with 
letrozole (Femar
®) in combination with ribociclib (Kisqali
®) 
for at least 6 months followed by surgery; premenopausal 
women will also be considered for this protocol (in 
combination with the LHRH-analogue goserelin in addition 
to letrozole and ribociclib). Indication for additional 
adjuvant therapy (chemotherapy and/or endocrine 
therapy) will be considered individually after surgery 

 
8
(without restrictions and according to national guidelines 
provided by the Norwegian Breast Cancer Group; 
www.nbcg.net). 
 
Diagnostics: 
All patients will perform a general staging prior to inclusion 
with CT-scans, MRI of the breasts and routine blood 
samples; some of the procedures will be repeated every 3 
months (i.e. blood samples, spectral-CT and MRI of the 
breast). A clinical tumor evaluation will also be completed 
every 4 weeks (prior to starting a new ribociclib-cycle). 
 
Samples: 
Blood samples/liquid biopsies, tissue samples and feces 
samples will be collected before (baseline) and during 
treatment (day 21, day 90 and day 180) according to the 
protocol (specified in details in: Appendix B: “laboratory 
procedures”).   
 
 
 
 
 
 

 
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BACKGROUND AND AIMS OF THE STUDY 
 
Neoadjuvant endocrine therapy (NET) offers a good treatment option to down-
size large (inoperable) primary breast tumors and / or advanced axillary lymph 
node metastasis prior to definite surgery. For selected patients (postmenopausal 
women with ER-positive breast cancer), primary endocrine therapy has been 
shown to be as effective as standard neoadjuvant chemotherapy 4. It is the 
general opinion that aromatase inhibitors of the "third-generation" (letrozole, 
anastrozole, exemestane) are the preferable drugs for neoadjuvant endocrine 
therapy in ER-positive, postmenopausal breast cancer patients 5. Anastrozole, 
letrozole and exemestane have shown to suppress total body aromatisation as 
well as plasma and tissue estrogen levels by > 90% in vivo 6-10. All three drugs 
are currently used as standard care in the neoadjuvant, adjuvant and metastatic 
setting worldwide, including Norway.  
In 2015, a new class of anti-cancer drugs was introduced, known as 
cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors 11,12. Following pivotal 
trials showing dramatic effects when combined with aromatase inhibitors 13-15 or 
steroidal antiestrogens like fulvestrant 16-20, these compounds are now well 
established in distinct combinations during therapy of metastatic breast cancer.  
All in all, the addition of a CDK4/6 inhibitor to standard anti-hormone 
therapy of breast cancer, doubled the time to disease progression and caused 
significant improvements in overall survival 1,21-23. Based on these findings, 
CDK4/6 inhibitors have also been tested in the neoadjuvant setting in clinical 
trials with very promising results 24-29. However, the combination of an 
aromatase inhibitor and a CDK4/6-inhibitor is currently not (yet) approved as 
standard neoadjuvant treatment in the algorithms for patients with locally 

 
10
advanced breast cancer (LABC) in Norway. Thus, the study described here, will 
make this highly promising drug combination available for all patients who may 
participate in this study. At the same time, the protocol will allow us to optimize 
the selection of patients benefitting from this treatment, highlight relevant 
biomarkers for personalized medicine and treatment, evaluate predictive 
markers and study the basic biology underlying treatment effects and resistance. 
In the recent years we have seen a growing body of evidence concerning 
the involvement of the immune system in the onset and prognosis of breast 
cancer both locally (in the tumor or connected tissues 30), but also through the 
involvement of the immune system as a whole 
31. Interestingly, the 
gastrointestinal (gut) microbiota seems to play an important role in determining 
whether the immune system is able to fight against several cancer types  2,32-37. 
Pre-clinical studies have shown that the microbes in the gut may influence the 
repertoire and activity of immune cells, such as T cells, potentially tailoring the 
immune system to allow cancer cell recognition and killing. This hypothesis is 
supported by the finding that treatment responses in a variety of cancer types to 
immune checkpoint inhibitors (ICIs) is depended on the distinct gut microbiome 
of an individual patient, as the efficacy of the ICIs rely on the presence of cancer-
recognizing T cells 3,38,39.  
In addition, recent findings indicate, that the emerging group of small 
molecules / targeting cancer therapies have immune altering properties. The 
MAPK kinase (MEK) inhibitors exemplify this by contributing to anti-tumor 
immunity through increasing the levels of cytotoxic T lymphocytes in preclinical 
model systems 40. Similar findings have recently been reported for both CDK4/6 
inhibitors and PI3K-inhibitors 41-43. Thus, CDK4/6 inhibitors are believed to 

 
11
increase the antigen presentation of cancer cells, to increase the activity of tumor 
infiltration by CD45+ cells and effector T cell activation, as well as to decrease 
the Treg cell proliferation 42,44,45. While these mechanisms have been well 
described in in vitro and animal models, they have to be studied in humans as 
soon as possible to understand them and to allow to take advantage of these 
novel aspects in clinical decision making.  In addition, an immunomodulatory 
effect of e.g. CDK4/6 inhibitors warrants further investigation and confirmation 
to allow for optimal drug combinations and enable concomitant anti-tumorigenic 
effects of drugs. These findings may also pave the way for microbiome and 
immune-related data as potential biomarkers for patient selection and response 
evaluations during therapy in the long run. 
In addition to the clinical effects as standard care for breast cancer 
patients suffering from locally advanced breast cancer, neoadjuvant therapy is 
widely used to study the endocrinology of breast cancer in general 46 and is 
recognized as one of the best model systems to predict responses in other 
clinical settings (early breast cancer or metastatic breast cancer) as well 47.  
 
Tumor biopsies obtained before initiation of treatment and following six 
months on therapy will allow correlations to the individual type of clinical 
response (partial responses vs complete responses etc.). We will focus on intra-
tumor mechanisms of adaption through tumor characterization using single cell 
technology which will allow us to follow both (i) the different cancer clones but 
also (ii) the evolution of different immune cells during therapy.  We will use 
novel approaches to evaluate liquid biopsies (measurement of cytokines levels, 
metabolites and cell free DNA-fragments etc.) during letrozole and ribociclib 
using state of the art laboratory methods at our hospital and in the laboratories 

 
12
of our listed collaborators. Finally, basic information on intra-tumor gene 
expression and regulation in individual breast cancer patients treated with both 
drugs in concert will become available in the planned spin-off-studies involving 
some of the leading cancer research teams in the world. 
Thus, all in all, the planned study will be able to contribute to our basic 
understanding of this very potent new drug combination that may be used in 
large groups of breast cancer patients in the future, including locally advanced 
breast cancer, hopefully reducing the use of traditional chemotherapy in this 
setting.  
 
 
PATIENTS AND TREATMENT 
 
 
Patients: 
Patients diagnosed with locally advanced, ER-positive (defined by ER-pos. in > 
50% of cancer cells), HER-2 negative, luminal-A/B breast cancer suitable for 
neoadjuvant anti-hormonal therapy ("pre-surgical downstaging") will be 
considered for this protocol. Locally advanced breast cancer according to this 
protocol is defined as either large T2 (> 3 cm in diameter) or T3-T4, and/or N2-3 
primary breast cancer. Patients with primary tumors above 3 cm in diameter 
(„large T2-tumors“) may be recruited in accordance with the international trend 
to provide neoadjuvant therapies to these patients in clinical trials. 
At least 100 patients should complete the study, with the aim of having an 
equal distribution between Luminal A and Luminal B. Thereafter, the protocol 
will be closed for further inclusion, but each patient included up to that time 
point will be completed according to the protocol procedures.  

 
13
All patients have to be postmenopausal (natural status or induced by 
treatment with the LHRH-analogue goserelin 3.6 mg implant s.c. given every 4 
weeks) to be able to benefit from aromatase inhibition (definition of natural 
postmenopausal status: age above 55 years or age above 50 years and at least 2 
years of amenorrhea in addition to LH-, FSH-, and plasma estradiol levels in the 
postmenopausal range). Patients with HER-2 positive disease, suitable for 
neoadjuvant therapy with trastuzumab, pertuzumab and taxanes, will be 
excluded from this protocol. The decision to include patients in this protocol will 
only be made following a comprehensive evaluation of each individual patient by 
a multidiscipline team of experienced breast cancer oncologists, pathologists, 
surgeons and radiologists. 
Triple-negative breast cancer is an exclusion criteria. Patients with 
distant metastasis of any type are also excluded from this trial.  
Patients who are in need of therapy with other strong CYP3A4 inhibitors 
should (if unavoidable additional therapy) reduce ribociclib to 400 mg daily (D1-
21). Patients who are using strong CYP3A4 inducers should only be considered if 
they may use alternative additional therapies with less potential to induce 
CYP3A4 48. Inclusion and exclusion criteria are summarized in table 1. 
 
 
 

 
14
Table 1:  Summary: Inclusion/Exclusion criteria for the NEOLETRIB-trial 
 
Key Inclusion Criteria: 
 
! The decision to include patients in this protocol will only be made 
following a comprehensive evaluation of each individual patient by 
experienced breast cancer oncologists 
! ≥ 18 years-old at the time of ICF signature 
! Histologically confirmed locally advanced breast carcinoma, defined as 
either large T2 (>3cm in diameter) or T3-T4, and/or N2-3 primary breast 
cancer. 
! ER-positive (defined by ER-pos. in > 50% of cancer cells) and HER-2 
negative, luminal A/B breast cancer  
! Postmenopausal status (natural status or induced by treatment with the 
LHRH-analogue goserelin 3.6 mg implant s.c. given every 4 weeks) 
– definition of natural postmenopausal status: age above 55 years or 
age above 50 years and at least 2 years of amenorrhea in addition 
to LH-, FSH-, and plasma estradiol levels in the postmenopausal 
range.  
! Patient has adequate bone marrow and organ function as defined by the 
following laboratory values (as assessed by central laboratory for 
eligibility): 
– Absolute neutrophil count ≥ 1.0 × 109/L 
– Platelets ≥ 100 × 109/L 
– Hemoglobin ≥ 9.0 g/dL 
– INR ≤1.5 (unless the patient is receiving anticoagulants and the 
INR is within the therapeutic range of intended use for that 
anticoagulant within 7 days prior to the first dose of study drug) 
– Estimated glomerular filtration rate (eGFR) ≥ 30 mL/min/1.73m2 
according to the Modification of Diet in Renal Disease (MDRD) 
formula 
– Total bilirubin < ULN except for patients with Gilbert’s syndrome 
who may only be included if the total bilirubin is ≤ 3.0 × ULN or 
direct bilirubin ≤ 1.5 × ULN. 
– Aspartate transaminase (AST) < 2.5 × ULN,  
– Alanine transaminase (ALT) < 2.5 × ULN,  

 
15
– Patient must have the following laboratory values within normal 
limits or corrected to within normal limits with supplements 
before the first dose of study medication: 
! Potassium 
! Magnesium 
! Total Calcium (corrected for serum albumin) 
! Standard 12-lead ECG values defined as the mean of the triplicate ECGs  
– QTcF interval at screening < 450 msec (QT interval using 
Fridericia’s correction) 
– Mean resting heart rate 50-90 bpm (determined from the ECG) 
! Performance Status: Eastern Cooperative Oncology Group (ECOG) score 
0-1 
! Ability and willingness to comply with study visits, treatment, testing and 
to comply with the protocol. 
 
Key Exclusion criteria: 
 
! Any prior treatment for primary invasive breast cancer. 
! Patient with a known hypersensitivity to any of the excipients of ribociclib or 
letrozole 
! Patient with known hypersensitivity to peanuts or soya-products; 
! Any evidence of distant metastasis;  
! Triple-negative breast cancer 
! HER-2 positive disease, suitable for neoadjuvant therapy with trastuzumab, 
pertuzumab and taxanes etc. 
! Other conditions rendering patients in need of other treatment options with 
immediate effect like chemotherapy 
! Concomitant medications that are known strong inducers of CYP3A4/5.  
! Clinically significant, uncontrolled heart disease and/or cardiac 
repolarization abnormality, including any of the following: 
– History of documented myocardial infarction (MI), angina pectoris, 
symptomatic pericarditis, or coronary artery bypass graft (CABG) 
within 6 months prior to study entry 
– Documented cardiomyopathy 

 
16
– Left Ventricular Ejection Fraction (LVEF) < 50% as determined by 
Multiple Gated acquisition (MUGA) scan or echocardiogram (ECHO) 
– Long QT syndrome or family history of idiopathic sudden death or 
congenital long QT syndrome, or any of the following: 
! Risk factors for Torsades de Pointe (TdP) including 
uncorrected hypocalcemia, hypokalemia or hypomagnesemia, 
history of cardiac failure, or history of clinically 
significant/symptomatic bradycardia 
! Concomitant medication(s) with a known risk to prolong the 
QT interval and/or known to cause Torsades de Pointe that 
cannot be discontinued or replaced by safe alternative 
medication (e.g., within 5 half-lives or 7 days prior to starting 
study drug) 
! Inability to determine the QTcF interval 
– Clinically significant cardiac arrhythmias (e.g., ventricular 
tachycardia), complete left bundle branch block, high-grade AV block 
(e.g., bifascicular block, Mobitz type II and third degree AV block)  
– Systolic Blood Pressure (SBP) >160 or <90 mmHg 
! Patient is currently receiving or has received systemic corticosteroids ≤ 2 
weeks prior to starting study drug, or who have not fully recovered from side 
effects of such treatment. Note: The following uses of corticosteroids are 
permitted: a short duration (<5 days) of systemic corticosteroids; any 
duration of topical applications (e.g. for rash), inhaled sprays (e.g., for 
obstructive airways diseases), eye drops or local injections (e.g., intra-
articular). 
! Pregnant or breast-feeding (lactating) women or women who plan to become 
pregnant or breast-feed during the trial 
! Women of child-bearing potential defined as all women physiologically 
capable of becoming pregnant, unless they are using highly effective methods 
of contraception during the study treatment and for 21 days after stopping 
the treatment. Highly effective contraception methods include: 
– Total abstinence (when this is in line with the preferred and usual 
lifestyle of the patient. Periodic abstinence (e.g., calendar, ovulation, 
symptothermal, post-ovulation methods) and withdrawal are not 
acceptable methods of contraception 
– Female sterilization (have had surgical bilateral oophorectomy with 
or without hysterectomy), total hysterectomy or tubal ligation at least 

 
17
6 weeks before taking study treatment. In case of oophorectomy 
alone, only when the reproductive status of the woman has been 
confirmed by follow up hormone level assessment 
– Male partner sterilization (at least 6 months prior to screening). For 
female patients on the study, the vasectomized male partner should be 
the sole partner for that patient and the success of the vasectomy 
must be medically confirmed as per local practice. 
– Placement of an intrauterine device (IUD). 
– Note: Use of oral (estrogen and progesterone), transdermal, injected, 
implanted, hormone containing intrauterine systems (IUS) or any 
other hormonal methods of contraception is not allowed in this study 
! Autoimmune disorders or significant allergies (i.e. rheumatoid arthritis, 
asthma, psoriasis etc).  
! Known HIV infection, Hepatitis B or C infection (testing not mandatory) 
! History of autoimmune celiac, inflammatory bowel disease, or other chronic 
GI disease 
! Recent use (within past month) of more than 3 days of antibiotics use 
! Current use of probiotic supplements 
! Taking proton pump inhibitors, steroids, other non-steroidal anti-
inflammatory drugs such as ibuprofen or acetyl salicylic acid; 
! Past bariatric surgery 
 
 
Treatment: 
Participating patients will receive neoadjuvant therapy with letrozole and  
ribociclib for at least 6 months. Patients will have a clinical tumor evaluation 
every 4 weeks and MRI-evaluations etc. every 3 months. The design of this trial 
is given briefly in figure 1. Letrozole will be given once daily by the oral route in 
the established dose of 2.5 mg daily. Ribociclib will be given as 600 mg once daily 
p.o. (standard starting dose) for a period of 21 days followed by 7 days without 
ribociclib. The general recommendations for drug dose reductions, blood 

 
18
sampling, ECG assessments etc. provided by the manufacturer (Novartis 
Pharmaceuticals) will be followed through the entire trial. ECG assessments will 
be performed at baseline (screening), day 1 and 15 of cycle 1 and 2, on day 1 of 
all following cycles and at the end of treatment. 
Premenopausal women will always start with goserelin from day 1 in 
addition to the aromatase inhibitor letrozole and ribociclib.  
Concerning the timing of surgery, it is planned to do surgery during one of 
the last days of the ongoing treatment cycle with ribociclib (surgery while on 
treatment). A check of the hematological profile will, however, be done directly 
prior to surgery and surgery will be postponed if needed (to guarantee sufficient 
peripheral blood cell counts on the day of surgery). 
 
 
 
 
The NEOLETRIB trial: 
Study design: Presurgical Treatment with Ribociclib and Letrozole in Patients 
with Locally Advanced, LUM-A/B Breast Cancer 
Diagnosis 
Day 1: 
Baseline 
samples*; 
MRI / CT (I); 
Start of treatm. 
Tumor-biopsy I. 
Day 90: 
Evaluation 
with: 
MRI / CT (II) 
Day 90 
samples* 
Day 180: 
Pre-surgical eval.: 
MRI / CT (III) 
Day 180-
samples* 
Tumor-biopsy III. 
Surgery 
*Samples: serum, plasma, feces etc. (for details see: laboratory manual); 
  
Day 21: 
Tumor-
biopsy II.  
+ Liquid 
biopsies 
etc* 
Figure 1. 

 
19
All cancer drugs used in this trial (letrozole, ribociclib, goserelin) are well-
established in MBC and have also proven high efficacy in previous neoadjuvant 
trials in locally advanced breast cancer, both as monotherapy and in combination 
4,49-58. Thus, it is known that most of the patients will have clinical benefit from 
these medications and many will be able to avoid neoadjuvant/adjuvant 
chemotherapy that is associated with considerable more serious side-effects 
compared to study medication. However, if patients do not profit sufficiently 
from the medication given in this trial, they will be taken off study medication 
and be treated with all available types of standard chemotherapy established for 
these patients (no restrictions caused by the present protocol). In addition, all 
patients will be considered for adjuvant therapy according to the final 
pathological report following surgery (yTNM-status) and according to the 
Norwegian breast cancer guidelines published by the Norwegian Breast Cancer 
Group (www.nbcg.net). 
 
 
MEDICAL EVALUATIONS and SAMPLING  
 
 
Clinical evaluations 
In general, patients will be followed at the outpatient clinics (Akershus 
University Hospital, Department of Oncology & Drammen Hospital, Department 
of Oncology) by experienced medical breast cancer oncologists at least every 4 
weeks during the entire study (including caliper measurements of maximum 
tumor diameters). In addition, measurement of maximum tumor diameters will 
be performed at study entry and during therapy every 3 months by advanced 
MRI (ax T1, T2, DWI, dynamic study and spectroscopy) and Spectral-CT scans at 

 
20
the Departments of Radiology, Akershus University Hospital and Drammen 
Hospital. All measurements (MRI, spectral-CT etc.) will be performed always 
prior to biopsies at baseline and before final surgery as usual to avoid that 
bleeding etc. may interfere with the diagnostic procedures. Patients will be 
informed about the opportunity to perform PET-scanning prior to treatment 
after protocol as a general screening procedure for distant metastasis (not 
mandatory). All patients will be screened for distant metastasis by CT-scanning 
(thoracic, abdominal and pelvic CT-scanning as well as a bone-scintigraphy) at 
study entry. All available additional diagnostic procedures may be used at the 
discretion of the responsible oncologists / breast cancer surgeons (not 
mandatory). 
 

 
21
Blood samples (scientific samples) 
 
Blood samples will be drawn preferably in the morning following an overnight 
fast (not mandatory but recommended) at the following time points:  
 
- 
1st.. blood sample: on days – 7 to 1 (but before the first treatment after 
protocol; day 1 = day of the first tablets) 
- 
2nd. blood samples: day 21; 
- 
3rd. blood sample: following 3 months on therapy (+/- 7 days); 
- 
4th. blood sample: following 6 months on therapy (+/- 7 days); 
(whenever possible during on of the last days of the ongoing ribociclib 
cycle); 
- 
Additional blood samples: every 3 months (if treatment > 6 months); 
- 
After surgery: once every year for 5 years; 
- 
At relapse/metastasis; 
 
Blood samples per timepoint include: 2 x 10 ml of heparin-plasma; 2 x 10 ml 
EDTA-plasma (cells will be kept in separate vials); 2 x 10 ml serum; 2 x 10 ml of 
citrate-plasma; 2 CPT vials and 4 x 10 ml of full-blood (EDTA). These scientific 
blood samples will be spinned as usual and stored at –80ºC at the individual 
research departments (EPIGEN-center, Akershus University Hospital; Research 
unit at Drammen Hospital) until processing. 
In addition, all standard established blood samples (liver samples, kidney-
function, bone-marrow function etc.) will be taken as usual and recommended 
for the treatment with aromatase inhibitors in combination with ribociclib. 

 
22
Detailed instructions for the handling of scientific blood samples are 
summarized in the appendix of this protocol. 
 
Tissue samples 
 
All in all, tumor tissue samples (each about 500-1000 mg of tumor tissue) will be 
obtained at three time points (at baseline, after 21 days on treatment and after 6 
months of treatment) and either stored or processed immediately for scRNA-seq. 
The tumor tissue will be divided in four portions as described below. One sample 
(ca. 250-500 mg) will be stored immediately in liquid nitrogen (transport 
medium to the research departments and will be primarily used for RNA and 
DNA extraction) before the sample is stored at –80ºC until processing otherwise. 
The second sample (ca. 250-500 mg) will be immediately (within 24h) 
dissociated at the single cell level for single cell RNA profiling and single cell T-
cell receptor sequencing. The third tissue sample will be prepared for histology 
assessments at the departments of pathology, (ca. 100-300 mg, stored in 
formalin before it will be paraffin-embedded as usual). A fourth sample will be 
stored in RNA-later over night at 4ºC and then transferred to -80ºC. The tissue 
samples will be prepared as quickly as possible (max. time: 5-10 minutes) 
preferably in the operational theatre, trimmed (if necessary) for fat and blood as 
usual. In general, all biopsies will be obtained as either open (surgical) biopsies 
or ultrasound guided biopsies using adequate local anesthesia. Vacuum-biopsies 
are also allowed. During final surgery, the scientific tumor biopsy will also be 
obtained through the skin whenever possible to avoid manipulation of the 
posterior surface of the breast (allowing the normal evaluation of free margins, 
distance to tumor tissue etc.). If patients need additional neoadjuvant treatment 

 
23
following 6 months of therapy according to this protocol, the biopsy scheduled 
after 6 months is giving critical information and should therefore be obtained for 
all patients. 
 
Pathology / Histology 
As described above, a baseline tissue biopsy will be used to ensure that the right 
patients are selected for the protocol and will provide all routinely needed 
standard information such as subtype of breast cancer, grading, ER-status, PGR-
status, HER-2 status and level of Ki67-expression. For this purpose, a formalin-
embedded sample will be prepared as usual at baseline and at all subsequent 
time points. All following (scientific / non-diagnostic) biopsies will be 
investigated by a pathologist to ensure that the biopsy is consisting of tumor 
tissue. 
 
Fecal samples 
Fecal samples will be collected at baseline and during follow up (day 21, day 90, 
day 180 and annually yr. 1-5; in addition, in case of relapse/metastasis). The 
fecal samples are sampled at home, placed in designated containers with DNA 
and RNA preserving additives (Stool DNA Stabilizer, Stratec Molecular GMBH, 
Berlin, Germany) and kept refrigerated before being delivered to Akershus 
University Hospital / Drammen Hospital, the biobank location, where it will be 
stored at -80oC.  
We will use a DNA-based approach to determine fecal microbial 
composition. DNA purification from fecal samples will be performed using PSP 
Spin Stool DNA Plus Kit (Stratec Molecular GMBH). Next generation amplicon 

 
24
sequencing targeting the V4 region of the 16S rRNA gene (DNA) will be applied 
to detect the members of the fecal microbiota.  
 
Study endpoints 
 
The longitudinal collection of biopsies and blood samples, at different time point 
of the neoadjuvant treatment regimen and after surgery (follow up with blood 
samples only) is carefully designed to answer several key questions: 
 
1. Study the microenvironment of the tumor and changes during therapy with the 
chosen AI/CDK4/6i -combination 
The immune microenvironment has been associated with response to 
neoadjuvant chemotherapy 30. The single cell RNA-seq profiling of tumors 
collected in this trial will allow us to accurately profile the composition of the 
immune microenvironment at diagnosis and after treatment. This will enable us 
to (i) find possible immune related biomarkers of response at the tumor site, and 
(ii) link the gut microbiota and the serum cytokines profiling to the immune 
tumor microenvironment. As described above scRNA-seq will be performed at 
Ullevål Hospital. 
 
In addition, we will sequence the T-cell receptors of the lymphocyte T 
cells found at the tumor site. Effective immune induced tumor eradication is 
conditioned by the recognition by immune cells of neoantigens which are 
created by the genetic aberration ongoing in tumor cells. Measuring both the 
tumor neoantigens and the ability of the T-cell receptor to recognize such tumor-
antigen will help understanding the ‘laws’ and molecular events underlying 
immune-related tumor eradication. T-cell receptor sequencing will be performed 

 
25
at Ullevål University Hospital at the same time as the scRNA-seq. Neoantigen 
detection will be performed by collaborators at Karolinska Institute, Stockholm 
University, Sweden (Prof. Lehtiö’s laboratory) using state of the art proteomics 
quantification and analysis 59. 
 
Based on recent results we will also evaluate whether responders vs non 
responders of this drug combination (AI + CDK4/6i) belong to a newly 
discovered immune-related breast cancer subtype 30. Subtyping for these 
immune-clusters will be performed by the inventor of the method. 
 
2. Study the mechanisms of adaptation and resistance to aromatase inhibitor 
(letrozole) in combination with the CDK4/6-inhibitor ribociclib. 
We will use the samples collected at diagnosis and after treatment (at surgery) to 
follow the changes in tumor and immune cell phenotypes. We will profile each 
cell of the tumor tissue using single-cell RNA-seq (scRNA-seq). This method 
allows us to profile thousands of individual cells and to build complex cellular 
atlases of whole tumors biomarkers for targeted cancer treatments 60. scRNA-seq 
will allow us to characterize which exact cancer cell phenotypes (clones) are 
eradicated by the treatment combination and find biomarkers of response while 
monitoring which cancer cells survive or are expanding under the treatment 
pressure. Being able to find the cancer cells surviving treatment will allow to 
pave the way to designing the next treatment options for the patients not 
responding to the here propose treatment regimen. We have established the 
scRNA-seq method on primary breast tumors (Figure 2), these data are the first 
scRNA-seq data performed on solid tumors in Norway.  

 
26
The experimentation will be conducted at Ullevål University Hospital using the 
10X GENOMICS platform for single cell analysis.  
 
Figure 2: scRNA-seq of primary breast tumor (preliminary results). 
A: tSNE clustering of ~ 5.000 cells from breast tumor. A dot represents a cell, A color, a cell type, we therefore identify 
several cell phenotypes in this breast tumor. 
B: On the respective t-SNE plot the expression of CD8 a marker of cytotoxic immune cells is plotted showing that the 
cells on the top left are T lymphocytes. 
 
 
Furthermore, to follow clonal evolution of the tumor based on mutations and 
genetic aberrations, whole genome sequencing of the tumor DNA is planned. 
Bulk transcriptomic of the tumor tissue will allow accurate molecular subtyping 
using gene expression and to obtain for example PAM50 subtypes and ROR 
scores 61. Such standard molecular profiling of the tumor using next generation 
sequencing will be performed either at Akershus or Ullevål University Hospital, 
where such methods are used routinely. 
 
3. To evaluate the immediate effects of letrozole/ribociclib treatment on the 
clinical presentation of the tumor in addition to surrogate markers (like 
suppression of Ki-67, influence on PEPI-score etc.) 
 
4. To study the relationship between treatment effect and the gut microbiota 
The gut microbiome in women with breast cancer differs from that of healthy 
women 38. How the gut microbiome may affect breast cancer risk is as not known 
and warrants further research. Proposing hypotheses on the relationship 
A
B

 
27
between 
breast 
cancer 
and 
gut 
microbiota 
involve 
both 
the 
microbiota/estrobolome (the collection of the enteric bacterial genes whose 
products metabolize estrogen and its metabolites) where perturbation could 
lead to elevated levels of estrogens and its metabolites, and the estrogen-
independent pathway where bacterial translocation from the gut to the 
mammary gland via an endogenous route could take place. The microbiota has 
been found to regulate the response to cancer chemotherapy by affecting 
mechanisms of action and toxicity, and regulate the effectiveness of ICI by 
modulating inflammation and adaptive immunity 39. In the present study we will 
explore the influence of the gut microbiota on the treatment effect and the 
immune system, as well as observing the direct effects of ribociclib and letrozole 
on the gut microbiome. We will perform next generation amplicon sequencing on 
the V4 region of the bacterial 16S rRNA gene at baseline and follow up. This gives 
the opportunity to identify and follow the composition and diversity of the gut 
microbiota associated with disease course, immune response and response to 
treatment. Changes in the intestinal microbiota during treatment and the 
possibility of the gut microbiota, as a community or certain members, to be a 
biomarker and prognostic tool will be explored. The DNA extraction and 
amplicon sequencing will be performed at Dept of Clinical Molecular Biology, 
Akershus University Hospital. 
 
5. Non-invasive biomarkers of response or relapse during therapy with the 
aromatase inhibitor letrozole in combination with the CDK4/6 inhibitor ribociclib 
a. Serum cytokine levels 

 
28
To seek for non-invasive biomarkers of response to therapy in relation to 
systemic and tumor-related immune reaction, we will measure cytokines serum 
levels at different time points. Cytokines are small proteins that are important in 
cell signaling. They are highly inducible peptides and mediate intercellular 
communication in the immune system 62. Alterations in specific cytokine 
expression patters have been detected in patients with breast cancer, and 
systemic inflammation has been associated with aggressive tumor growth, 
invasion and metastasis 63. Therefore, serum cytokines levels represent 
promising non-invasive biomarkers to monitor patient’s response to therapy and 
will be measured in this study. Measurement of cytokines will be performed at 
Akershus University Hospital using the Luminex platform as we previously 
described 64. 
 
b. Serum metabolite levels 
Metabolites are small chemical compounds that are intermediates or products of 
metabolism. It is well known that cancer cells have a reprogrammed metabolism 
to convert nutrients to biomass while maintaining energy production. This 
phenomenon is increasingly recognized as a potential target for treatment, but 
also as a source for biomarkers that can be used for risk stratification, 
prognostication and therapy monitoring. In breast cancer, significant changes in 
the metabolism have been described, both in tumor tissue 65 and in biofluids 66.  
Our assessment of metabolites in the serum of the patients will therefore allow 
for the discovery of biomarkers as well as finding a connection between the 
microbiota and the host / systemic host metabolism. Indeed, the gut microbiota 
is involved in the regulation of numerous physiological pathways, among others 

 
29
the energy metabolism is of particular interest. Metabolites will be measured 
using mass spectrometry as previously by collaborator in Trondheim (NTNU) as 
previously described 65. 
 
c. Circulating free tumor DNA (cftDNA) 
Detection of circulating free tumor DNA (cftDNA) during follow-up after initial 
treatment for early breast cancer was associated with a high risk of relapse 67. 
This gives the rationale to collect blood samples after surgery and end of 
treatment as it has strong potential to early detect the patients relapsing and or 
at risk of relapsing. Circulating free DNA will be extracted and sequenced at 
Akershus University Hospital. 
 
RECRUITMENT TIME 
 
Starting on December 1st. 2020 (or when the protocol is approved by local 
authorities and at the hospital level) until all patients are recruited (estimated 
recruitment period:  2 years). 
 
SUBSEQUENT THERAPY 
 
All patients treated following this protocol will be evaluated by an experienced 
breast surgeon for the best local surgical treatment option (breast conserving 
surgery or mastectomy including necessary procedures including the ipsilateral 
axilla). 
After surgery, all patients will be considered for adjuvant treatment (both 
endocrine options and chemotherapy but also zoledronic acid etc.) according  to 

 
30
the 
national 
recommendations 
(Norwegian 
Breast 
Cancer 
Group; 
www.nbcg.net). 
Patients with progressive diseases (at any time point) during neoadjuvant 
therapy or stable diseases following 3 months of therapy according to this 
protocol, will be able to obtain additional therapies (chemotherapy etc.) without 
any restrictions to allow down-staging prior to surgery by other means. 
 
PROTOCOL AMENDMENTS AND SAE REPORTING 
If necessary, protocol amendments will be done in collaboration with all 
involved parties and the regional ethical committee will be informed about all 
changes prior to implementation.  
 
ADVERSE EVENT (AE) REPORTING 
AE definition 
An adverse event (AE) is defined as the appearance (or worsening in the case of 
pre-existing conditions) of any undesirable sign, symptom or disease that arises 
after having obtained the patient's informed consent, regardless of if it is 
considered to be related to the study drug. Any abnormal analytical values or 
test results occurring after obtaining informed consent shall only be reported in 
the eCRF under adverse events when there are clinical signs or symptoms, they 
are considered clinically relevant, they require treatment (for example, a 
hematological abnormality that requires transfusions or supportive therapy with 
hematopoietic stem cells) or require the study medication to be modified.  
AEs that appear or worsen after obtaining the informed consent should be 
recorded in the eCRF under adverse events. Diseases that were already present 
at the time of granting informed consent must be recorded in the eCRF under 
medical history. Continuous monitoring for AEs will be carried out for at least 30 
days after the last dose of the study treatment. The AEs (including the abnormal 

 
31
analytical values constituting AEs) must be described by mentioning a diagnosis 
to the extent possible, instead of the underlying signs and symptoms. When no 
clear diagnosis can be identified, every sign or symptom must be reported as an 
independent AE. 
The AEs shall be assessed in accordance with the Common Terminology Criteria 
for Adverse Events (CTCAE), version 4.03, whenever possible. In the event that a 
grade for an AE is not included in the CTCAE, the severity of mild, moderate, 
severe, potentially fatal and death shall be used, corresponding to grades 1 to 5. 
The onset of AEs should be investigated by asking the patient open-ended 
questions during the screening process after the signing of the informed consent 
and at each study visit. AEs can also be detected by spontaneous reporting by the 
patient during the screening process or between visits, as well as through the 
physical examination, analyses or other assessments. To the extent possible, all 
AEs must be assessed to determine: 
1. Grade of severity (CTCAE grades 1 to 5). 
2. Duration (dates of onset and finalization). 
3. Relationship with the study treatment (reasonable likelihood that the AE 
is related: No/ Yes; causality will be assessed individually for each study 
treatment). 
4. Measure adopted with regard to the study treatment (none, dose 
adjustment, temporary discontinuation, permanent discontinuation, 
unknown, not applicable). 
5. Administration of medication or treatment (absence of concomitant 
medication or nonpharmacological treatment, concomitant medication or 
non-pharmacological treatment). 
6. Outcome (no recovery/no resolution, recovery/resolution, in 
recovery/being resolved, recovery/resolution with sequelae, fatal, 
unknown). 
7. Seriousness, in accordance with the definition of serious adverse event 
(SAE) indicated in the section under. 
All AEs must be treated appropriately. When a concomitant medication or non-
pharmacological treatment is administered, this circumstance must be recorded 
in the eCRF under AE. 

 
32
Once an AE is detected, it must be monitored until its resolution or until it is 
considered permanent; at each of the visits (or more frequently if necessary) the 
variation in the severity, the supposed relationship with the study treatment, the 
interventions needed to treat it and the outcome will be assessed. 
With regard to determination of causality mentioned in point 3, the sponsor will 
subsequently classify the adverse event, on the basis of causality with the drug, 
according to the algorithm of Karch and Lasagna (1977), as: 
• Definitive: there is a reasonable temporal sequence between 
administration of the drug and the appearance of the adverse event. This 
event coincides with the adverse event described for the drug, improves 
when it is interrupted and reappears after readministration and cannot 
be explained by alternative causes. 
• Probable: there is a reasonable temporal sequence between 
administration of the drug and the appearance of the adverse event. This 
event coincides with the adverse event described for the drug, improves 
when it is interrupted and cannot be explained by alternative causes. 
• Possible: there is a reasonable temporal sequence between 
administration of the drug and the appearance of the adverse event. This 
event coincides with the adverse reaction described for the drug, but may 
be explained by alternative causes. 
• Conditional or unlikely: there is a reasonable temporal sequence between 
administration of the drug and the appearance of the adverse event. This 
event does not coincide with the adverse reaction described for the drug, 
and may be explained by alternative causes. 
• Unrelated: there is no reasonable temporal sequence between 
administration of the drug and the appearance of the adverse event. This 
event does not coincide with the adverse reaction described for the drug, 
and may be explained by alternative causes. 
 
Serious adverse events 
Definitions 
Serious adverse event (SAE) is defined as one of the following: 
• Is fatal or life-threatening 

 
33
• Results in persistent or significant disability/incapacity 
• Constitutes a congenital anomaly/birth defect 
• Is medically significant, i.e., defined as an event that jeopardizes the 
patient or may require medical or surgical intervention to prevent one of 
the outcomes listed above 
• Requires inpatient hospitalization or prolongation of existing 
hospitalization, 
 
Note that hospitalizations for the following reasons should not be reported as 
serious adverse events: 
• Routine treatment or monitoring of the studied indication, not associated 
with any deterioration in condition (i.e. to perform study related 
assessments) 
• Elective or pre-planned treatment for a pre-existing condition that is 
unrelated to the indication under study and has not worsened since 
signing the informed consent 
• Social reasons and respite care in the absence of any deterioration in the 
patient’s general condition 
Note that treatment on an emergency outpatient basis that does not result in 
hospital admission and involves an event not fulfilling any of the definitions of a 
SAE given above is not a serious adverse event 
 
Reporting 
AEs and SAEs  
All reported adverse events and serious adverse events will be reported as 
defined in the previous section and recorded in the patient's eCRF.  
SAEs must be reported by the investigator to national coordinating investigator 
as outlined in the ISF within 24 hours after the site has gained knowledge of the 
SAE. Every SAE must be documented by the investigator on the SAE pages in the 
eCRF, generating an automatic message to the national coordinating investigator 
and sponsor. The initial report shall promptly be followed by detailed, written 
reports if necessary. The initial and follow-up reports shall identify the trial 
subjects by unique code numbers assigned to the latter.  

 
34
The sponsor keeps detailed records of all AEs and SAEs reported by the 
investigators and performs an evaluation with respect to seriousness, causality 
and expectedness.  
 
Suspected Unexpected Serious Adverse Reaction (SUSARs)  
SUSARs will be reported to the Competent Authority according to national 
regulation. The following timelines should be followed:  
The sponsor will ensure that all relevant information about suspected serious 
unexpected adverse reactions that are fatal or life-threatening is recorded and 
reported as soon as possible to the Competent Authority and Ethics Committee, 
and in any case no later than seven (7) days after knowledge by the sponsor of 
such a case, and that relevant follow-up information is subsequently 
communicated within an additional eight (8) days.  
All other suspected serious unexpected adverse reactions will be reported to the 
Competent Authority concerned and to the Ethics Committee concerned as soon 
as possible but within a maximum of fifteen (15) days of first knowledge by the 
sponsor.  
SUSARs will be reported using the Council for International Organizations of 
Medical Sciences (CIOMS) form  
The Sponsor is responsible for informing all investigators about SUSARSs 
occurring in the study on a regular basis.  
 
 
Pregnancies 
To ensure patient safety, each pregnancy in a patient on study treatment must be 
reported to the Sponsor within 24 hours of learning of its occurrence. The 
pregnancy should be followed up to determine outcome, including spontaneous 
or voluntary termination, details of the birth, and the presence or absence of any 
birth defects, congenital abnormalities, or maternal and/or newborn 
complications. 
Pregnancy should be recorded on a Clinical Trial Pregnancy Form and send to 
the Sponsor. 

 
35
Pregnancy follow-up should be recorded on the same form and should include an 
assessment of the possible relationship to the study treatment of any pregnancy 
outcome. Any SAE experienced during pregnancy must be reported on the SAE 
Report Form. 
Pregnancy outcomes must be collected for the female partners of any males who 
took study treatment in this study. Consent to report information regarding 
these pregnancy outcomes should be obtained from the mother. 
 
Study drug misuse or abuse 
Any misuse or abuse of the Study drug must be reported to the Sponsor within 
24 hours of learning of its occurrence. 
 
Annual Safety Report  
Once a year throughout the clinical trial, the sponsor will provide the Competent 
Authority with an annual safety report. The format will comply with national 
requirements. 
 
ETHICAL CONSIDERATIONS AND APPROVALS 
The protocol will be submitted to the regional ethical committee for approval 
prior to initiation of the study. There are no ethical conflicts connected to this 
protocol. The treatment provided has been tested in previous protocols and is 
known to be very effective in selected patients (as described in this protocol). 
The intention of the present protocol is solely to study the unknown mechanisms 
of tumor cell adaption during therapy in relation to the two drugs involved. As 
mentioned before, both drugs have been evaluated as highly potent and effective 
given in combination to breast cancer patients. 
The design of the NEOLETRIB-trial has already been discussed with the 
Norwegian drug approval authority (Statens legemiddelverk – SLV) and we have 

 
36
received the final evaluation from the responsible committee, confirming that 
NEOLETRIB may be initiated without further discussions with the SLV (due to 
the fact that all used drugs are approved in Norway for the treatment of breast 
cancer and no randomization is planned in NEOLETRIB). 
 
DATA FLOW AND HANDLING 
 
For all analyses to be conducted, patients will be recognized by a code number 
not allowing personal identification. The manual identifying each patient 
number by name will be kept locked in the Department of Oncology and is the 
responsibility of the principal corresponding investigator. 
 
 
PUBLICATION OF DATA 
 
All results will be published in relevant medical journals. Qualification for co-
authorship will be decided by the principal investigator in collaboration with all 
involved partners in accordance with general practice (Vancouver-guidelines 
etc.). 
 
FUNDING AND INVOLVED PARTIES 
This trial is a research collaboration between the involved parties (UiO, AHUS 
and Novartis Norway AS), which involves study design, steering group 
participation and sharing of cost as regulated by the contracted agreement 
between the parties. 
 
 
 

 
37
APPENDIX 
Single cell RNA and T cell and B cell receptor sequencing 
In the surgery theatre needle or open biopsies will be immediately immerged in 
culture media to allow optimal transport to Ullevål Hospital on ice. At Ullevål, the 
breast tumor tissue will be dissociated at the single cell level using enzymatic 
and mechanical means and red blood cells removed. Approximatively 10.000 live 
dissociated cells will be processed through the 10X genomics chromium 
controller to embed each cell with a gel bead which contains a unique bar code 
which will allow to recognize the cell from which the mRNA molecules are 
captured from. Following library preparation and sequencing, the molecular 
RNA profiling of each of the 10.000 cells will be obtained to allow in depth 
characterization of the composition of the tumor tissue. In addition, though 
enrichment and amplification of the genes coding for T cell and B cell receptors, 
we will have unprecedent knowledge of the clones of immune cell present at the 
tumor. 
 
Cytokines measurement 
Serum from 4 mL EDTA blood tubes will be obtained by centrifugation the blood 
30 min after collection at 15000 x g for 10 minutes in a refrigerated centrifuge. 
The serum will be aliquoted in clean polypropylene tubes and stored at -80C. 
Aliquots will be thawed the day cytokines levels need to be measured using the 
54-plex cytokine panel (Bio-Rad Laboratories) and will be analyzed with the 
Luminex xMAP 200 platform (Luminex Corporation, Austin, TX, USA). The assay 
includes a series of known concentrations to generate standard curves. All 
samples will be analyzed in duplicates. 

 
38
DNA and RNA extraction from fresh frozen tissue 
Tumor biopsies will immediately be immerged in liquid nitrogen for instant 
freezing to preserve nucleic acids (DNA and RNA). Fresh frozen biopsies will be 
stored at -80C until they will be further processed. Fresh frozen tumor biopsies 
are dissected into small pieces, mixed, and divided into amounts suitable for 
DNA and RNA extraction. For RNA extraction using the RNeasy kit for RNA 
extraction (Qiagen) or DNA extraction with the DNeasy for tissue extraction kit 
(Qiagen). 
 
RNA sequencing for Molecular profiling of biopsies 
1 μg of starting total RNA will be processed for library construction and 
sequencing. poly(A) mRNA is isolated from the total RNA. First strand cDNA 
synthesis is performed using random hexamers and standard dNTP mix, second 
strand cDNA synthesis is performed using dUTP. The cDNA is end-repaired and 
A-tailed, and diluted TruSeq adapters with barcodes are ligated. Adapter-ligated 
cDNA is then size-selected Typically, 10 to 24 barcoded libraries are included in 
a pool and each pool is sequenced in at least one lane across dual flowcells. 
Paired-end sequencing of 50 bp read-length is performed on an Illumina HiSeq 
2000 instrument. 
 
DNA sequencing for mutation and clonality analysis 
gDNA isolated is normalized to 1-3 μg. Libraries are prepared using the Agilent 
SureSelectXT Target Enrichment Library protocol and Agilent SureSelectXT 
Human All Exon v4 enrichment capture library.  Captured Whole Exome final 
libraries passing the final QC step are normalised to 2nM and pooled for 

 
39
sequencing on the HiSeq 2500 instrument. Dual HiSeq SBS v4 runs at 101bp 
paired-end reads generated the data for analysis. Target coverage was 400-500x 
for the tumour regions and 100-200x for the associated normal. 
 
DNA sequencing of the gut microbiota 
DNA purification from fecal samples will be performed using PSP Spin Stool DNA 
Plus Kit (Stratec Molecular GMBH, Berlin, Germany). The bacterial taxonomic 
composition of the fecal microbiota will be determined using amplicon 
sequencing of the bacterial 16S rRNA variable 4 (V4) region and the Illumina 
MiSeq technology following published protocol (ref). A MiSeq instrument is 
available at the Dept of Clinical Molecular Biology, Akershus University Hospital. 
The sequencing data will be analyzed using QIIME2 (ref) and the R computing 
environment (ref).  
 
APPENDIX 
A. Detailed study design (see figure 1); 
B. Laboratory Procedures 
C Primary timelines: 
It is planned to start enrolment of patients in December 2020 at two 
Norwegian Centers. The last patients should be enrolled after ca. 2 
years. 
D. Budget (separate document has been provided); 
 
 

 
40
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1. 
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Vetizou, M., et al. Anticancer immunotherapy by CTLA-4 blockade relies 
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Gopalakrishnan, V., et al. Gut microbiome modulates response to anti-PD-
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Semiglazov, V.F., et al. Phase 2 randomized trial of primary endocrine 
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Geisler, J., et al. Letrozole is Superior to Anastrozole in Suppressing Breast 
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Geisler, J., et al. In vivo inhibition of aromatization by exemestane, a novel 
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Geisler, J., et al. Influence of anastrozole (Arimidex), a selective, non-
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Gnant, M., Steger, G.G. & Bartsch, R. CDK4/6 inhibitors in luminal breast 
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Hortobagyi, G.N., et al. Updated results from MONALEESA-2, a phase III 
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Oncol 29, 1541-1547 (2018). 
14. 
Hortobagyi, G.N., et al. Updated results from MONALEESA-2, a phase III 
trial of first-line ribociclib plus letrozole versus placebo plus letrozole in 
hormone receptor-positive, HER2-negative advanced breast cancer. Ann 
Oncol 30, 1842 (2019). 

 
41
15. 
Finn, R.S., et al. Palbociclib and Letrozole in Advanced Breast Cancer. N 
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