Clinical trial NCT02536742
Conditions: Metastatic Breast Cancer
Keywords: Breast Cancer Metastatic HER2 negative Palbociclib
Sponsor: ETOP IBCSG Partners Foundation
Start date: 2016-08-30; Completion date: 2022-12-22
Enrollment: 124 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This international, multicenter, prospective single arm Phase II biomarker discovery clinical trial with the primary objective of assessing the association of PFS with gene mutations, gene copy number aberrations and gene signatures in post-menopausal women with hormone receptor positive, HER2-negative metastatic or locally relapsed breast cancer whose disease has progressed after prior adjuvant endocrine therapy or one line systemic treatment, i.e., endocrine treatment or chemotherapy, administered for metastatic disease.

Detailed description:
Patients will be treated with the combination of palbociclib and fulvestrant. The primary objective is to assess the association of the primary endpoint progression-free survival (PFS) with potential markers.

The trial is included in the AURORA program conducted by the Breast International Group (BIG), an international study aiming to collect and characterize biological samples, including metastatic tissue, from patients with advanced breast cancer.

The primary aim of the PYTHIA study is to discover potentially innovative biomarkers for the selection of patients to Palbociclib/Fulvestrant treatment. The strength of the trial lies in its conduct in conjunction with the AURORA study, which systematically evaluates a panel of biomarkers in tissue and blood, in a certified central lab. Stemming from this association, an abundance of molecular profiling information will become available for different biological samples. Additional molecular and functional imaging assessments performed within the context of the PYTHIA study increase its scientific merit, since it will represent a prospective, systematic effort to identify biomarkers for patient stratification, integrating several molecular profiling assessments.

Arms: arm 1: Palbociclib plus Fulvestrant
Interventions: intervention 1: Palbociclib intervention 2: Fulvestrant
Intervention descriptions: intervention 1: 125 mg, orally, daily for 3 weeks followed by 1 week off; repeated at every 28 days cycle until progression, lack of tolerability, or patient declines further protocol treatment. intervention 2: 500mg, intramuscularly on days 1 and 15 of cycle 1, then on day 1 (+/- 3 days) of every 28 days cycle until progression, lack of tolerability, or patient declines further protocol treatment.

Protocol documents:
Prot_000.pdf: 
IBCSG 53-14 / BIG 14-04 PYTHIA 
 
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V 2.0  Amendment 1   26 January 2017 
Coordinating Center Effingerstrasse 40 CH-3008 Bern Switzerland www.ibcsg.org 
 
 
 
A Phase II Study of Palbociclib plus Fulvestrant for pretreated 
patients with ER+/HER2- Metastatic Breast Cancer 
 
Palbociclib in molecularly characterized ER-positive/HER2-
negative metastatic breast cancer: the PYTHIA study 
 
 
 
EudraCT number: 
2014-005387-15 
 
Pfizer number: 
WI198393 
 
Sponsor:   
International Breast Cancer Study 
Group (IBCSG) 
 
 
IBCSG Coordinating Center, Effingerstrasse 40, CH- 3008 Bern 
Phone: +41 31 389 93 91 
Fax: +41 31 389 93 92 
 
 
 
Amendment 1 
 
Protocol Version 2.0 
 
 
 
  
 
 
 
 
 
 
 
 
 
INTERNATIONAL BREAST 
CANCER STUDY GROUP 
IBCSG 53-14 / BIG 14-04 
 
PYTHIA 
 
 
 
 
 


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Trial Specific Contacts 
 
Trial Chair 
Luca Malorni, MD, PhD 
USL4 Hospital of Prato, Italy 
Email: lmalorni@usl4.toscana.it 
Tel: +39 (0574) 802523 
Fax: +39 (0574) 802903 
BIG Headquarters representative 
Dimitrios Zardavas, MD 
Email: 
Dimitrios.Zardavas@bigagainstbc.org 
Tel: +32 2 486 16 64 
 
Chair of PET substudy  
Patrick Flamen, MD, PhD 
Head Dpt. Nuclear Medicine 
Institut Jules Bordet 
Email: patrick.flamen@bordet.be 
Tel: +32 2 541 3240 
Fax: +32 2 541 3224 
 
 
 
Trial Statistician 
Meredith Regan, ScD 
 
Email: mregan@jimmy.harvard.edu 
Tel: +1 617-632-3012 
Fax: +1 617-632-5444 
 
 
 
Trial Data Manager 
 
Trial Coordinator 
 
Adriana Mora de Karausch 
Poonam Jani 
IBCSG Data Management Center 
 
Email: ibcsg53_PYTHIA@fstrf.org 
Tel: +1 716-834-0900 (general) 
Fax:  +1 716-833-3990 
Translational Research 
Coordinator 
 
Rosita Kammler 
 
Email: rosita.kammler@ibcsg.org 
 
AURORA Laboratory 
AURORA lab 
OncoDNA s.a. 
25 avenue George Lemaître 
6041 Gosselies, Belgium 
 
Email: infos@oncodna.com 
Tel: +32 71 34 78 99 
Fax:  
Orilab (PET substudy) 
Thomas Guiot 
Camilo Garcia, MD 
Orilab 
Institut Jules Bordet 
Email: pythia.trial@orilab.org 
Tel: +32 2 541 3996 
Fax: +32 2 541 3224 
 
IBCSG and BIG Contacts 
 
BIG Headquarters 
 
 
Theodora Goulioti, MD 
Scientific Director 
Email: 
Theodora.Goulioti@bigagainstbc.org 
Tel: +32 2 541 7246 
Fax:   
 
Coordinating Center 
 
 
Anita Hiltbrunner 
Barbara Ruepp, PharmD 
Manuela Rabaglio, MD 
Rudolf Maibach, PhD 
Linda Fritzsche / Sabrina Ribeli-
Hofmann  
Rosita Kammler 
IBCSG Coordinating Center 
Effingerstrasse 40 
CH-3008  Bern, Switzerland 
Email: anita.hiltbrunner@ibcsg.org  
Email: barbara.ruepp@ibcsg.org 
Email: manuela.rabaglio@ibcsg.org 
Email: rudolf.maibach@ibcsg.org 
Email: drugsupply@ibcsg.org 
 
Email: rosita.kammler@ibcsg.org 
Tel: +41 31 511 94 00 
Fax: +41 31 511 94 012 
 
 
 


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Data Management Center 
Lynette Blacher 
Karolyn Scott 
Holly Shaw 
FSTRF 
4033 Maple Rd.  
Amherst, NY  14226 USA 
Email: blacher.lynette@fstrf.org 
Email: scott.karolyn@fstrf.org 
Email : hshaw@fstrf.org 
Tel: +1 716 834-0900 
Fax: +1 716 833-3990 
 
Data Quality Control Office 
 
IBCSG Data Management Center 
 
 
Email: ib.dqc@fstrf.org 
 
Center Training Office 
 
IBCSG Data Management Center 
E-mail: ibcsg.training@fstrf.org 
 
Statistical Center 
Prof. Richard D. Gelber 
Meredith M. Regan, ScD 
Email: gelber@jimmy.harvard.edu 
Email: mregan@jimmy.harvard.edu 
 
 
 
 
Dept. of Biostatistics and 
Computational Biology, CLSB 11007 
Dana-Farber Cancer Institute 
450 Brookline Ave. 
Boston, MA 02215  USA 
Tel: +1 617-632-3012 
Fax: +1 617-632-5444 
 
 
 
Scientific Committee Chair 
Dr. Marco Colleoni  
European Institute of Oncology 
Milan, Italy 
Email: marco.colleoni@ieo.it 
Tel: +39 02 574 89 439 
Fax: +39 02 574 89 581 
 
 
 
In collaboration with Pfizer Inc.  
 
 


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Protocol Signature Page 
 
IBCSG 53-14 
 
PYTHIA 
 
 
 
 
 
Approved by: 
Group Statistician, International Breast Cancer Study Group 
Dr. M.M. Regan 
 
 
 
___________________________________________________ 
______________ 
 
 
 
Date 
 
Approved by: 
Director, International Breast Cancer Study Group 
Anita Hiltbrunner 
 
 
 
___________________________________________________ 
______________ 
 
 
 
Date 
 
 
Approved by: 
Associate Scientific Director, Breast International Group 
Dr. Dimitrios Zardavas 
 
 
 
___________________________________________________ 
______________ 
 
 
 
Date 
 
 
Approved by: 
Trial Chair; Medical Director, U.O. Oncologia Medica "Sandro Pitigliani", Prato ITA 
Dr. Luca Malorni 
 
 
 
___________________________________________________ 
______________ 
 
 
 
Date 




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Principal Investigator Protocol Signature Page 
Amendment 1 
 
IBCSG 53-14 
PYTHIA 
 
I have read the protocol and agree that it contains all necessary details for conducting this 
trial. I will conduct the trial as outlined in the following protocol and in compliance with GCP, 
and will apply due diligence to avoid protocol deviations. I will provide copies of the protocol 
and all drug information relating to pre-clinical and prior clinical experience furnished to me 
by IBCSG, to all physicians responsible to me who participate in this trial. I will discuss 
this material with them to assure that they are fully informed regarding the drugs and the 
conduct of the trial. I agree to keep records on all patient information (Case Report Forms and 
patient's Informed Consent statement), drug-shipment and return forms, and all other 
information collected during the trial for a minimum period of 15 years. 
 
Name of Principal Investigator:___________________________________________ 
 
 
_____________________________________________________ ______________ 
 
 
Signature 
Date 
 


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TABLE OF CONTENTS  
 
Section 
Page 
1. 
Protocol Summary and Schema .......................................................................... 10 
2. 
Trial schedule ...................................................................................................... 19 
3. 
List of abbreviations............................................................................................ 21 
4. 
Background and scientific rationale .................................................................... 23 
4.1. 
Breast cancer ........................................................................................................ 23 
4.2. 
Molecular characterization .................................................................................. 23 
4.3. 
CDK inhibition .................................................................................................... 23 
4.4. 
Palbociclib ........................................................................................................... 24 
4.5. 
Fulvestrant ........................................................................................................... 29 
4.6. 
Biomarkers for palbociclib activity ..................................................................... 30 
4.7. 
Overall risk-benefit assessment ........................................................................... 30 
4.8. 
Rationale for the trial design ............................................................................... 31 
4.9. 
Rationale for the FDG-PET substudy .................................................................. 32 
5. 
Trial objectives and endpoints ............................................................................ 33 
5.1. 
Primary objective ................................................................................................. 33 
5.2. 
Primary endpoint ................................................................................................. 33 
5.3. 
Secondary objectives ........................................................................................... 33 
6. 
Trial design, duration and termination ................................................................ 34 
6.1. 
Trial design .......................................................................................................... 34 
6.2. 
Trial schema ........................................................................................................ 34 
6.3. 
Treatment ............................................................................................................. 34 
6.4. 
Clinical evaluations ............................................................................................. 35 
6.5. 
Biological evaluations ......................................................................................... 35 
6.6. 
Sample size and trial duration ............................................................................. 35 
7. 
Patient selection .................................................................................................. 35 
7.1. 
Inclusion criteria .................................................................................................. 35 
7.2. 
Exclusion criteria ................................................................................................. 37 
8. 
Enrollment ........................................................................................................... 39 
8.1. 
Patient registration ............................................................................................... 39 
8.2. 
Registration Help Desk ........................................................................................ 39 
8.3. 
AURORA Registration Confirmation ................................................................. 40 
9. 
Trial drugs formulation and handling ................................................................. 40 
9.1. 
Palbociclib ........................................................................................................... 40 
9.2 
Fulvestrant ........................................................................................................... 41 
10 
Treatment ............................................................................................................ 41 
10.1 
Trial treatments .................................................................................................... 41 
10.2 
Treatment Administration .................................................................................... 42 
10.3 
Dose modifications and delays for fulvestrant .................................................... 42 
10.4 
Dose delays and modifications for palbociclib .................................................... 43 
10.5 
Concomitant therapy............................................................................................ 45 
10.6 
Stop of trial treatment .......................................................................................... 47 
10.7 
Removal from the trial ......................................................................................... 48 
11 
Safety .................................................................................................................. 48 
11.1 
Adverse reactions to fulvestrant .......................................................................... 48 
11.2 
Adverse reactions to palbociclib .......................................................................... 49 
11.3 
Potential drug induced liver injury ...................................................................... 50 
11.4 
Drug-drug interactions ......................................................................................... 51 


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Adverse event and serious adverse event reporting ............................................ 52 
12.1 
Adverse event reporting ....................................................................................... 52 
12.2 
Targeted adverse events ....................................................................................... 54 
12.3 
Adverse events of special interest (AESIs) ......................................................... 54 
12.4 
Otherwise reportable events ................................................................................ 54 
12.5 
Serious adverse event (SAE) ............................................................................... 55 
12.6 
Reporting SAEs ................................................................................................... 56 
12.7 
Occupational exposure ........................................................................................ 57 
13 
Disease assessment, response and progression (RECIST 1.1) ............................ 57 
13.1 
Introduction ......................................................................................................... 57 
13.2 
Methods of assessment ........................................................................................ 57 
13.3 
Measurability of tumor at baseline ...................................................................... 58 
13.4 
Selection of target lesions .................................................................................... 59 
13.5 
Selection of non-target lesions ............................................................................ 59 
13.6 
Evaluation of target lesions (measurable disease) ............................................... 60 
13.7 
Evaluation of non-target lesions .......................................................................... 60 
13.8 
Determination of new lesions .............................................................................. 61 
13.9 
Additional considerations .................................................................................... 61 
13.10 
Determination of time point response ................................................................. 61 
13.11 
Determination of best overall response ............................................................... 62 
13.12 
Progression-free Survival .................................................................................... 62 
13.13 
Storage of imaging............................................................................................... 63 
14 
Clinical and laboratory evaluations and follow-up ............................................. 63 
14.1 
Screening ............................................................................................................. 63 
14.2 
Day 1 of every treatment cycle ............................................................................ 64 
14.3 
Treatment day 15 (cycle 1) .................................................................................. 64 
14.4 
Tumor assessments .............................................................................................. 65 
14.5 
After discontinuation of trial treatment ............................................................... 65 
14.6 
Follow-up prior to documented disease progression ........................................... 65 
15 
Biological evaluations ......................................................................................... 66 
15.1 
Introduction and overview ................................................................................... 66 
15.2 
Translational research .......................................................................................... 66 
16 
FDG-PET substudy ............................................................................................. 67 
16.1 
Introduction ......................................................................................................... 67 
16.2 
Study objectives ................................................................................................... 68 
16.3 
Study design ........................................................................................................ 68 
16.4 
Definitions ........................................................................................................... 69 
16.5 
ORILAB – Imaging CoreLAB: ........................................................................... 70 
17 
Data submission .................................................................................................. 70 
17.1 
Case report forms schedule .................................................................................. 71 
17.2 
Signing and submitting forms.............................................................................. 72 
17.3 
Data management ................................................................................................ 72 
17.4 
Investigator Site File ............................................................................................ 72 
17.5 
Authorization log ................................................................................................. 74 
17.6 
Patient identification log ...................................................................................... 74 
18 
Statistical considerations ..................................................................................... 74 
18.1 
Primary Objective ................................................................................................ 74 
18.2 
Secondary Objectives .......................................................................................... 76 
18.3 
Definitions of Trial Populations .......................................................................... 76 
18.4 
Interim Analyses .................................................................................................. 76 
18.5 
Accrual ................................................................................................................. 76 


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18.6 
Data and Safety Monitoring ................................................................................ 77 
19 
Ethical aspects, regulatory approval, and patient informed consent ................... 77 
19.1 
Ethical Review Board/Ethics Committee ............................................................ 77 
19.2 
Regulatory approval procedures .......................................................................... 77 
19.3 
Protection of human patients ............................................................................... 77 
19.4 
Informed Consent ................................................................................................ 78 
19.5 
Premature withdrawal .......................................................................................... 78 
20 
Governance and Administrative Considerations ................................................. 79 
20.1 
Insurance .............................................................................................................. 79 
20.2 
Steering Committee ............................................................................................. 79 
20.3 
Data and Safety Monitoring Committee (DSMC) ............................................... 79 
20.4 
Publication of trial results .................................................................................... 79 
20.5 
Premature discontinuation of the trial ................................................................. 80 
20.6 
Quality Assurance................................................................................................ 80 
20.7 
Protocol adherence .............................................................................................. 80 
20.8 
Data protection .................................................................................................... 80 
20.9 
Record Retention ................................................................................................. 81 
21 
Confidentiality .................................................................................................... 81 
22 
References ........................................................................................................... 81 
Appendices  
1. 
Requirements for Informed Consent  
 
Index of Tables 
Table 1. 
ECOG Performance Status.................................................................................. 38 
Table 2. 
NYHA functional classification .......................................................................... 38 
Table 3. 
Palbociclib capsule characteristics ...................................................................... 41 
Table 4. 
Palbociclib dose reduction/delay for hematological toxicities ........................... 43 
Table 5. 
Palbociclib dose reduction/delay for non-hematological toxicities .................... 44 
Table 6. 
Available dose levels .......................................................................................... 44 
Table 7. 
Adverse reactions based on pooled dataset from 3 randomized studies  ............ 49 
Table 8. 
Measurable Disease - Overall Response ............................................................. 61 
Table 9. 
Non-Measurable Disease - Overall Response ..................................................... 62 
Table 10. Confidence Intervals (CI) for AE Rates (%) in Experimental Group  ................ 76 
 
 


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1. 
Protocol Summary and Schema 
 
Title 
A Phase II Study of Palbociclib plus Fulvestrant for pretreated patients 
with ER+/HER2- Metastatic Breast Cancer (PYTHIA trial) 
Sponsor 
International Breast Cancer Study Group (IBCSG) 
Pharma 
Partner 
Pfizer 
Clinical phase 
Phase II 
Population 
Patients with endocrine-resistant metastatic or locally relapsed, 
ER+/HER2-negative breast cancer not amenable to treatment with a 
curative intent enrolled in the AURORA study.  
Treatment 
Patients will receive: 
 Palbociclib plus fulvestrant 
Fulvestrant 500mg will be administered intramuscularly on days 1 and 
15 of cycle 1, then on day 1 of every 28 days cycle (+/- 3 days). 
Palbociclib will be administered orally at a dose of 125 mg per day 
continuously for 3 weeks followed by 1 week off; repeated at each 
subsequent cycle of 28 days. Patients will receive the treatment until 
progression, lack of tolerability, or patient declines further protocol 
treatment. 
 
 


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Trial Schema 
Rationale 
This is an international, multicenter, prospective single arm Phase II 
biomarker discovery clinical trial with the primary objective of assessing 
the association of PFS with gene mutations, gene copy number aberrations 
and gene signatures in post-menopausal women with hormone receptor 
positive, HER2-negative metastatic or locally relapsed breast cancer whose 
disease has progressed after prior adjuvant endocrine therapy or one line of 
systemic 
treatment, 
i.e., 
endocrine 
treatment 
or 
chemotherapy, 
administered for metastatic disease. This trial is included in the AURORA 
program conducted by the Breast International Group (BIG), an 
international study aiming to collect and characterize biological samples, 
including metastatic tissue, from patients with advanced breast cancer. 
Detailed molecular information (including somatic mutations, copy 
number variation, gene expression profiling and circulating biomarkers), as 
well as functional image data for a subset of patients, will be available to 
identify putative biomarkers of response to the trial regimen. 
Palbociclib is a potent, highly selective, reversible, orally active, inhibitor 
of cyclin dependent kinases 4 and 6 (CDK 4/6). This compound prevents 
progression of the cell cycle from G1 into the S phase, therefore inhibiting 
cell growth. Estrogen receptor is a key driver of the proliferation and 
growth of breast cancer cells and it is a well-known predictive factor for 
endocrine therapy response. The proliferative program driven by the 
estrogen receptor requires the transcriptional activation of the Cyclin D1 
gene leading to formation of the cyclin D1-CDK4/6-Rb complex, which 
∙∙∙until  PD
MBC, ER pos, Her2 neg, 
endocrine 
pretreated, 1st or 2nd line
15
28
day 1
Palbociclib (p.o)
Fulvestrant (i.m)
Substudy 30 patients: FDG-PET
EoT
+ 30 days 
CT scan or MRI (every 12 weeks until PD)
cycle 3
day 1
cycle 4
day 1
cycle 5
day 1
cycle 6
day 1
cycle 7
day 1
∙∙∙
∙∙∙
Biological samples for translational research through AURORA:
Mandatory (if available): FFPE blocks from primary tumor and metastatic lesion
Mandatory: Whole blood sample for pharmacogenomics and ctDNA
Mandatory: Serial plasma & serum samples for biomarker analysis: at screening, every 6 months and at progression
Serum
for TK1 
Enrollment  in PYTHIA
And
Registration in
AURORA program and 
molecular 
characterization


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facilitates the G1 to S phase transition.  
The introduction of cyclin-dependent kinases 4 and 6 (CDK4/6) inhibition 
in conjunction with endocrine treatment represents an important treatment 
advancement for patients with hormone receptor (HR) positive, human 
epidermal growth factor receptor 2 (HER2) negative metastatic breast 
cancer (O’Leary B, 2016). In particular, when palbociclib was administered 
in combination with letrozole to treat patients with newly diagnosed 
metastatic breast cancer in the randomized phase II PALOMA-1 study, 
progression-free survival (PFS) was significantly improved compared with 
that in patients treated with letrozole alone (10.2 months (95% CI 5.7-12.6) 
for the letrozole group and 20.2 months (13.8-27.5) for the palbociclib plus 
letrozole group, hazard ratio 0.488, 95% Confidence Interval, CI, 0.319-
0.748; one-sided p=0.0004) (Finn RS, 2015). Additionally, in the 
PALOMA-3 randomized phase III trial, the efficacy of fulvestrant plus 
palbociclib was compared with that of fulvestrant plus placebo in 521 
premenopausal and postmenopausal patients with HR-positive, HER2-
negative metastatic breast cancer whose disease had relapsed or progressed 
with previous endocrine therapy. The combination of fulvestrant plus 
palbociclib was associated with longer PFS than was fulvestrant plus 
placebo (9.2 versus 3.8 months respectively, hazard ratio for disease 
progression or death, 0.42; 95% CI, 0.32 to 0.56; P<0.001) (Turner NC, 
2015). Abemaciclib, another potent selective CDK4/6 blocking agent, has 
been recently granted Food and Drug Administration (FDA) breakthrough 
therapy designation for patients with pretreated HR-positive metastatic 
breast cancer, on the basis of antitumor activity exerted as monotherapy in 
a phase I study that reported a clinical benefit rate (CBR) of 61.1% in 
patients with heavily pretreated metastatic breast cancer (Patnaik A, 2016). 
The above mentioned results substantiate the notion that the CDK4/6 
blockade will become an essential treatment option in the therapeutic 
armamentarium for patients with advanced luminal breast cancer. 
Nevertheless, a particular subgroup of patients who are likely to respond 
better than others to the combination of CDK4/6 blockade with endocrine 
treatment has not been identified. Indeed, the attempts to the present day to 
identify predictive biomarkers to guide patient selection for this type of 
systemic treatment have failed. Preclinical evidence had shown that 
increased expression of cyclin D1 and phosphorylated Rb and decreased 
expression of p16 were associated with response to palbociclib (Finn RS, 
2009). On the basis of this data, the 165 patients in the PALOMA-1 trial 
were enrolled into two cohorts, one of which included patients with 
amplification of cyclin D1 or loss of p16, or both; no predictive relevance 
of these aberrations was found (Finn RS, 2015). Similarly, attempts to 
correlate the efficacy of the fulvestrant plus palbociclib combination with 
either the degree of endocrine sensitivity assessed clinically (i.e., previous 
endocrine response) or pathologically (i.e., levels of expression of estrogen 


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and progesterone receptors, ER and PgR respectively) or to PIK3CA 
mutational status failed within the context of the PALOMA-3 trial 
(Cristofanilli M, 2016). 
Fulvestrant is a potent antiestrogen drug that binds to and induces 
degradation of ER. Recent data with fulvestrant administered at the dose of 
500 mg/month to patients with recurrent hormone receptor positive, HER2-
negative breast cancer indicate significant antitumor activity after both 
antiestrogen and aromatase inhibitor (AI) failures. Fulvestrant is currently 
indicated for the treatment of postmenopausal women with metastatic 
hormone receptor positive breast cancer following the failure of 
antiestrogen therapy.  
The growing data about the activity of fulvestrant after antihormonal 
therapy failure together with emerging efficacy data of palbociclib support 
their combined use in women with hormone receptor positive, HER2-
negative metastatic breast cancer whose disease has progressed after prior 
endocrine therapy.  
The primary aim of the PYTHIA study is to discover potentially innovative 
biomarkers for the selection of patients to palbociclib/fulvestrant treatment. 
The strength of the trial lies in its conduct in conjunction with the 
AURORA study, which systematically evaluates a panel of biomarkers in 
tissue and blood, in a certified central lab (Zardavas D, 2014). Stemming 
from this association, an abundance of molecular profiling information will 
become available for different biological samples. Additional molecular 
and functional imaging assessments performed within the context of the 
PYTHIA study increase its scientific merit, since it will represent a 
prospective, systematic effort to identify biomarkers for patient 
stratification, integrating several molecular profiling assessments. 
References: 
Cristofanilli M, Turner NC, Bondarenko I, et al. Fulvestrant plus 
palbociclib versus fulvestrant plus placebo for treatment of hormone-
receptor-positive, HER2-negative metastatic breast cancer that progressed 
on previous endocrine therapy (PALOMA-3): final analysis of the 
multicentre, double-blind, phase 3 randomised controlled trial. Lancet 
Oncol. 2016 Apr;17(4):425–39 
Finn RS, Dering J, Conklin D, et al. PD 0332991, a selective cyclin D 
kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen 
receptor-positive human breast cancer cell lines in vitro. Breast Cancer Res 
BCR. 2009;11(5):R77 
Finn RS, Crown JP, Lang I, et al. The cyclin-dependent kinase 4/6 inhibitor 
palbociclib in combination with letrozole versus letrozole alone as first-line 
treatment of oestrogen receptor-positive, HER2-negative, advanced breast 
cancer (PALOMA-1/TRIO-18): a randomised phase 2 study. Lancet Oncol. 


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2015 Jan;16(1):25–35 
O’Leary B, Finn RS, Turner NC. Treating cancer with selective CDK4/6 
inhibitors. Nat Rev Clin Oncol. 2016 Jul;13(7):417–30 
Patnaik A, Rosen LS, Tolaney SM, et al. Efficacy and Safety of 
Abemaciclib, an Inhibitor of CDK4 and CDK6, for Patients with Breast 
Cancer, Non-Small Cell Lung Cancer, and Other Solid Tumors. Cancer 
Discov. 2016 Jul;6(7):740–53 
Turner NC, Ro J, André F, et al. Palbociclib in Hormone-Receptor-Positive 
Advanced Breast Cancer. N Engl J Med. 2015 Jul 16;373(3):209–19 
Zardavas D, Maetens M, Irrthum A, et al. The AURORA initiative for 
metastatic breast cancer. Br J Cancer. 2014 Nov 11;111(10):1881–7 
 
Eligibility 
Inclusion criteria: 
 Histologically confirmed breast adenocarcinoma that is metastatic or 
locally relapsed disease not amenable to curative therapy. 
 Female gender 
 Age ≥ 18 years 
 Postmenopausal, defined as women with:  
- Prior bilateral surgical oophorectomy; or 
- Amenorrhea and age ≥ 60 years; or 
- Age < 60 years and amenorrhea for 12 or more consecutive months 
in the absence of alternative pathological or physiological cause 
(including chemotherapy, tamoxifen, toremifene, or ovarian 
suppression) and FSH and serum estradiol levels within the 
laboratory’s reference ranges for postmenopausal women. 
 Endocrine resistant disease, defined as one of: 
- Relapse while on adjuvant endocrine therapy; 
- Relapse within 12 months after completion of adjuvant endocrine 
therapy; 
- Progression of disease under first line endocrine therapy for 
metastatic and/or loco-regionally advanced breast cancer. 
Note: Patient may have received one prior chemotherapy for advanced 
or metastatic breast cancer.   
 ER positive and HER2-negative tumor, as assessed locally  
 Eastern Cooperative Oncology Group (ECOG) Performance Status  
0-1. 
 Measurable or non-measurable but evaluable disease according to 
RECIST 1.1. Bone only disease is allowed. Previously irradiated 
lesions are deemed measurable only if progression is documented at 


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the site after completion of radiation.  
 Written Informed Consent (IC) for screening procedures and trial 
participation must be signed and dated by the patient and the 
Investigator prior to screening. 
 Written informed consent to participate in the AURORA program of 
BIG. 
 The patient has been informed of and agrees to data transfer and 
handling, in accordance with national data protection guidelines. 
  Life expectancy >3 months. 
 Hematological status: 
- Absolute neutrophil count ≥ 1.5 × 109/L (without growth factor 
support); 
- Platelet count ≥ 100 × 109/L (no transfusion allowed within 2 weeks 
prior to assessment); 
- Hemoglobin ≥ 9 g/dL (transfusion permitted). 
 Hepatic status: 
- Serum total bilirubin ≤ 1.5 × upper limit of normal (ULN). In the 
case of known Gilbert’s syndrome, a higher serum total bilirubin (< 
3 × ULN) is allowed. 
- AST and ALT ≤ 2.5 × ULN; if the patient has liver metastases, ALT 
and AST must be ≤ 5 × ULN. 
 Glucose in normal range, or well-controlled diabetes defined as an 
HbA1c level ≤ 7.5%. 
 Renal status:  
- Creatinine ≤ 1.5 ×ULN or creatinine clearance > 60 ml/min. 
 International Normalized Ratio (INR) or Prothrombin Time (PT) ≤1.5 
× ULN unless patient is receiving anticoagulant therapy as long as PT 
or PTT is within therapeutic range of intended use of anticoagulant. 
 Ability to swallow oral medication. 
Exclusion criteria: 
 Prior use of fulvestrant or any CDK inhibitor. 
 More than one prior line of chemotherapy for metastatic or locally 
relapsed disease. 
 Previous or current non-breast malignancies within the last 5 years, 
with the exception of in situ carcinoma of the cervix, and adequately 
treated basal cell or squamous cell carcinoma of the skin. 
 Known active uncontrolled or symptomatic CNS metastases, 
carcinomatous meningitis or leptomeningeal disease as indicated by 
clinical symptoms, cerebral edema, and/or progressive growth. 
Patients with a history of CNS metastases or cord compression are 
eligible if they have been definitively treated with local therapy (e.g., 
radiotherapy,) and are clinically stable off anticonvulsants and steroids 


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for at least 4 weeks before enrollment. 
 Any of the following in the previous 6 months: myocardial infarction, 
severe/unstable angina pectoris, ongoing cardiac dysrhythmias of NCI 
CTCAE grade ≥2, atrial fibrillation of any grade, coronary/peripheral 
artery bypass graft, symptomatic congestive heart failure (NYHA 
functional classification ≥ III), cerebrovascular accident including 
transient ischemic attack, or symptomatic pulmonary embolism. 
 QTc exceeding 480msec, family or personal history of long or short 
QT syndrome, Brugada syndrome or known history of QTc 
prolongation, or Torsade de Pointes (TdP). 
 Uncontrolled electrolyte disorders that can reinforce the QT-
prolonging effect of the drug (e.g., hypocalcemia, hypokalemia, 
hypomagnesemia). 
 Known history of HIV seropositivity. HIV screening is not required at 
baseline. 
 Uncontrolled diabetes defined as HbA1c level > 7.5%. 
 Concurrent disease or familial, sociological or geographical condition 
that would make the patient inappropriate for trial participation or any 
serious medical disorder that would interfere with the patient’s safety. 
 Dementia, altered mental status, or any psychiatric condition that 
would prevent the understanding or rendering of Informed Consent. 
 Known abnormalities in coagulation such as bleeding diathesis, or 
treatment with anticoagulants precluding intramuscular injections of 
fulvestrant. 
 Treatment with an investigational agent in the 4 weeks before 
enrollment. 
 Concurrent treatment with any of the drugs not permitted, i.e., strong 
CYP3A inhibitors/inducers and drugs known to cause QT interval 
prolongation. 
 Adverse events (except alopecia) from previous systemic cancer 
therapy, radiotherapy or surgery have not recovered to CTCAE v4.0 
grade 1 or resolved prior to enrollment. 
Trial 
Objectives and 
Endpoints 
Primary objective 
The primary objective of this single-arm phase II trial is to interrogate in a 
prospective manner a series of different molecular aberrations, assessed at 
a central lab, as well as functional imaging data both at baseline and after 
one cycle of treatment for a subset of 30 patients; all those potential 
biomarkers will be assessed for their associations with the progression-free 
survival of patients receiving fulvestrant plus palbociclib treatment and 
they can be listed as follows: 
a. Gene mutations in a panel of genes assessed on ctDNA, as 
determined by the AURORA platform 


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b. Gene mutations and gene copy number aberrations of an extended 
panel of cancer-related genes measured on primary tumors 
and/or tissue from metastatic biopsies, as determined by the 
AURORA platform.  
c. Baseline levels of activity of TK1 as well as early changes in TK1 
levels measured during and after one cycle of therapy.  
d. Gene signatures as inferred by RNA sequencing that will be 
performed through the AURORA program. In particular, in 
addition to other gene signatures measuring the activational 
status of several oncogenic signaling pathways, the following gene 
signatures with potential predictive utility in terms of sensitivity 
to CDK4/6 blockade will be assessed, namely: 
i. A gene signature called ‘RBsig’ consisting of 87 genes that is 
indicative of loss-of-function of the Retinoblastoma (Rb) 
pathway.  
ii. A gene signature developed at Université libre de Bruxelles 
consisting of 11 genes that is indicative of the phosphorylation 
status of CDK4.  
Secondary objectives 
To evaluate: 
 Safety and tolerability, as documented according to NCI CTCAE v4.0. 
 Disease control, based on RECIST 1.1 criteria.  
FDG-PET 
substudy 
An FDG-PET substudy will be conducted on a subset of 30 patients from 
selected sites. While the substudy is too small to draw definitive 
conclusions, FDG-PET early response may be a promising tool particularly 
for clinical development of new drugs or combinations. 
Translational 
research 
From the AURORA program, detailed molecular information (including 
somatic mutations, copy number variation, gene expression profiling and 
circulating biomarkers) will be available. In particular, the following four 
types of biospecimens will be collected and analyzed: 
 Archived primary tumor samples  
 Metastatic tumor tissue  
 A plasma sample at baseline  
 A whole blood sample at baseline 
There will be a targeted gene sequencing for all biospecimens perfomed 
using a panel of 411 cancer related genes for the tissue samples and a panel 
of 27 cancer related genes for the plasma sample. For successful inclusion 
in the AURORA program, the successful sequencing of at least 2 out of 
these 3 biospecimens is required. 


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These will be correlated to the endpoints of the present trial to identify 
putative biomarkers of response to the trial regimen.  
In the AURORA program, a whole blood and a plasma sample for 
pharmacogenomics will be taken at the time of inclusion of the patient; 
plasma and serum samples will be taken every 6 months and at time of 
disease progression to monitor the disease and to better understand the 
contribution of ctDNA to the evolution of the disease. 
Translational research proposals not outlined in this protocol will be 
assessed by a trial-specific research committee for merit and feasibility. 
Statistical 
considerations 
The primary objective of this single arm phase II trial is to assess the 
association of the primary endpoint progression-free survival (PFS) with 
biomolecular markers. 120 patients will be included and treated with 
palbociclib plus fulvestrant.   
The median PFS of patients treated with palbociclib plus fulvestrant is 
expected to be in the range of 9 to 10.5 months, with about 67% of patients 
having documented PFS events (~80 events) at the time of analysis. For 
sample size and power considerations we take the simplified situation 
wherein PFS is compared between groups of patients with and without a 
certain biomarker. The table below shows the detectable hazard ratios with 
80% power for alternative scenarios in which all 120 patients have 
biomarker data or conservatively in which ~80% (N=90) patients have 
biomarker data; and on the basis of nominal two-sided α=0.05 level test and 
α=0.001 level test as a rough consideration of multiple testing. The sample 
size of 120 was selected to have 80% power to detect a HR of 2.0 for 
biomarker with 30-50% prevalence (two-sided α=0.05). 
 
 
Analyzable 
Sample Size 
2-sided 
α-level 
Biomarker Prevalence 
 
5% 
10% 
15% 
20% 
30% 
50% 
 
Detectable HR 
N=120 
0.05 
4.2 
2.85 
2.4 
2.2 
2.0 
1.9 
0.001 
8.3 
4.65 
3.65 
3.2 
2.75 
2.5 
N=90 
0.05 
5.25 
3.35 
2.75 
2.5 
2.2 
2.1 
0.001 
>10 
5.9 
4.5 
3.8 
3.2 
2.9 
Number of 
patients 
This trial will enroll 120 patients.   
Duration of 
trial 
Enrollment is expected to be completed within 12 months. The final trial 
analysis is expected 30-36 months after the inclusion of the first patient 
under the amendment. The end of the trial is defined as last patient last visit 
and is expected to be 36 months after enrollment of the first patient under 
amendment 1. 
 


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2. 
Trial schedule 
(See section 14 for detailed examinations schedule) 
 
≤28 days 
prior to 
enroll-
ment 
Day 1 of 
every 28 
days 
cycle 2 
Day 15 
of 
cycle 1 
Weeks 12, 
24 etc 
(every 12 
weeks) 
Within 30 
days after 
end of 
treatment 7 
Follow-up 
every 12 
weeks 
until PD 15 
at pro-
gression 
Informed consent 1 
x 
 
 
 
 
 
 
Medical history 
x 
 
 
 
 
 
 
Physical examination,  
ECOG PS, height, weight 3 
x 
x 
x 3 
 
x 
x 
 
Post-menopausal status 4 
x 
 
 
 
 
 
 
Baseline symptoms and 
adverse events 5 
x 
 
 
 
 
 
 
Adverse events 5 
 
x 
x 
 
x 
 
 
Concomitant medications 
continuously from 28 days prior to enrollment until 28 
days after treatment stop 
 
 
Treatment 
 
 
 
 
 
 
 
Fulvestrant 
 
x 
x 
 
 
 
 
Palbociclib 
 
x 6 
 
 
 
 
 
Laboratory tests 
 
 
 
 
 
 
 
Complete blood count 8 
x 
x 
√ 
 
x 
 
 
Biochemistry 9 
x 
x 
 
 
x 
 
 
Coagulation profile 10 
x 
√ 
√ 
 
√ 
 
 
Tumor Evaluation 
 
 
 
 
 
 
 
Clinical 11 
x 
 
 
x 
 
x 
x 
CT or MRI 11 
x 
 
 
x 
x 13 
x 
x 
Bone scan 11 
√ 
 
 
√ 
 
√ 
√ 
FDG PET 12 
√ 
 
 
√ 
 
√ 
√ 
Cardiac evaluation 14 
 
 
 
 
 
 
 
Electrocardiogram 
x 
√ 
 
 
x 
 
 
Research assay 
 
 
 
 
 
 
 
Serum/plasma sample 16 
(x) 
x 
x 
 
 
 
(x) 
Registration into 
AURORA 
x 
 
 
 
 
 
 
x = mandatory √ = if medically indicated 
Legend for trial schedule: 
1. Informed consent may be obtained earlier than within 28 days before enrollment. 
2. Cycle visits should take place every 28 days, +/- 3 days 
3. Physical examination should be performed according to local standards. Height needs to be 
recorded only at baseline. Day 15 of cycle 1: physical examination only. 
4. Postmenopausal, defined as  
- 
Prior bilateral surgical oophorectomy; or 
- 
Amenorrhea and age ≥ 60 years; or 
- 
Age < 60 years and amenorrhea for 12 or more consecutive months in the absence of alternative 
pathological or physiological cause (including chemotherapy, tamoxifen, toremifene, or 
ovarian suppression) and FSH and serum estradiol levels within the laboratory’s reference 
ranges for postmenopausal women 
5. Baseline symptoms and adverse events should be recorded from signature of informed consent to 
prior to start of treatment. Adverse events occurred during the previous cycle should be checked at 


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day 1 visit of next cycle, at end of treatment visit and up to progression. Serious adverse events will 
be collected from day of signature of informed consent until 28 days following the end of treatment. 
Treatment 
6. Dispense palbociclib to patient for next 3 weeks of this cycle. In case of toxicity, refer to section 
10.4 for guidance on delaying or reducing the dose of palbociclib. Collect and check patient diary 
of previous cycle; hand out patient diary for next cycle. 
7. End-of-treatment visit within 30 days after last dose, or at the time of decision to stop the trial 
treatment if the decision is taken >30 days after last dose 
Laboratory tests 
8. Hemoglobin, platelet count, white blood cell count including differential (absolute neutrophil 
count). To be repeated on day 1 of every treatment cycle prior to start of treatment, if not done 
within 3 days prior to this day. 
9. Biochemistry: serum potassium, HbA1c, glucose, sodium, calcium, LDH, ;  
Liver function tests: albumin, total bilirubin, ALT, AST, ALP, GGT;  
Kidney function tests: urea, creatinine. 
To be repeated on day 1 prior to start of treatment if not done within 3 days prior to this day 
10. Coagulation function: prothrombin time (PT), partial thromboplastin time (PTT), and international 
normalized ratio (INR). 
Tumor evaluation 
11. Clinical and radiological (by CT scan or MRI) tumor assessments will be performed at baseline 
(preferentially within 28 days, but at the maximum 42 days before enrollment), then every 12 weeks 
(+/- 2 weeks) from enrollment until progression. Bone scan will be performed if clinically indicated 
at the same time points.  
12. Per RECIST criteria, FDG-PET/CT is not foreseen for regular response assessments (see section 
13.2). The FDG-PET substudy will be done on a subgroup of patients from selected sites with an 
FDG-PET done at baseline, maximum 2 weeks prior to start of treatment, and after cycle 1, on 
day 25-28. 
13. To be performed if not done already within 30 days prior to this visit. 
Cardiac evaluation 
14. Electrocardiogram will be done at baseline, thereafter if medically indicated, and again at end of 
treatment visit. 
Follow-up schedule 
15. Follow-up visits should be scheduled every 12 +/- 2 weeks at same time as imaging for tumor 
evaluation and documentation of any anti-tumor therapy between EoT and tumor progression. in 
case trial treatment was stopped prior to objective disease progression, follow-up continues until 
progression is documented, or for a maximum of 12 months after treatment stop 
Research assay 
16. Two serum samples for TK1 assay should be taken on day 15 (+/- 2 days) of cycle 1 and between 
day 26 of cycle 1 and before any cycle 2 trial treatment, at the latest on day 35. Serum samples at 
baseline and at progression and marked as “(x)” are taken through the AURORA program, see note 
below. 
A plasma sample for ctDNA must be taken at baseline (= AURORA screening sample) 
Note on biological studies (see section 15) 
As part of AURORA, two formalin-fixed, paraffin-embedded (FFPE) tumor blocks, one from the 
original diagnosis of the primary breast cancer and one from the diagnostic core or excisional biopsy of 
a loco-regional or metastatic lesion will be available in the AURORA biobank.  
As part of AURORA, serum and plasma samples for biomarker analyses will be available in the 
AURORA biobank. They will be collected at screening into AURORA and every 6 months thereafter, 
as well as at time of tumor progression. 
 


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3. 
List of abbreviations 
AE 
Adverse event 
AESI 
Adverse event of special interest 
AI 
Aromatase Inhibitor 
ALP 
Alkaline phosphatase 
ALT 
Alanine transaminase 
ANC 
Absolute Neutrophil Count 
AST  
Aspartate transaminase 
BIG 
Breast International Group  
CDK 
Cyclin Dependent Kinase 
CI 
Confidence interval 
CNS 
Central Nervous System 
ctDNA 
Circulating tumor DNA 
CR 
Complete Response 
CRF 
Case Report Form 
CT 
Computed tomography 
CTCAE 
Common toxicity criteria for adverse events 
CYP3A 
Cytochrom P450 3A4 
DC 
Disease control  
DDPLS 
Dedifferentiated Liposarcomas 
DLT 
Dose-limiting toxicity  
DMC 
Data Management Center 
DSMC 
Data Safety Monitoring Committee 
ECG 
Electrocardiogram 
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
Electronic case report form 
EEA 
European Economic Area 
EORTC 
European Organization for Research and Treatment of Cancer 
EoT 
End of Treatment 
ER 
Estrogen receptor 
ERB 
Ethical Review Board 
ESR1 
Estrogen receptor 1 
FDG 
Fluoro-D-Glucose 
FFPE 
Formalin-fixed paraffin-embedded 
FISH 
Fluorescence in situ hybridization 
FLT 
Fluorothymidine 
FSH 
Follicle-Stimulating Hormone 
FSTRF 
Frontier Science and Technology Research Foundation 
GBM 
Glioblastoma 
GCP 
Good clinical practice 
GGT  
Gamma-Glutamyl Transferase 
HbA1c 
Glycated haemoglobin 
HDPE 
High Density Polyethylene 
HER2 
human epidermal growth factor receptor 2 
HIV 
Human Immunodeficiency Virus 
HR 
Hazard Ratio 
IB 
Investigator’s Brochure 
IBCSG 
International Breast Cancer Study Group 
IC 
Informed consent 
IC50 
Half Maximal Inhibitory Concentration 
ICH 
International Conference on Harmonization 


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iDF 
iDataFax 
IHC 
Immunohistochemistry 
INR 
International Normalized Ratio 
IMP 
Investigational Medicinal Product 
IRB 
Institutional Review Board 
LD 
Lethal Dose 
MBC 
Metastatic breast cancer 
MRI 
Magnetic resonance imaging 
MTD 
Maximum tolerated dose 
mTOR 
Mechanistic Target of Rapamycin 
NCI 
National Cancer Institute 
NE 
Not Evaluable 
NIMP 
Non-Investigational Medicinal Product 
NYHA 
New York Heart Association 
OHRP 
Office for Human Research Protection 
ORR 
Overall response rate 
PD 
Progressive Disease 
PET 
Positron Emission Tomography 
PFS 
Progression free survival 
PgR 
Progesterone receptor 
PI 
Principal Investigator 
PIS/IC 
Patient information sheet / informed consent 
PR 
Partial response 
pRb 
Phosphorylated Retinoblastoma 
 
PS 
Performance status 
PT 
Prothrombin Time 
PTT 
Partial Thromboplastin Time 
QD 
Once daily (Quaque Die) 
QTc 
Corrected QT Interval 
QTcF 
Corrected QT Interval using the Fridericia correction 
Rb 
Retinoblastoma 
RECIST 
Response Evaluation Criteria in Solid Tumors 
RP2D 
Recommended phase II dose 
SAE 
Serious adverse event 
SCID 
Severe Combined Immunodeficiency 
SD 
Stable disease 
shRNA 
Small hairpin RNA 
SPC 
Summary of Product Characteristics 
SPIM 
Standard Procedures Imaging Manual 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
SUV(max) 
Maximum Standardized Uptake Value 
TEV 
Tumor Evaluation Form 
TdP 
Torsade de Pointes 
ULN 
Upper limit of normal 
 
 


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4. 
Background and scientific rationale 
4.1. 
Breast cancer 
Breast cancer represents a major health concern worldwide, with approximately 1,000,000 
newly diagnosed cases each year[1]. To the present day, therapeutic decisions are being taken 
on the basis of the expression status of three biomarkers, namely: estrogen receptor (ER), 
progesterone receptor (PgR) and human epidermal growth factor receptor (HER2). Extensive 
efforts have been made to molecularly characterize this common disease, with several 
molecular subtypes being identified[2-6]. An extensive repertoire of pathogenic molecular 
aberrations driving tumor cell growth have been identified[7-11], with the hope being that 
therapeutic targeting of these aberrations will improve the clinical outcome of patients with 
breast cancer[12]. 
4.2. 
Molecular characterization 
Despite this molecular heterogeneity governing breast cancer, dysregulation of normal cell 
cycle control seems to be a ubiquitous feature to many different subtypes[13]. Under normal 
conditions the cell cycle is a tightly regulated step-wise process, consisting of distinct phases. 
One important step is the transition from G1 to S phase, regulated by the interaction between 
the cyclin dependent kinases (CDKs) and the cyclin proteins, with the former being 
serine/threonine kinases[14]. Shortly, CDK4 and CDK6 mediate hyperphosphorylation of the 
retinoblastoma (Rb) gene product in early G1. This results in Rb inactivation, with the 
consecutive release of transcription factors inducing progression to the S phase.  
4.3. 
CDK inhibition 
Accumulating preclinical evidence supports CDK inhibition as a promising therapeutic 
strategy for human breast cancer. Indeed, a wide number of molecular aberrations affecting 
CDKs and their corresponding cyclins have been identified. Cyclin D1 (CCND1) gene 
amplification and overexpression of the encoded cyclin D1 protein have been found in up to 
50% of breast cancer cases[15] and cyclin E over-expression correlates strongly with poor 
clinical outcome across all subtypes of early stage breast cancer[16], luminal breast 
cancer[17] and a cohort of untreated patients with HER2-overexpressing disease[18]. 
Additionally, dysregulation of the network of CDKs and cyclins has been shown to mediate 
trastuzumab resistance in HER2-overexpressing breast cancer cells and tumour xenograft 
models, through cyclin E overexpression[19]. 
Importantly, strong preclinical evidence supports that CDKs and cyclin network 
dysregulations mediate endocrine resistance: high expression of cyclin E2 (CCNE2) was 
strongly predictive of shorter distant metastasis-free survival following endocrine 
therapy[20]. In the same study expression of either cyclin E1 or E2 in T-47D breast cancer 
cells conferred acute antiestrogen resistance, suggesting that cyclin E overexpression 
contributes to the antiestrogen resistance of tamoxifen-resistant cells. Moreover, a different 
study in vitro showed that PD0332991 (palbociclib), a selective CDK4/6 inhibitor, shows 
increased antitumor activity preferentially in luminal ER positive human breast cancer cell 
lines[21]. In this study, palbociclib showed synergy with tamoxifen in ER-positive breast 
cancer cell lines.  


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Despite this striking clinical activity, the identification of predictive biomarkers of sensitivity 
and/or resistance to CDK inhibition for luminal breast cancer patients is of paramount 
importance. To this end, solid understanding of the cell cycle regulation by the CDKs-cyclins’ 
network and results generated by preclinical research can be valuable. In the study showing 
the preferential in vitro activity of palbociclib against luminal ER positive human breast 
cancer cell lines, potential biomarkers were identified: Rb and cyclin D1 were elevated and 
CDKN2A (p16) was decreased in the most sensitive cell lines[21]. Another study showed that 
chronic loss of Rb was specifically associated with emergence of a CDK4/6 independent state 
and ultimately resistance to palbociclib in a panel of breast cancer cell lines[22]. Loss of Rb 
has been associated with resistance to CDK inhibition in the setting of other cancer cell types, 
thus indicating that it could serve as a major biomarker of resistance.  
Loss of Rb function can be mediated by inactivating mutations that have been reported, albeit 
at low frequencies (0.4% and 3% for luminal A and B breast cancer cases respectively)[8]. 
However, additional mechanisms have been reported in human breast cancer resulting in Rb 
functional inactivation, namely: loss of heterozygosity at the RB1 gene locus[23], 
overexpression of cyclin D1 and/or amplification of the cyclin D1 gene (CCND1)[15] and 
gene silencing of the CDK-inhibitor p16ink4a contributing to the deregulation of Rb 
phosphorylation[24]. A way to capture the functional status of the Rb pathway is through gene 
expression profiling. Indeed, a gene signature of Rb loss consisting of 159 genes has been 
reported to be a negative prognostic factor in ER-positive breast cancer[25]. Such a gene 
signature could be of predictive value for patient selection that will respond to CDK 
inhibition.  
From the above mentioned information, it becomes clear that detailed molecular 
characterization of tumor samples will be of paramount importance to identify the molecular 
subgroup of luminal breast cancer patients that will derive benefit from the CDK inhibition 
therapeutic strategy. To this end, AURORA, the most recent research initiative of Breast 
International Group (B.I.G.) focusing on metastatic breast cancer offers a unique opportunity: 
By applying next generation sequencing for a panel of 411 cancer-related genes to blood, as 
well as both primary tumors and their matched metastatic lesions, AURORA will generate an 
abundance of molecular profiling information that can be mined to identify potential 
predictive biomarkers associated with sensitivity and/or resistance to the CDK4/6 inhibition 
plus fulvestrant treatment utilized in the present study 
4.4. 
Palbociclib 
4.4.1. 
In vitro data 
Palbociclib is a highly selective inhibitor of CDK4/cyclinD1 kinase activity (IC50 = 11 nM; 
Ki = 2 nM). Palbociclib is selective for CDK4/6, with little or no activity against a large panel 
of 274 other protein kinases including other CDKs and a wide variety of tyrosine and 
serine/threonine kinases. CDK6, another enzyme that also complexes with cyclin-D subunits, 
is also commonly expressed in mammalian cells and tumors. CDK6 is highly homologous to 
CDK4 and can perform the same function by phosphorylating Rb, thus potentially creating a 
redundant mechanism to promote cell cycle progression. Consequently, inhibition of both 
enzymes is necessary to ensure complete suppression of Rb phosphorylation and the greatest 


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possible spectrum of antitumor activity. Results indicate that palbociclib inhibits CDK6 with 
equivalent potency to CDK4. 
The only known natural substrate for CDK4/cyclinD1 is the retinoblastoma gene product, Rb. 
Specific CDK4 phosphorylation sites on Rb include serine-780 and serine-795. Therefore, the 
phosphorylation status of Rb at these specific sites in treated tumors can serve as an 
appropriate biomarker for target modulation by palbociclib. The IC50 for reduction of Rb 
phosphorylation at serine-780 in the MDA-MB-435 breast carcinoma cell line was 0.066 µM. 
Palbociclib was equally effective at reducing Rb phosphorylation at serine-795 in this tumor 
cell line with an IC50 of 0.063 µM. Similar effects on serine-780 and serine-795 
phosphorylation were obtained in the Colo-205 colon carcinoma cell line.   
Palbociclib inhibits cellular proliferation and prevents cellular DNA synthesis by preventing 
cells from entering S phase of the cell cycle. Palbociclib inhibited thymidine incorporation 
into the DNA of a panel of Rb-positive human breast, colon, and lung carcinomas, with IC50 
values ranging from 0.040 to 0.17 µM. Palbociclib was also effective in preventing cell cycle 
progression in human leukemias and in nontransformed human epithelial cells and fibroblasts 
and was equally effective in suppressing cell division in human tumor cell lines. 
A selective CDK4/cyclinD inhibitor should cause a specific accumulation of cells in G1, but 
have no effect on other phases of the cell cycle, in which cells should continue to progress 
and eventually decline in number. MDA-MB-453 breast carcinoma cells that were exposed 
to various concentrations of palbociclib for 24 hours show a significant increase in the 
percentage of cells in G1 in the presence of as little as 0.04 µM palbociclib with a concomitant 
decline in other phases of the cell cycle. Finally, to provide further evidence of the selectivity 
of palbociclib, the compound was tested against Rb-negative tumor cells, which should not 
be sensitive to a specific CDK4 inhibitor. Palbociclib was tested against the MDA-MB-468 
human breast carcinoma and the H2009 human non-small cell lung carcinoma, both of which 
have deleted Rb. The compound had no anti-proliferative activity on these cells when assayed 
at 3 µM (highest concentration tested), which is 1 to 2 orders of magnitude higher than the 
concentration necessary to inhibit Rb-positive tumor cells. 
Published reports demonstrate palbociclib produced a potent G1 cell cycle arrest and induced 
senescence in a panel of 16 Rb-positive glioblastoma (GBM) cell lines, whereas 5 Rb-
negative cell lines were resistant[26]. shRNA knockdown of Rb expression conferred 
resistance of GBM cells to palbociclib. Palbociclib was effective against intracranial GBM 
xenograft tumors, including those that had recurred after temozolomide therapy. Palbociclib 
combined favorably with radiation against GBM xenografts[26]. 
4.4.2. 
In vivo data 
A comprehensive survey of molecular alterations was conducted in 207 patient samples 
representing seven major subtypes of soft tissue sarcoma[27]. Among prominent alterations 
discovered were mutations in PIK3CA, TP53, and NF1, along with a complex pattern of 
amplification of chromosome 12q in 90% of dedifferentiated liposarcomas (DDPLS). The 
most strongly amplified gene among these 12q amplifications in DDLPS was CDK4. shRNA 
knockdown of CDK4 inhibited proliferation of two DDLPS cell lines, LPS141 and 
DDLS8817, as did treatment with palbociclib. 


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The MTD in SCID mice was 150 mg/kg/day when administered orally, once a day, for 14 
days. The MTD was defined as the highest dose that was nonlethal (<LD10). At the MTD on 
this regimen, palbociclib has significant antitumor efficacy against multiple human tumor 
xenograft models. The Colo-205 model is exquisitely sensitive to palbociclib. At doses as low 
as 12.5 mg/kg, a 13-day growth delay was obtained, indicating a 90% inhibition of tumor 
growth rate. Palbociclib was inactive against the H23 lung and the SW-620 colon carcinomas. 
The lack of response may be associated with the presence of oncogenic K-ras mutations in 
SW-620 and H23; none of the xenografts sensitive to palbociclib had such mutations. 
Further evidence that the anti-tumor activity observed in Rb-positive tumors is due to 
inhibition of CDK4/CDK6 protein kinase activity was obtained by testing palbociclib in the 
MDA-MB-468 breast carcinoma and the DU-145 prostate tumor models. These are Rb-
negative tumors; neither of which responded to this compound. The lack of efficacy in Rb-
negative tumors is consistent with the lack of anti-proliferative activity observed in vitro. 
Taken together, these results support the proposed mechanism of palbociclib (inhibition of 
CDK4/6-mediated Rb phosphorylation) and the specificity of the compound demonstrated in 
enzyme activity tests. 
Further studies investigated whether continuous daily dosing of palbociclib was needed for 
optimal efficacy. Four dosing schedules were employed against the MDA-MB-435 breast 
carcinoma model over 14 days of treatment, including continuous daily, every other day, 
every third day, and 3 courses of 3 days dosing followed by 4-day drug holidays. The design 
of this experiment was such that the total compound administered over the 2-week period was 
identical for each treatment schedule. The results show that a similar degree of efficacy was 
attained with all schedules, implying that an intermittent regimen is feasible without 
compromising activity. Similar experiments were conducted against the Colo-205 colon 
carcinoma model. Again, intermittent schedules were as efficacious as daily dosing, with 
tumor regressions occurring during all dosing regimens. 
During the 14-day treatment period employed for most of the efficacy experiments, no cures 
were documented, and the tumors grew back after therapy. It is possible that a tumor variant 
had selectively grown back and acquired resistance to the compound. To address this 
possibility, Colo-205 colon tumors that had initially significantly regressed in response to 
treatment with palbociclib were harvested and reimplanted into naive mice. After the tumors 
grew to 100 to 150 mg, these tumor-bearing mice were treated with palbociclib with a dose 
and dosing schedule identical to the original experiment. The tumors responded with equal 
sensitivity to the drug and fully regressed, indicating that no resistance had developed during 
the initial treatment. A similar result was observed with retreated MDA-MB-435 tumors. 
4.4.3. 
Prior phase I/II/III non-breast cancer trials 
The first in-human study of palbociclib was conducted among 33 patients with Rb-positive 
advanced solid tumors or non-Hodgkin's lymphoma refractory to standard therapy, exploring 
a 2 weeks on, 1 week off schedule with once daily administration (QD)[28]. Six patients had 
dose-limiting toxicities (DLT) (18%; four receiving 200mg QD; two receiving 225mg QD); 
the maximum tolerated dose (MTD) was 200mg QD. Treatment-related, non-hematological 
adverse events occurred in 29 patients (88%) during cycle 1 and in 27 patients (82%) 


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thereafter. Adverse events were generally mild to moderate. In terms of antitumor activity, 
there were 31 patients out of the total 33 that completed at least one post-treatment tumor 
assessment and were thus considered evaluable for response[28]. One patient with non-
seminomatous testicular cancer, previously treated with bleomycin, cisplatin and etoposide in 
the adjuvant setting, had a PR with palbociclib administered at 200 mg QD on Schedule 2/1. 
An additional nine patients (29%) experienced stable disease (SD) lasting ≥2 cycles. Of note, 
no patient with breast cancer was included in this first in-human trial. 
Another phase I study was conducted among 41 patients with Rb-positive advanced tumors 
to identify the DLT and MTD of palbociclib in six dose-escalation cohorts following a 
standard 3 + 3 design[29]. DLTs were observed in five patients (12%) overall; at the 75, 125, 
and 150 mg once daily dose levels. The MTD and recommended phase II dose of palbociclib 
was 125 mg once daily. Neutropenia was the only dose-limiting effect. After cycle 1, grade 3 
neutropenia, anemia, and leukopenia occurred in five (12%), three (7%), and one (2%) 
patient(s), respectively. The most common non-hematologic adverse events included fatigue, 
nausea, and diarrhea. In terms of antitumor activity, none of the 37 patients evaluable for 
response met RECIST guidelines for partial response [29]. However, 13 patients (35%) 
maintained SD for at least 2 cycles, with one cycle corresponding to a period of 3 weeks on 
followed by 1 week off, one among whom had breast cancer. SD lasted ≥4 cycles in 10 
patients (27.0%) and ≥10 cycles in 6 patients (16.2%). Regarding the latter, there was one 
patient with breast cancer receiving 50 mg once daily, with high levels (>80%) of Rb-positive 
cells.   
Subsequent studies focusing on specific cancer diagnoses have been performed and reported. 
A phase I pharmacodynamic study of palbociclib was conducted in 17 patients with relapsed 
mantle cell lymphoma, using 2-deoxy-2-[(18)F] fluoro-D-glucose (FDG) and 3-deoxy-
3[(18)F] fluorothymidine (FLT) positron emission tomography (PET) to study tumor 
metabolism and proliferation, respectively, in concert with pre- and on-treatment lymph node 
biopsies to assess Rb phosphorylation and markers of proliferation and apoptosis[30]. The 
patients received 125 mg of palbociclib monotherapy QD, 3 weeks on, 1 week off. Substantial 
reductions in the summed FLT-PET maximal standard uptake value [SUV(max)], as well as 
in Rb phosphorylation and Ki-67 expression, occurred after 3 weeks in most patients, with 
significant correlations among these end points. Concerning antitumor activity, 5 patients 
achieved PFS time exceeding 1 year (range, 14.9-30.1+ months), with 1 CR and 2 PRs (18% 
objective response rate; 90% confidence interval, 5%-40%). These patients demonstrated 
> 70%, > 90%, and ≥ 87.5% reductions in summed FLT SUV(max) and expression of pRb 
and Ki67, respectively, parameters necessary but not sufficient for long-term disease control.  
A phase II monotherapy trial has been conducted in molecularly selected patients with 
advanced liposarcoma[31]. A total of 30 patients with Rb-positive disease at the IHC level 
and CDK4 amplified as assessed by fluorescence in situ hybridization (FISH) received 200mg 
palbociclib QD for 14 consecutive days in 3-week cycles; 12-week PFS was the primary 
endpoint. Promising antitumor activity was noted, with 12-week PFS reaching 66% (90% CI, 
51% to 100%), median PFS being 18 weeks and one case of PR at 74 weeks[31]. In terms of 
toxicities, grade 3 to 4 events included anemia (17%), thrombocytopenia (30%), neutropenia 


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(50%), and febrile neutropenia (3%). Dose reduction for hematologic toxicity was required 
for 24% of patients. 
4.4.4. 
Prior trials in metastatic breast cancer 
In the setting of metastatic breast cancer, the final results of PALOMA-1 (also known as Study 
1003 and TRIO-18), a randomized phase II trial, were recently reported [32]. This study 
evaluated the palbociclib 125 mg QD for 3 weeks followed by 1 week off plus Letrozole 
combination as 1st line treatment for post-menopausal patients with ER-positive/HER2-
negative metastatic breast cancer as compared to Letrozole monotherapy. The study consisted 
of two parts: Part 1 enrolled patients with no further biomarker assessment besides 
ER+/HER2– biomarkers while Part 2 enrolled patients additionally screened for CCND1 gene 
amplification and/or loss of p16. The primary endpoint was investigator-assessed PFS defined 
as time from randomization to objective progression or death. Secondary endpoints included 
OR, OS, safety, and correlative biomarker studies. A total of 165 patients were randomized 
in PALOMA-1; 66 patients in Part 1 and 99 patients in Part 2. Baseline characteristics were 
balanced between treatment arms. The final analysis of primary endpoint showed a 
statistically significant improvement in PFS for the investigational arm (20.2 months) vs. the 
Letrozole arm (10.2 months) with hazard ratio (HR)=0.488 (95% CI: 0.319, 0.748) and 1-
sided p=0.0004[32]. The treatment effects were also demonstrated when Part 1 and Part 2 
were analyzed separately (HR=0.299 [95% CI: 0.156, 0.572]; 1-sided p=0.0001 for Part 1 and 
HR=0.508 [95% CI: 0.303, 0.853]; 1-sided p=0.0046 for Part 2). The OS analysis with 61 
events demonstrated a trend favoring the palbociclib plus Letrozole combination vs. Letrozole 
(37.5 months vs. 33.3 months, respectively; HR=0.813; p=0.2105). In terms of toxicities 
observed, the most common adverse events in the palbociclib plus Letrozole arm were 
neutropenia, leukopenia, fatigue, and anemia, consistent with the toxicity profile of 
palbociclib previously reported. Intriguingly, the benefit derived from the palbociclib and 
letrozole combination was mainly driven by the population not having a CCND1 gene 
amplification and/or loss of p16 (HR for the biomarker-positive and biomarker-negative 
patient population was 0.37 and 0.19, respectively, for the comparison of palbociclib plus 
letrozole versus letrozole arms)[32]. 
These data were recently confirmed in a larger, double-blind, randomized, phase III 
registration trial with the same design of PALOMA-1, the PALOMA-2 trial. In this trial, 666 
postmenopausal women with ER-positive, HER2-negative BC, who had not received prior 
treatment for advanced disease, were randomized in a 2:1 fashion to receive palbociclib plus 
letrozole or placebo plus letrozole. The primary endpoint was PFS, as assessed by the 
Investigators. Results showed that the median progression-free survival was 24.8 months 
(95% confidence interval [CI], 22.1 to not estimable) in the palbociclib plus letrozole group, 
as compared with 14.5 months (95% CI, 12.9 to 17.1) in the placebo plus letrozole group (HR, 
0.58; 95% CI, 0.46 to 0.72; p<0.001). Subgroup analyses did not reveal any group of patients 
selectively receiving benefit from the combination. Adverse events in PALOMA-2 confirmed 
the overall very favourable toxicity profile of palbociclib seen in the PALOMA-1 trial. The 
most common grade 3 or 4 adverse events were neutropenia (occurring in 66.4% of the 
patients in the palbociclib plus letrozole group vs. 1.4% in the placebo plus letrozole group), 
leukopenia (24.8% vs. 0%), anemia (5.4% vs. 1.8%), and fatigue (1.8% vs. 0.5%). Febrile 


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neutropenia was reported in 1.8% of patients in the palbociclib plus letrozole group and in 
none of the patients in the placebo plus letrozole group. Permanent discontinuation of any 
trial treatment as a result of adverse events occurred in 43 patients (9.7%) in the palbociclib 
plus letrozole group and in 13 patients (5.9%) in the placebo plus letrozole group [33]. 
In the setting of endocrine pre-treated metastatic breast cancer, results from the phase III 
PALOMA-3 trial have recently been published [34, 35]. This trial involved patients with ER-
positive/HER2-negative metastatic breast cancer that had relapsed or progressed during prior 
endocrine therapy. Patients were randomized in a 2:1 ratio favoring the combination arm, to 
receive palbociclib and fulvestrant or placebo and fulvestrant. Premenopausal or 
perimenopausal women were eligible and also received goserelin. Patients who had received 
one line of prior chemotherapy for the treatment of metastatic disease were eligible for this 
trial. A total of 521 patients were randomized in this study, 347 in the palbociclib and 
fulvestrant arm and 174 in the palbociclib and placebo arm. The majority of the patients had 
a post-menopausal status (79.3% in both arms) and received one or more prior lines of therapy 
for metastatic disease (patients who received no prior treatment for metastatic disease 
represented 24.2% and 25.9% of the trial population in the palbociclib+fulvestrant and the 
placebo+fulvestrant arms, respectively). The combination of fulvestrant plus palbociclib was 
associated with longer PFS than was fulvestrant plus placebo (9.2 versus 3.8 months 
respectively, hazard ratio for disease progression or death, 0.42; 95% CI, 0.32 to 0.56; 
P<0.001)[36] . Toxicities were mostly in line with previous data. The rate of discontinuation 
due to adverse events was 2.6% with palbociclib and 1.7% with placebo. Overall survival data 
are still immature and require longer follow up.  
A detailed quality of life analysis in patients participating in the PALOMA-3 study was 
recently published. This analysis showed that patients randomized in the palbociclib plus 
fulvestrant group reported a significant improvement in the global quality of life scores. Also, 
a greater improvement in pain from baseline was observed in this group with a significantly 
delayed deterioration in global quality of life and pain compared with fulvestrant alone [37]. 
4.4.5. 
Prior Pharmacokinetic Studies 
In the aforementioned first in-human phase I trial, additional pharmacokinetic experiments 
were performed: Palbociclib was slowly absorbed (mean Tmax 4.2h) and eliminated (mean 
half-life time 26.7h). Volume of distribution was large (mean 3241 L) with dose-proportional 
exposure[28]. In the second phase I dose-finding study, similar pharmacokinetic 
characteristics were observed: Palbociclib was slowly absorbed (median Tmax was found to 
be 5.5 hours), and slowly eliminated (mean half-life was 25.9 hours) with a large volume of 
distribution (mean, 2,793 L)[29]. The area under the concentration–time curve increased 
linearly with dose.    
4.5. 
Fulvestrant 
Fulvestrant is a potent anti-estrogen drug that binds to and induces degradation of ER. Recent 
data with fulvestrant administered at the dose of 500 mg/monthly to patients with recurrent 
hormone receptor positive, HER2-negative breast cancer, indicate significant antitumor 
activity after both antiestrogen and aromatase inhibitor (AI) failures[38]. Fulvestrant is 
currently indicated and remains the standard of care for the treatment of postmenopausal 


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women with metastatic hormone receptor positive breast cancer following the failure of 
antiestrogen therapy.  
4.6. 
Biomarkers for palbociclib activity 
Currently, there is a lack of biomarkers to identify patients more likely to achieve a response 
to CDK4/6 inhibitors. Efforts have been made towards the identifications of single biomarkers 
in the CyclinD1/CDK4-6/RB1 pathway itself or in other co-activating pathways such as the 
PI3K pathway, but have proven unsuccessful so far. This suggests that single biomarkers may 
not capture the complex biology of resistance to CDK4/6 inhibitors and that a more thorough 
evaluation of the pathway activity may be necessary. 
Genetic loss of RB1 is a marker of primary resistance to CDK4/6 inhibitors but it is 
uncommon in HR+ or HER2+ subtypes. Recent studies have shown that functional loss of the 
CyclinD1/CDK4-6/Rb pathway can be measured by gene-expression studies. Several gene-
signatures of RB-loss have been developed which have shown to be prognostic in breast 
cancer subtypes as well as predictive of response to neoadjuvant chemotherapy [39, 40]  
The above mentioned results substantiate the notion that the CDK4/6 blockade will become 
an essential treatment option in the therapeutic armamentarium for patients with advanced 
luminal breast cancer. Nevertheless, a particular subgroup of patients who are likely to 
respond better than others to the combination of CDK4/6 blockade with endocrine treatment 
has not been identified. Indeed, the attempts to the present day to identify predictive 
biomarkers to guide patient selection for this type of systemic treatment have failed. 
Preclinical evidence had shown that increased expression of cyclin D1 and phosphorylated 
Rb and decreased expression of p16 were associated with response to palbociclib [21]. On the 
basis of this data, the 165 patients in the PALOMA-1 trial were enrolled into two cohorts, one 
of which included patients with amplification of cyclin D1 or loss of p16, or both; no 
predictive relevance of these aberrations was found [32]. Similarly, attempts to correlate the 
efficacy of the fulvestrant plus palbociclib combination with either the degree of endocrine 
sensitivity assessed clinically (i.e., previous endocrine response) or pathologically (i.e., levels 
of expression of estrogen and progesterone receptors, ER and PgR respectively) or to PIK3CA 
mutational status failed within the context of the PALOMA-3 trial [35].  
We have recently developed a gene signature of functional loss of Rb (RBsig) that is 
prognostic in luminal breast cancer subtypes and can predict response to the CDK4/6 inhibitor 
palbociclib in cell line models of breast cancer [41]. In particular, cell lines with increased 
levels of RBsig (RBsig HIGH) are among the most resistant to palbociclib treatment. 
4.7. 
Overall risk-benefit assessment 
The metastatic or advanced setting was chosen to evaluate this new therapy combination. The 
setting of incurable disease is acceptable to justify the evaluation of a potential new 
combination in breast cancer. As these patients are resistant to previous endocrine therapy, an 
investigational therapy that could potentially reverse this resistance is of high interest in this 
patient population. The available data provide scientific rationale for the combination of 
palbociclib and fulvestrant in this patient population. The tolerability of the combination in 
this population has been shown to be acceptable in the PALOMA-3 trial. Safety will be 
carefully monitored throughout the trial. 


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4.8. 
Rationale for the trial design 
This is an international, multicenter, phase II clinical trial for post-menopausal women with 
hormone receptor positive, HER2-negative metastatic or locally relapsed breast cancer whose 
disease has progressed after prior endocrine therapy (1st or 2nd line). Patients will be treated 
with the combination of palbociclib and fulvestrant.  The primary objective is to assess the 
association of the primary endpoint progression-free survival (PFS) with potential markers. 
This trial is included in the AURORA program, an international study aiming to collect and 
characterize biological samples, including metastatic tissue, from patients with advanced 
breast cancer. Therefore, for patients participating in the current trial, detailed molecular 
information (including somatic mutations, copy number variation, gene expression profiling 
and circulating biomarkers) will be available for retrospective studies aiming to identify 
putative biomarkers of response to the study regimen. 
Palbociclib is a potent, highly selective, reversible, orally active, inhibitor of cyclin dependent 
kinases 4 and 6 (CDK 4/6). This compound prevents progression of the cell cycle from G1 
into the S phase, therefore inhibiting cell growth. ER is a key driver of the proliferation and 
growth of breast cancer cells and it is a well-known predictive factor for endocrine therapy 
response. The proliferative program driven by the ER requires the transcriptional activation 
of the cyclin D1 gene leading to formation of the cyclin D1-CDK4/6-Rb complex which 
facilitates the G1 to S phase transition.  
The introduction of cyclin-dependent kinases 4 and 6 (CDK4/6) inhibition in conjunction with 
endocrine treatment represents an important treatment advancement for patients with 
hormone receptor (HR) positive, human epidermal growth factor receptor 2 (HER2) negative 
metastatic breast cancer [32, 36, 42]. Abemaciclib, another potent selective CDK4/6 blocking 
agent, has been recently granted Food and Drug Administration (FDA) breakthrough therapy 
designation for patients with pretreated HR-positive metastatic breast cancer, on the basis of 
antitumor activity exerted as monotherapy in a phase I study that reported a clinical benefit 
rate (CBR) of 61.1% in patients with heavily pretreated metastatic breast cancer [43]. 
The primary aim of the PYTHIA study is to discover potentially innovative biomarkers for 
the selection of patients to Palbociclib/Fulvestrant treatment. The strength of the trial lies in 
its conduct in conjunction with the AURORA study, which systematically evaluates a panel 
of biomarkers in tissue and blood, in a certified central lab [44]. Stemming from this 
association, an abundance of molecular profiling information will become available for 
different biological samples. Additional molecular and functional imaging assessments 
performed within the context of the PYTHIA study increase its scientific merit, since it will 
represent a prospective, systematic effort to identify biomarkers for patient stratification, 
integrating several molecular profiling assessments. 
At present, there is lack of data regarding predictive biomarkers to palbociclib in the clinical 
setting. Pre-clinical data suggest that aberrations in genes and proteins related to the CDK4-
6/Cyclin D1 pathway might be predictive of response to palbociclib, but clinical validation 
has failed so far to confirm these hypotheses analyzing primary tumor tissue samples. A major 
obstacle to the development of predictive biomarkers for metastatic breast cancer patients is 
the unavailability for molecular studies of tissue samples from the metastatic site where it is 
possible to capture the current biology of the disease. The present trial will provide an 


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important addition to the current knowledge in the field by providing extensive molecular 
characterization of the metastatic disease and of circulating biomarkers that might be better 
suited for retrospective biologic correlative studies. 
In its meeting of November 7, 2016, the Data and Safety Monitoring Committee (DSMC) for 
this trial discussed the currently available information about the safety and efficacy of the 
combination of palbociclib and fulvestrant in this patient population. The DSMC concluded 
that a randomization between palbociclib plus fulvestrant vs. placebo plus fulvestrant was not 
justified anymore, and that all patients should receive the active drug. Study Partners, i.e. 
IBCSG and BIG, have therefore decided to convert the trial into a single arm phase II. 
4.9. 
Rationale for the FDG-PET substudy 
Up to the present day, there has been a shortage of data regarding the evaluation of the clinical 
utility of FDG-PET (fluorodeoxyglucose positron emission tomography) for patients with 
advanced breast cancer, which is even more pronounced in the setting of treatment with 
targeted agents. There have been small studies with populations of less than 20 patients that 
reported promising results with regards to the predictive potential of FDG-PET among 
chemotherapy-treated patients with advanced breast cancer[45, 46]. 
A study assessing the early metabolic response to neoadjuvant letrozole as defined by FDG-
PET examination was conducted among 11 patients with hormone receptor positive early-
stage breast cancer. This study demonstrated the correlation between FDG-PET metabolic 
response at 4 weeks and change in tumor size as measured by ultrasound, pathological 
response, and change in Ki-67 labelling index as compared to lack of such an FDG-PET 
metabolic response[47]. Two additional studies provided further evidence supporting the 
notion that metabolic response assessed by FDG-PET in patients with metastatic breast cancer 
is predictive of the PFS[48, 49]; one among 22 patients with hormone receptor positive 
metastatic disease treated with endocrine therapy and the second among 25 patients with 
early-stage disease treated with neoadjuvant letrozole and bevacizumab. These studies 
showed that FDG-PET metabolic response was correlated with clinical and/or pathological 
outcomes. 
An exploratory sub-study of the NeoALTTO (Neoadjuvant Lapatinib and/or Trastuzumab 
Treatment Optimization) study, a neoadjuvant trial evaluating combinations of trastuzumab 
and/or lapatinib with paclitaxel among women with HER2-positive breast cancer, reviewed 
the role of FDG-PET as a possible biomarker in HER2-positive disease in 77 of the 455 
patients included in NeoALTTO[50]. Metabolic changes in the primary tumor in patients with 
invasive operable breast cancer treated with HER2 blockade were detected as early as after 
2 weeks of treatment. Additionally, pathological complete response (pCR) rates were twice 
as high in PET responders compared to non-responders according to EORTC criteria (41% 
versus 21% at week 2 and 43% versus 19% at week 6). While this substudy is too small to 
draw definitive conclusions, FDG-PET early response may be a promising tool particularly 
for clinical development of new drugs or combinations[51]. Additional evidence for a 
potential role of FDG-PET as a predictive biomarker has been generated for targeted agents 
as well, mostly assessing this role in the setting of mTOR inhibition in different tumor 
types[52-55]. 


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5. 
Trial objectives and endpoints 
5.1. 
Primary objective 
This is a single arm phase II trial for postmenopausal patients receiving fulvestrant and 
palbociclib for estrogen receptor positive (ER+) / human epidermal growth factor receptor 2 
negative (HER2-) metastatic or locally advanced breast cancer who have progressed under 
previous endocrine therapy. The primary objective is to interrogate in a prospective manner a 
series of potential biomarkers, which will be assessed for their association with PFS, 
specifically: 
a. gene mutations in a panel of genes assessed on ctDNA, as determined by the 
AURORA platform 
b. gene mutations and gene copy number aberrations of an extended panel of cancer-
related genes measured on primary tumors and/or tissue from metastatic biopsies, 
as determined by the AURORA platform.  
c. baseline levels of activity of TK1 as well as early changes in TK1 levels measured 
during and after one cycle of therapy.  
d. gene signatures as inferred by RNA sequencing that will be performed through the 
AURORA program. In particular, in addition to other gene signatures measuring the 
activational status of several oncogenic signaling pathways, the following gene 
signatures with potential predictive utility in terms of sensitivity to CDK4/6 
blockade will be assessed, namely: 
i. a gene signature called ‘RBsig’ consisting of 87 genes that is indicative of loss-
of-function of the Retinoblastoma (Rb) pathway[41].  
ii. A gene signature developed at Université libre de Bruxelles consisting of 11 
genes that is indicative of the phosphorylation status of CDK4.  
5.2. 
Primary endpoint 
Progression-free survival (PFS) is defined as time from treatment initiation until documented 
disease progression according to RECIST 1.1 criteria (Section 13.12) or death, whichever 
occurs first.  
5.3. 
Secondary objectives 
To evaluate: 
 
Safety and tolerability, as documented according to NCI CTCAE v4.0 
 
Disease control, based on RECIST 1.1 criteria 
Secondary endpoints are defined in section 18.2. 


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6. 
Trial design, duration and termination 
6.1. 
Trial design 
This is an international, multi-center, phase II trial that will enroll postmenopausal women 
with ER-positive, HER2-negative metastatic or locally relapsed breast cancer progressing 
under previous endocrine treatment to receive the combination of palbociclib plus fulvestrant..  
One line of prior chemotherapy treatment for advanced disease is allowed. 
An FDG-PET substudy will be conducted at selected sites on the potential association 
between metabolic response as assessed through FDG-PET and PFS for 30 patients enrolled 
in the present study. This FDG-PET evaluation will take place at baseline (max. 2 weeks prior 
to start of trial treatment), as well as at the end of week 4 (day 25-28). 
Patients have to be registered in the AURORA program, which means that tumor samples of 
both primary and metastatic disease (if available) are being secured and will be centralized in 
the AURORA bio-repository (Integrated Biobank of Luxembourg (IBBL), with registered 
office at 6, rue Nicolas Ernest Barblé, L-1210, Luxembourg).  
6.2. 
Trial schema 
 
 
6.3. 
Treatment 
Fulvestrant 500mg (2 injections of 250mg) will be administered intramuscularly on days 1 
and 15 of cycle 1, and then on day 1 (+/- 3 days) of each subsequent 28 days cycle. Palbociclib  
will be administered orally at a dose of 125 mg per day continuously for 3 weeks followed by 
∙∙∙until  PD
MBC, ER pos, Her2 neg, 
endocrine 
pretreated, 1st or 2nd line
15
28
day 1
Palbociclib (p.o)
Fulvestrant (i.m)
Substudy 30 patients: FDG-PET
EoT
+ 30 days 
CT scan or MRI (every 12 weeks until PD)
cycle 3
day 1
cycle 4
day 1
cycle 5
day 1
cycle 6
day 1
cycle 7
day 1
∙∙∙
∙∙∙
Biological samples for translational research through AURORA:
Mandatory (if available): FFPE blocks from primary tumor and metastatic lesion
Mandatory: Whole blood sample for pharmacogenomics and ctDNA
Mandatory: Serial plasma & serum samples for biomarker analysis: at screening, every 6 months and at progression
Serum
for TK1 
Enrollment  in PYTHIA
And
Registration in
AURORA program and 
molecular 
characterization


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1 week off; repeated at each subsequent cycle of 28 days. Patients will receive the treatment 
until progression, lack of tolerability, or patient declines further protocol treatment.  
6.4. 
Clinical evaluations 
Tumor response will be evaluated according to RECIST version 1.1 (see section 13) using 
computed tomography (CT) or magnetic resonance imaging (MRI) of the thorax, abdomen 
and pelvis. For each patient, the same technique must be used to evaluate each lesion through 
the trial. 
Tumor assessments will be performed at screening (prior to start of protocol therapy), and 
every 12 weeks from start of treatment until progressive disease is observed. Bone scans 
should be performed as clinically indicated. All patients who are discontinued from the 
protocol therapy for any reason other than disease progression will continue to have tumor 
assessments as per the schedule and until the patient has documented disease progression, for 
up to a maximum of 12 months. Any other radiological assessment should be performed as 
clinically indicated according to the medical judgment of the Investigator.  
6.5. 
Biological evaluations 
Patients that have been consented for the AURORA program for metastatic breast cancer, 
conducted by BIG, will be candidates for enrollment in the current trial. FFPE material from 
primary breast tumor and one metastatic lesion will be subjected to next generation 
sequencing for an extended panel of cancer-related genes through the AURORA program, 
making available extensive molecular background information.  
Blood samples for biomarker analyses will be taken through the AURORA program. In 
addition, a serum sample will be taken at two weeks and at the end of cycle 1 in view of the 
thymidine kinase-1 (TK1) assay DiviTum . 
6.6. 
Sample size and trial duration 
The trial will enroll a total of 120 patients.   
Patients will be enrolled by approximately 22 AURORA sites in Belgium, Italy and the United 
Kingdom. 
The enrollment is expected to be completed within 12 months after activation of Amendment 
1. Individual patients’ trial participation ends with the End of Treatment visit, or a maximum 
of 12 months after EoT in the absence of tumor progression (Section 10.6). Clinical visits are 
expected to span approximately 36 months after enrollment of the first patient. The final trial 
analysis is expected 30-36 months after the inclusion of the first patient under Amendment 1. 
The end of the trial is defined as last patient last visit and is expected to be 36 months after 
enrollment of the first patient under amendment 1. 
7. 
Patient selection 
7.1. 
Inclusion criteria  
7.1.1. 
Histologically confirmed breast adenocarcinoma that is metastatic or locally relapsed 


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disease not amenable to curative therapy. 
7.1.2. 
Female gender 
7.1.3. 
Age ≥ 18 years 
7.1.4. 
Postmenopausal, defined as women with:  
- Prior bilateral surgical oophorectomy; or 
- Amenorrhea and age ≥ 60 years; or 
- Age < 60 years and amenorrhea for 12 or more consecutive months in the absence 
of alternative pathological or physiological cause (including chemotherapy, 
tamoxifen, toremifene, or ovarian suppression) and FSH and serum estradiol 
levels within the laboratory’s reference ranges for postmenopausal women. 
7.1.5. 
Endocrine resistant disease, defined as one of: 
- Relapse while on adjuvant endocrine therapy; 
- Relapse within 12 months after completion of adjuvant endocrine therapy; 
- Progression of disease under first line endocrine therapy for metastatic and/or 
loco-regionally advanced breast cancer. 
Note: Patient may have received one prior chemotherapy for advanced or metastatic 
breast cancer.   
7.1.6. 
ER positive tumor and HER2-negative tumor, as assessed locally.  
7.1.7. 
Eastern Cooperative Oncology Group (ECOG) Performance Status 0-1 (see Table 1 
below). 
7.1.8. 
Measurable or non-measurable but evaluable disease according to RECIST 1.1. Bone 
only disease is allowed. Previously irradiated lesions are deemed measurable only if 
progression is documented at the site after completion of radiation.  
7.1.9. 
Written Informed Consent (IC) for screening procedures and trial participation must 
be signed and dated by the patient and the Investigator prior to screening. 
7.1.10. Written informed consent to participate in the AURORA program of BIG. 
7.1.11. The patient has been informed of and agrees to data transfer and handling, in 
accordance with national data protection guidelines. 
7.1.12.  Life expectancy >3 months. 
7.1.13. Hematologic status: 
- Absolute neutrophil count ≥ 1.5 × 109/L (without growth factor support), 
- Platelet count ≥ 100 × 109/L (no transfusion allowed within 2 weeks prior to 
assessment), 
- Hemoglobin ≥ 9 g/dL (transfusion permitted) 
7.1.14. Hepatic status: 


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- Serum total bilirubin ≤ 1.5 × upper limit of normal (ULN). In the case of known 
Gilbert’s syndrome, a higher serum total bilirubin (< 3 × ULN) is allowed. 
- AST and ALT ≤ 2.5 × ULN; if the patient has liver metastases, ALT and AST 
must be ≤ 5 × ULN. 
7.1.15. Glucose in normal range, or well-controlled diabetes defined as an HbA1c  
level ≤ 7.5%. 
7.1.16. Renal status:  
- Creatinine ≤ 1.5 ×ULN or creatinine clearance > 60 ml/min. 
7.1.17. International Normalized Ratio (INR) or Prothrombin Time (PT) ≤1.5 × ULN unless 
patient is receiving anticoagulant therapy as long as PT or PTT is within therapeutic 
range of intended use of anticoagulant. 
7.1.18. Ability to swallow oral medication. 
7.2. 
Exclusion criteria 
7.2.1. 
Prior use of fulvestrant or any CDK inhibitor. 
7.2.2. 
More than one prior line of chemotherapy for metastatic or locally relapsed disease. 
7.2.3. 
Previous or current non-breast malignancies within the last 5 years, with the 
exception of in situ carcinoma of the cervix, and adequately treated basal cell or 
squamous cell carcinoma of the skin. 
7.2.4. 
Known active uncontrolled or symptomatic CNS metastases, carcinomatous 
meningitis or leptomeningeal disease as indicated by clinical symptoms, cerebral 
edema, and/or progressive growth. Patients with a history of CNS metastases or cord 
compression are eligible if they have been definitively treated with local therapy 
(e.g., radiotherapy,) and are clinically stable off anticonvulsants and steroids for at 
least 4 weeks before enrollment. 
7.2.5. 
Any of the following in the previous 6 months: myocardial infarction, 
severe/unstable angina pectoris, ongoing cardiac dysrhythmias of NCI CTCAE grade 
≥2, atrial fibrillation of any grade, coronary/peripheral artery bypass graft, 
symptomatic congestive heart failure (NYHA functional classification ≥III, see 
Table 2 below), cerebrovascular accident including transient ischemic attack, or 
symptomatic pulmonary embolism. 
7.2.6. 
QTc exceeding 480msec, family or personal history of long or short QT syndrome, 
Brugada syndrome or known history of QTc prolongation, or Torsade de Pointes 
(TdP). 
7.2.7. 
Uncontrolled electrolyte disorders that can reinforce the QT-prolonging effect of the 
drug (e.g., hypocalcemia, hypokalemia, hypomagnesemia). 
7.2.8. 
Known history of HIV seropositivity. HIV screening is not required at baseline. 
7.2.9. 
Uncontrolled diabetes defined as HbA1c level > 7.5%. 


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7.2.10. Concurrent disease or familial, sociological or geographical condition that would 
make the patient inappropriate for trial participation or any serious medical disorder 
that would interfere with the patient’s safety. 
7.2.11. Dementia, altered mental status, or any psychiatric condition that would prevent the 
understanding or rendering of Informed Consent. 
7.2.12. Known abnormalities in coagulation such as bleeding diathesis, or treatment with 
anticoagulants precluding intramuscular injections of fulvestrant; see section 10.5.1 
for details. 
7.2.13. Treatment with an investigational agent in the 4 weeks before enrollment. 
7.2.14. Concurrent treatment with any of the drugs not permitted, i.e. strong CYP3A 
inhibitors/inducers and drugs known to cause QT interval prolongation; see section 
10.5.1 for details. 
7.2.15. Adverse events (except alopecia) from previous systemic cancer therapy, 
radiotherapy or surgery have not recovered to CTCAE v4.0 grade 1 or resolved prior 
to enrollment. 
 
Table 1. 
ECOG Performance Status 
PS 0 
Fully active, able to carry on all pre-disease performance without restriction. 
PS 1 
Restricted in physically strenuous activity but ambulatory and able to carry out work 
of a light or sedentary nature, e.g., light housework, office work. 
PS 2 
Ambulatory and capable of all self-care but unable to carry out any work activities. 
Up and about more than 50% of waking hours. 
PS 3 
Capable of only limited self-care, confined to bed or chair more than 50% of waking 
hours. 
PS 4 
Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair. 
 
Table 2. 
NYHA functional classification 
Class 
Functional Capacity: How a patient with cardiac disease feels during physical 
activity 
I 
Patients with cardiac disease but resulting in no limitation of physical activity. 
Ordinary physical activity does not cause undue fatigue, palpitation, dyspnea or 
anginal pain. 
II 
Patients with cardiac disease resulting in slight limitation of physical activity. 
They are comfortable at rest. Ordinary physical activity results in fatigue, 
palpitation, dyspnea or anginal pain. 
III 
Patients with cardiac disease resulting in marked limitation of physical activity. 
They are comfortable at rest. Less than ordinary activity causes fatigue, 
palpitation, dyspnea or anginal pain. 


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Class 
Functional Capacity: How a patient with cardiac disease feels during physical 
activity 
IV 
Patients with cardiac disease resulting in inability to carry on any physical 
activity without discomfort. Symptoms of heart failure or the anginal syndrome 
may be present even at rest. If any physical activity is undertaken, discomfort 
increases. 
8. 
Enrollment 
This trial will use a web-based enrollment system. Specific details for enrollment are in the 
“IBCSG Registration/Randomization Procedures Manual” which is available on the IBCSG 
website (www.ibcsg.org). 
8.1. 
Patient registration 
8.1.1. 
Verify that the patient has given written informed consent to the AURORA program. 
8.1.2. 
Patient needs to give informed consent to trial participation and screening procedures 
for the PYTHIA trial. 
8.1.3. 
Verify eligibility (see section 7). Screening procedures need to be done within 28 
days before enrollment (maximum within 42 days for imaging). 
8.1.4. 
Access the IBCSG Registration/Randomization System (Registration: Step 1) and 
provide the requested information as indicated on the Confirmation of Registration 
(53-A1) Form. The date the Informed Consent Form was signed by the patient and 
the date signed by the Investigator are both required to complete registration. 
The IBCSG Registration/Randomization System will provide the Patient ID 
(Registration Number) via e-mail. 
8.1.5. 
Submit the Confirmation of Registration (53-A) electronic case report form (eCRF) 
via iDataFax. 
8.2. 
Registration Help Desk 
The IBCSG Data Management Center (located at Frontier Science and Technology Research 
Foundation (FSTRF)) is responsible for developing and maintaining the IBCSG 
Registration/Randomization System. The Help Desk includes technical personnel and 
administrators of the registration programs at the Data Management Center in Amherst, NY, 
USA. 
The Help Desk is available around the clock 7 days per week, except for New Year's Eve, 
Memorial Day, Independence Day, Thanksgiving Day, Christmas Day. 
FSTRF Randomization Help Desk 
Frontier Science & Technology Research Foundation (FSTRF) 
4033 Maple Rd, Amherst, NY 14226 USA 
Phone: +1 716 834 0900 Extension 7301 
Fax: +1 716 832-8437 
Email: bc.helpdesk@fstrf.org  
 


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8.3. 
AURORA Registration Confirmation 
Participation in the PYTHIA trial requires co-registration in the AURORA Trial.   
8.3.1. 
Register the patient to the AURORA program and obtain the AURORA Patient ID 
Number (for details on the AURORA registration procedures, please refer to the 
AURORA protocol). 
8.3.2. 
Complete the AURORA Registration Confirmation Form (Form 53-AUR).  The 
AURORA Patient ID Number, date of registration, and AURORA Center Code are 
required to complete the form. 
8.3.3. 
Submit this form via iDataFax within two weeks of enrollment to PYTHIA. 
9. 
Trial drugs formulation and handling 
Palbociclib is the Investigational Medicinal Product (IMP) used in this trial; IMP will be 
supplied. 
Following the marketing authorization for palbociclib in November 2016, and once available 
in the respective countries, commercial product labelled for clinical trial use may be supplied. 
Fulvestrant will also be supplied. 
Complete details of the trial drug logistics, distribution, packaging, labeling, storage and 
handling as well as accountability are described in a separate Drug Supply Manual. This 
document is available for reference by the pharmacist and trial personnel. 
9.1. 
Palbociclib 
9.1.1 Name and Chemical Information: 
Palbociclib (also known as PD-0332991) 
9.1.2 Chemical Structure:  
6-Acetyl-8-cyclopentyl-5-methyl-2-{[5-(1-piperazinyl)-2-pyridinyl]amino}pyrido[2,3-
d]pyrimidin-7(8H)-one 
9.1.3 Mechanism of Action: 
Selective inhibitor of the cyclin-dependent kinases CDK4 and CDK6 
9.1.4 Formulation 
Refer to the current version of the PD-0332991 Investigator’s Brochure and the SPC 
(Summary of Product Characteristics) for pharmaceutical formulation information. 
Palbociclib will be supplied as capsules containing 75 mg, 100 mg, or 125 mg equivalents of 
palbociclib free base. Vendor will supply the oral drug formulation to sites in High Density 
Polyethylene (HDPE) bottles containing 75 mg, 100 mg, or 125 mg capsules.  The capsules 
can be differentiated by their size and color (see below). 


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Table 3. 
Palbociclib capsule characteristics 
Dosage 
Capsule color 
Capsule length 
75 mg 
Sunset Yellow/Sunset Yellow 
17.3 mm length 
100 mg  
Caramel/Sunset Yellow 
19 mm length 
125 mg 
Caramel/Caramel 
21.2 mm length 
9.1.5 Packaging and labeling 
Clinical supplies will be affixed with a clinical label in accordance with regulatory 
requirements. 
9.1.6 Storage and handling 
Clinical supplies must be stored at 20°C to 25°C in a secure, limited-access location under the 
storage conditions specified on the label. The Investigator shall take responsibility for and 
shall take all steps to maintain appropriate records and ensure appropriate supply, storage, 
handling, distribution and usage of investigational product in accordance with the protocol 
and any applicable laws and regulations.  
9.2 Fulvestrant 
9.2.1 Presentation 
Fulvestrant is used in accordance with the currently approved SPC and regarded as non-
investigational medicinal product (NIMPs), in accordance with the applicable EU legislation. 
Commercial fulvestrant (Faslodex®) will be supplied.  
Each vial contains 250 mg. 
9.2.2 Storage and handling 
Store fulvestrant in refrigerated conditions (2°C – 8°C). All drugs will be stored and handled 
as per the current version of the product’s SPC and the standard hospital procedures. 
Pharmacy will maintain temperature logs of all storage conditions and comply with hospital 
pharmacy standard operating procedures.  
10 Treatment 
10.1 
Trial treatments 
Trial treatment should start within one week after enrollment. Trial treatments will be 
administered in 4-week (28-day) cycles until progression, lack of tolerability, or until further 
protocol treatment is declined (see section 10.6). Fulvestrant can be continued in case of 
palbociclib cessation due to toxicity. If fulvestrant has been ceased for whatever reasons, 
palbociclib has to be stopped as well. 


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Treatment administration should comply with the protocol; compliance will be monitored by 
the Monitoring Team or Data Management Center. Complete details of dispensation and 
dosing are recorded on the eCRF.  
10.2 
Treatment Administration 
10.2.1 Fulvestrant administration 
Fulvestrant will be administered  
 500 mg intramuscularly on days 1 and 15 of cycle 1, and then  
 500 mg intramuscularly on day 1 of each subsequent 28 day cycle (+/- 3 days). 
Fulvestrant 500 mg will be administered intramuscularly into the buttocks slowly (1-2 
minutes per injection) as two 250 mg / 5 mL injections, one in each buttock.  
Planning the time of fulvestrant administration (e.g., time of the week for first administration; 
time of the day for each administration) should take trial visit procedures into consideration. 
10.2.2 Palbociclib administration 
Palbociclib will be administered orally at a dose of 125 mg per day continuously dosed for 3 
weeks followed by 1 week off; repeated at each subsequent cycle. The duration of a cycle of 
treatment is 28 days. A deviation of 1-3 days from this schedule is permitted. On treatment 
day 1, patients will be provided with trial treatment for self-administration at home. Sufficient 
capsules should be provided to cover administration until next scheduled visit. The patients 
will be instructed to take the trial treatment exactly as prescribed, promoting compliance. All 
dosages prescribed and dispensed to the patient and all dose changes, if applicable, during the 
trial must be recorded on the Treatment Form (53-PT). 
Patients should be instructed to take the palbociclib with food. Concomitant intake of proton 
pump inhibitors does not significantly affect the overall exposure to palbociclib taken with 
food. Patients should be instructed to swallow palbociclib capsules whole and not to chew 
them prior to swallowing. No capsule should be ingested if it is broken, cracked, or otherwise 
not intact. Patients should be encouraged to take their dose at approximately the same time 
each day. Patients should be instructed to record daily administration of the trial drug in the 
Patient Diary, and to bring the diary to the clinic at each visit. The Investigator should keep a 
copy in the patient file. 
10.3 
Dose modifications and delays for fulvestrant 
Refer to standard of care guidance or current clinical practice or currently approved 
fulvestrant European SPC for patients receiving fulvestrant treatment. 
No dose adjustment for fulvestrant is permitted. A fulvestrant dose can be skipped a maximum 
of two times during the whole trial duration for an individual patient. In case a fulvestrant 
dose cannot be delivered for a third time or two times consecutively then the patient should 
definitely stop all trial treatment. Treatment delay for fulvestrant-related toxicities will be 
performed as per the Investigator’s best medical judgment, but by no more than 7 days. In the 
event of a toxicity requiring dosing delay of palbociclib, fulvestrant can also be delayed. 


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Fulvestrant should not be administered if the platelet count is <50’000/mm3, and should be 
delayed until satisfactory recovery of the platelet count, preferably by not more than 7 days.  
Every effort should be made to synchronize day 1 of palbociclib and fulvestrant 
administration. 
Patients discontinuing fulvestrant treatment are not allowed to receive palbociclib according 
to trial protocol.  
10.4 
Dose delays and modifications for palbociclib 
In the event of significant treatment-related toxicity, palbociclib dosing may be interrupted or 
delayed and/or reduced as described below. In the event of multiple toxicities, dose 
modification should be based on the worst toxicity observed. Patients are to be instructed to 
notify Investigators at the first occurrence of any adverse sign or symptom. 
Every effort should be made to synchronize day 1 of palbociclib and fulvestrant 
administration. 
Table 4. 
Palbociclib dose reduction/delay for hematological toxicities 
Note: Table is adapted from SPC table 2, and applies to all hematological adverse reactions 
except lymphopenia (unless associated with clinical events, e.g. opportunistic infections) 
ANC = absolute neutrophil count 
CTCAE 4.0 Grade 
Dose modification / delay 
Grade 1 or 2 
No dose adjustment is required 
Grade 3 
Day 1 of cycle: 
Withhold palbociclib, repeat complete blood count within 1 
week. When recovered to Grade ≤ 2, start next cycle at the 
same dose 
Day 14 of first 2 cycles: 
Continue palbociclib at current dose to complete cycle. 
Repeat complete blood count on day 21. 
Consider dose reduction in case of prolonged (> 1 week) 
recovery from Grade 3 neutropenia or recurrent Grade 3 
neutropenia in subsequent cycles.  
Grade 3 ANC 
(500 – <1000 / mm3) 
+ fever ≥ 38°C  
and/or infection 
Withhold palbociclib until recovery to Grade ≤ 2.  
Resume at next lower dose. 
Grade 4 
Withhold palbociclib until recovery to Grade ≤ 2.  
Resume at next lower dose. 


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Table 5. 
Palbociclib dose reduction/delay for non-hematological toxicities 
(table adapted from SPC table 3) 
CTCAE 4.0 Grade 
Dose modification / delay 
Grade 1 or 2 
No dose adjustment is required 
Grade ≥ 3 
Withhold until symptoms resolve to 
1. Grade ≤ 1 
2. Grade ≤ 2 (if not considered a safety risk for the patient)  
Resume at next lower dose 
 
Patients requiring more than two dose reductions will be allowed to receive 75mg/day for 2 
weeks after 2 weeks off trial treatment (if, per the Investigator’s judgment, such schedule is 
manageable and preferred; this must be discussed with IBCSG beforehand by contacting 
ibcsg53_pythia@fstrf.org). Taking palbociclib according to recommendation (i.e., with food) 
should be reinforced and confirmed. Alternatively, the patient will be discontinued from the 
trial and entered into the follow-up phase of the trial. All dose modifications/adjustments must 
be clearly documented in the patient's source notes and the Protocol Therapy Form 53-PT.  
Once a dose has been reduced for a given patient, all subsequent cycles should be administered 
at that dose level, unless further dose reduction is required. Dose re-escalation is not allowed. 
Patients discontinuing palbociclib treatment due to treatment-related toxicity may continue 
on the active treatment phase of the trial receiving fulvestrant monotherapy at the 
Investigator’s discretion.  
Table 6. 
Available dose levels 
Dose Level 
Palbociclib for 3 out of 4 weeks 
Fulvestrant on day 1 of every 28 
days cycle 
Starting dose 
125 mg/d 
2x 250 mg/injection 
-1 
100 mg/d 
2x 250 mg/injection 
-2 
75 mg/d* 
2x 250 mg/injection 
-3 (for 
selected cases 
only) 
Discontinue palbociclib treatment or consider schedule of 75mg/d 2 
weeks on / 2 weeks off. This must be discussed with IBCSG beforehand 
by contacting ibcsg53_pythia@fstrf.org.  
If 2 weeks on/2 weeks off is not tolerated, then discontinue palbociclib. 


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* Palbociclib dose de-escalation below 75 mg/d is not allowed but if considered appropriate the schedule may 
move to 75mg/day two weeks on followed by two weeks off. 
10.5 Concomitant therapy 
The following rules should be followed: 
 Anti-cancer (cytotoxic or endocrine therapy, other than fulvestrant provided 
within the trial protocol) or investigational therapy is not permitted while 
patients are on trial therapy.  
 Administration of radiotherapy concurrently with trial medications is not 
allowed, except for palliative radiotherapy as specified below:  
For patients with bone involvement, it is suggested to institute palliative 
radiotherapy before trial initiation if possible and clinically appropriate. However, 
palliative radiotherapy is allowed exclusively for the treatment of painful bone 
lesions provided that the lesions were known to be present at baseline and the 
Investigator clearly documents that the need for palliative radiotherapy is not 
indicative of disease progression. 
In view of the current lack of data about the interaction of palbociclib with 
radiotherapy, palbociclib treatment should be interrupted during palliative 
radiotherapy, stopping 1 day before and resuming treatment 1 week after. 
 Concomitant surgery: 
Caution is advised on theoretical grounds for any surgical procedures during the 
trial. The appropriate interval of time between surgery and palbociclib required to 
minimize the risk of impaired wound healing and bleeding has not been 
determined. Based on the available pharmacokinetic data, stopping palbociclib is 
recommended at least 7 days prior to elective surgery. Postoperatively, the 
decision to reinitiate palbociclib treatment should be based on a clinical 
assessment of satisfactory wound healing and recovery from surgery. 
 Hormone replacement therapy, topical estrogens (including any intra-vaginal 
preparations), megestrol acetate and selective estrogen-receptor modulators (e.g., 
raloxifene) are prohibited. 
 The following guidelines are to be followed with respect to administration of 
bisphosphonates and/or denosumab in patients enrolled in the trial: 
o Chronic concomitant bisphosphonate/denosumab therapy for the prevention of 
bone metastases is not permitted during the trial.  
o Bisphosphonate/denosumab therapy for the treatment of osteoporosis of any 
grade is permitted during the trial. 
o Bisphosphonate/denosumab therapy at the time of enrollment for the 
management of bone metastases is recommended as standard of care.    
 Other supportive or palliative measures are permitted while patients are on trial 
therapy, as per physician’s choice, including hematopoietic growth factors.  


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The use of other concomitant medication/therapy judged by the Investigator to be necessary 
for the care of the patient is permitted. The Investigator should instruct the patient to notify 
the trial site about any new medications she takes after the start of the trial drug. 
The exclusion criteria in section 7.2 describe other medications which are prohibited in this 
trial. There are no prohibited therapies during the post-treatment follow-up phase.  
10.5.1 Prohibited medications: 
The following treatments are prohibited throughout the duration of the active treatment phase: 
 Strong CYP3A inhibitors/inducers: Palbociclib is metabolized to multiple 
metabolites in a qualitatively similar manner in rat, dog and human liver 
microsomes. In vitro, palbociclib is primarily metabolized by CYP3A4 enzymes. 
Co-administration with drugs that are CYP3A inhibitors and inducers may change 
the plasma concentrations of palbociclib in humans.  
The concurrent use of CYP3A inhibitors, including amprenavir, atazanavir, 
boceprevir, clarithromycin, conivaptan, delavirdine, diltiazem, erythromycin, 
fosamprenavir, indinavir, itraconazole, ketoconazole, lopinavir, mibefradil, 
miconazole, nefazodone, nelfinavir, posaconazole, ritonavir, saquinavir, 
telaprevir, telithromycin, verapamil, voriconazole, and grapefruit, grapefruit juice 
or any product containing grapefruit, are not allowed in the trial. 
The concurrent use of CYP3A inducers, including carbamazepine, felbamate, 
nevirapine, phenobarbital, phenytoin, primidone, rifabutin, rifampicin, rifampin, 
rifapentin, and St. John’s wort, are not allowed in the trial. 
 Drugs known to cause QT interval prolongation are prohibited during the active 
treatment phase (refer to Error! Reference source not found.). 
 Anticoagulants: if patient is receiving anticoagulants, refer to the Faslodex Product 
information[56]. 
10.5.2 Medications not recommended 
The following treatments are not recommended throughout the duration of the active 
treatment phase. Alternative therapies should be considered whenever possible. 
 Moderate CYP3A Inducers: The concurrent use of moderate CYP3A inducers 
such as bosentan, efavirenz, etravirine, modafinil, and nafcillin, as well as 
dexamethasone, is not recommended. 
 CYP3A Substrates: Palbociclib and its oxidative metabolite, PF-05089326, 
demonstrated little or no inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C9, 
CYP2C19, or CYP2D6 enzyme activities and thus, showed low potential for CYP-
mediated pharmacokinetic drug interactions. However, palbociclib and PF-
05089326 caused time-dependent inhibition of CYP3A midazolam 1’-
hydroxylase and testosterone 6-hydroxylase activities with Ki and kinact values for 
PD-0332991 of 10 µM, 0.036 min-1 and 19 µM, 0.087 min-1 and for PF-05089326 
of 7.0 µM, 0.094 min-1 and 6.4 µM, 0.15 min-1, respectively. Therefore, 


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palbociclib and its metabolite may have the potential for pharmacokinetic drug 
interactions with compounds for which CYP3A-mediated metabolism constitutes 
the primary mechanism of clearance. While the clinical significance of this 
inhibitory effect is yet to be investigated, caution must be exercised in patients 
receiving palbociclib in combination with drugs that are predominantly 
metabolized by CYP3A. In particular, co-administration palbociclib with 
CYP3A4 substrates with narrow therapeutic index including, but not limited to 
alfentanil, aripiprazole, cyclosporine, ergotamine, fentanyl, halofantrine, 
pimozide, quinidine, sirolimus, tacrolimus, triazolam, astemizole, cisapride, and 
terfenadine are not recommended during the active treatment phase of the trial. 
Alternative therapies should be used when available. 
 Chronic immunosuppressive therapies should be avoided, including systemic 
corticosteroids. Steroids given for physiological replacement, as anti-emetics or 
inhaled as well as short course of oral/topical steroids given for allergic reactions 
or asthma flares are allowed. 
 The use of herbal medicine is not recommended during the active treatment phase. 
10.6 
Stop of trial treatment  
Duration of therapy will depend on individual response, evidence of disease progression and 
tolerance.  
The treatment of the individual patient will be discontinued in case of: 
 Disease progression according to RECIST 1.1. as defined in section 13. 
 Unacceptable adverse event(s). 
 Intercurrent illness that prevents further administration of trial treatment. 
 Patient demonstrates an inability or unwillingness to comply with the treatment 
regimen and/or trial requirements. 
 General or specific changes in the patient’s condition which render her unacceptable 
for further trial treatment in the opinion of the treating Investigator. 
 Patient withdraws consent to continue trial treatment. 
Note that in the unlikely case the patient does not fulfill AURORA screening requirements 
(due to failure of more than one sequencing of primary tumor tissue, metastatic tumor tissue 
and ctDNA), the patient continues trial treatment. 
Patients who discontinue treatment for any reason other than objective disease progression 
will continue to be followed every 12 weeks until documented disease progression, or in the 
absence of progression, for a maximum of 12 months, as per Section 14.6, and have the end 
of treatment visit as per Section 14.5. 
After the discontinuation of protocol therapy, future therapeutic decisions are at the discretion 
of the Investigator, with no restrictions. The End of Treatment (EoT) is defined as the last 
date that the patient has taken trial treatment. In the absence of tumor progression, the patient 
will continue to be followed for safety and efficacy after this time until documented disease 


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progression or a maximum of 12 months after stop of treatment. An end of treatment visit will 
be conducted within 30 days after EoT to report on any adverse events during this period 
(safety data).  
10.7 
Removal from the trial 
After a patient has been enrolled, she becomes part of the clinical trial population and cannot 
be removed from the trial for any reason other than a decision by the patient to decline any 
further participation with the trial requirements and/or to decline further collection of data 
(see Section 19.5). If the patient discontinues treatment for any of the reasons listed in the 
prior subsection other than disease progression, she should continue to be followed according 
to the protocol (see Section 10.6) and eCRFs should be completed as described in Section 
17.1. 
Patients who have been enrolled but never received any trial treatment for whatever reason 
(refusal, medical condition etc.) will have to be documented with an end of treatment visit 
(see section 14.5) and may be replaced. 
11 Safety 
11.1 
Adverse reactions to fulvestrant 
 
Adverse reactions by system organ class and frequency 
Infections and infestations  
Common  
Urinary tract infections  
Immune system disorders  
Common  
Hypersensitivity reactions  
Metabolism and nutrition disorders  
Common  
Anorexiaa 
Nervous system disorders  
Common  
Headache  
Vascular disorders  
Common  
Venous thromboembolisma, hot flushes  
Gastrointestinal disorders  
Very common  
Nausea  
Common  
Vomiting, diarrhea  
Hepatobiliary disorders  
Very common  
Increased hepatic enzymes (ALT, AST, ALP)a 
Common  
Elevated bilirubina 
Uncommon  
Hepatic failurec, hepatitisc, elevated GGT  
Skin and subcutaneous tissue disorders  Common  
Rash  
Musculoskeletal and connective tissue 
disorders  
Common  
Back paina 
Reproductive system and breast 
disorders  
Uncommon  
Vaginal moniliasis, leukorrhea, vaginal 
hemorrhage  
General disorders and administration 
site conditions  
Very common  
Astheniaa, injection site reactionsb 
Uncommon  
Injection site hemorrhage, injection site hematoma  
a Includes adverse drug reactions for which the exact contribution of Faslodex cannot be assessed due to the 
underlying disease. 
b The term injection site reactions does not include the terms injection site hemorrhage and injection site 
hematoma. 
c The event was not observed in major clinical studies (CONFIRM, FINDER 1, FINDER 2, NEWEST). The 
frequency has been calculated using the upper limit of the 95% confidence interval for the point estimate. 
This is calculated as 3/563 (where 563 is the number of patients in the major clinical studies), which equates 
to a frequency category of 'uncommon'. 


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11.2 
Adverse reactions to palbociclib 
Table 7, taken from the SPC table 4, reports the adverse reactions from the pooled dataset of 
3 randomized studies. The median duration of palbociclib treatment across the pooled dataset 
was 12.7 months. The adverse reactions are listed by system organ class and frequency 
category. Frequency categories are defined as: very common (≥1/10), common (≥1/100 to 
<1/10), and uncommon (≥1/1,000 to <1/100). 
The effect of palbociclib on the QT interval corrected for heart rate (QTc) interval was 
evaluated using time matched electrocardiogram (ECG) change from baseline and 
pharmacokinetic data in 77 patients with breast cancer. At the recommended dose, no 
palbociclib relevant effects on QT have been observed. 
Table 7. 
Adverse reactions based on pooled dataset from 3 randomized studies 
(N=872) 
System Organ Class 
 
Frequency 
 
 
Preferred Term 
All grades 
% 
Grade 3 
N (%) 
Grade 4 
N (%) 
Infections and infestations 
 
Very common 
 
 
Infections b 
 
 
54.7 
 
 
39 (4.5) 
 
 
6 (0.7) 
Blood and lymphatic system disorders 
 
Very common 
 
 
Neutropenia c 
 
 
Leukopenia d 
 
 
Anemia e 
 
 
Thrombocytopenia f 
 
Common  
 
 
Febrile neutropenia 
 
 
80.6 
45.2 
27.6 
19.0 
 
1.6 
 
 
55.3 
26.1 
4.4 
1.6 
 
1.1 
 
 
10.1 
0.6 
0.2 
0.3 
 
0.1 
Metabolism and nutrition disorders 
 
Very common 
 
 
Decreased appetite 
 
 
15.8 
 
 
0.8 
 
 
0.0 
Nervous system disorders 
 
Common 
 
 
Dysgeusia 
 
 
8.5 
 
 
0.0 
 
 
0.0 
Eye disorders 
 
Common 
 
 
Vision blurred 
 
 
Lacrimation increased 
 
 
Dry eye 
 
 
4.4 
5.7 
3.6 
 
 
0.1 
0.0 
0.0 
 
 
0.0 
0.0 
0.0 
Respiratory, thoracic and mediastinal 
disorders 
 
Common 
 
 
Epistaxis  
 
 
 
8.4 
 
 
 
0.0 
 
 
 
0.0 
Gastrointestinal disorders 
 
Very common 
 
 
Stomatitis g 
 
 
Nausea 
 
 
Diarrhea 
 
 
Vomiting 
 
 
28.9 
34.2 
24.5 
17.1 
 
 
0.7 
0.3 
1.0 
0.5 
 
 
0.0 
0.0 
0.0 
0.0 


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System Organ Class 
 
Frequency 
 
 
Preferred Term 
All grades 
% 
Grade 3 
N (%) 
Grade 4 
N (%) 
Skin and subcutaneous tissue disorders 
 
Very common 
 
 
Rash h 
 
 
Alopecia 
 
Common 
 
 
Dry skin 
 
 
16.5 
25.9 
 
9.4 
 
 
0.7 
Not applic. 
 
0.0 
 
 
0.0 
Not applic. 
 
0.0 
General disorders and administration 
site conditions 
 
Very common 
 
 
Fatigue 
 
Common 
 
 
Asthenia 
 
 
Pyrexia 
 
 
 
39.2 
 
12.8 
12.4 
 
 
 
2.3 
 
1.4 
0.1 
 
 
 
0.2 
 
0.0 
0.0 
Investigations 
 
Common 
 
 
ALT increased 
 
 
AST increased 
 
 
8.0 
8.6 
 
 
1.7 
2.5 
 
 
0.1 
0.0 
a Preferred Terms (PTs) are listed according to MedDRA 17.1. 
b Infections includes all PTs that are part of the System Organ Class Infections and 
infestations. 
c Neutropenia includes the following PTs: Neutropenia, Neutrophil count decreased. 
d Leukopenia includes the following PTs: Leukopenia, White blood cell count decreased. 
e Anemia includes the following PTs: Anemia, Hemoglobin decreased, Hematocrit decreased. 
f Thrombocytopenia includes the following PTs: Thrombocytopenia, Platelet count 
decreased. 
g Stomatitis includes the following PTs: Aphthous stomatitis, Cheilitis, Glossitis, 
Glossodynia, Mouth ulceration, Mucosal inflammation, Oral pain, Oropharyngeal 
discomfort, Oropharyngeal pain, Stomatitis. 
h Rash includes the following PTs: Rash, Rash maculo-papular, Rash pruritic, Rash 
erythematous, Rash papular, Dermatitis, Dermatitis acneiform, Toxic skin eruption. 
 
11.3 
Potential drug induced liver injury 
Abnormal values in aspartate transaminase (AST) and/or alanine transaminase (ALT) 
concurrent with abnormal elevations in total bilirubin that meet the criteria outlined below in 
the absence of other causes of liver injury are considered potential cases of drug-induced liver 
injury (potential Hy’s Law cases) and should always be considered important medical events. 
The threshold for laboratory abnormalities in the case of potential drug-induced liver injury 
depends on the patient’s individual baseline values and underlying conditions. Patients who 
present with the following laboratory abnormalities should be evaluated further to definitively 
determine the etiology of the abnormal laboratory values:  
 Patients with AST or ALT and total bilirubin baseline values within the normal 
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o who subsequently present with AST or ALT ≥3 times the upper limit of normal 
(× ULN)  
concurrent with  
o a total bilirubin ≥2 × ULN with no evidence of hemolysis and an alkaline 
phosphatase ≤2 × ULN or not available.  
 Patients with pre-existing AST or ALT baseline values above the normal range: 
o  AST or ALT ≥2 × the baseline values and ≥3 × ULN, or ≥8 × ULN (whichever 
is smaller) 
concurrent with  
o total bilirubin ≥2 × ULN and increased by 1 × ULN over baseline or ≥3 × ULN 
(whichever is smaller) with no evidence of hemolysis and an alkaline 
phosphatase ≤2 × ULN or not available 
The patient should return to the investigational site and be evaluated as soon as possible, 
preferably within 48 hours from awareness of the abnormal results. This evaluation should 
include laboratory tests, detailed history and physical assessment. In addition to repeating 
AST and ALT, laboratory tests should include albumin, creatine kinase, total bilirubin, direct 
and indirect bilirubin, gamma-glutamyl transferase, prothrombin time (PT)/INR, and alkaline 
phosphatase. A detailed history, including relevant information, such as review of ethanol, 
acetaminophen, recreational drug and supplement consumption, family history, occupational 
exposure, sexual history, travel history, history of contact with a jaundiced subject, surgery, 
blood transfusion, history of liver or allergic disease, and work exposure, should be collected. 
Further testing for acute hepatitis A, B, or C infection and liver imaging (e.g., biliary tract) 
may be warranted. All cases confirmed on repeat testing as meeting the laboratory criteria 
defined above, with no other cause for liver function test abnormalities identified at the time 
should be considered potential Hy’s Law cases irrespective of availability of all the results of 
the investigations performed to determine etiology of the abnormal liver function tests. Such 
potential Hy’s Law cases must be reported as SAEs. 
11.4 
Drug-drug interactions 
Drug interaction studies have not been conducted in humans. Palbociclib is metabolized in 
vitro primarily via CYP3A4. See sections 10.5.1 and 10.5.2 for prohibited and not 
recommended medications. 
The potential for a clinically significant drug-drug interaction between palbociclib and 
fulvestrant is expected to be very low. There are no known drug-drug interactions.  
Palbociclib is metabolized to multiple metabolites in a qualitatively similar manner in rat, dog 
and human liver microsomes. In vitro, palbociclib is primarily metabolized by Cytochrome 
P-450 (CYP) 3A4 enzymes. 
The routes of elimination for fulvestrant include combinations of different biotransformation 
pathways including oxidation, aromatic hydroxylation, conjugate with glucuronic acid and/or 
sulphate. Fulvestrant does not significantly inhibit any of the major CYP isoenzymes, 
including CYP 1A2, 2C9, 2C19, 2D6, and 3A4 in vitro. Studies of co-administration of 
fulvestrant with midazolam indicate that therapeutic doses of fulvestrant have no inhibitory 
effects on CYP 3A4 or alter blood levels of drug metabolized by that enzyme. According to 


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Fulvestrant product information, studies of interaction with rifampicin (CYP3A4 inducer) or 
ketokonazole (a potent inhibitor of CYP3A4) did not show any clinically relevant 
modification of fulvestrant PK. Therefore, dosage adjustment is not necessary in patients co-
prescribed CYP 3A4 inhibitors or inducers. 
Both fulvestrant and palbociclib display a favorable toxicity profile. Therefore, the likelihood 
that the combination of fulvestrant and palbociclib could cause clinically significant 
unexpected toxicities is low. 
The PALOMA 3 trial has yielded detailed information on adverse events under the 
combination of fulvestrant and palbociclib, and has shown a good tolerability of the 
combination. Toxicity will be carefully monitored during trial. 
12 Adverse event and serious adverse event reporting 
12.1 
Adverse event reporting 
The main criterion for tolerability is the occurrence of toxicities and adverse events. The 
severity and causality will be classified according to the NCI CTCAE Version 4.0. The 
CTCAE 
is 
available 
for 
downloading 
on 
the 
internet 
at 
http://evs.nci.nih.gov/ftp1/CTCAE/About.html. An interactive version can be found at 
http://safetyprofiler-ctep.nci.nih.gov/CTC/CTC.aspx. 
An adverse event is defined as any untoward medical occurrence that occurs from the first 
dose of trial medication until 28 days after all treatment discontinuation, regardless of whether 
it is considered related to a medication. 
Any grade of any observed adverse event should be reported on the Adverse Event Log (53-
AE) in iDataFax. Symptoms of the targeted cancer (if applicable) should not be reported as 
adverse events. 
12.1.1 Severity / intensity 
The adverse event severity grade provides a qualitative assessment of the extent or intensity 
of an adverse event, as determined by the Investigator or as reported by the patient. The 
severity grade does not reflect the clinical seriousness of the event, only the degree or extent 
of the affliction or occurrence (e.g., severe nausea, mild seizure), and does not reflect the 
relationship to trial drug. 
Severity grade for other adverse events not covered in the toxicity grading scale: 
Grade 1 = Mild – transient or mild discomfort; no limitation in activity; no medical 
intervention/therapy required 
Grade 2 = Moderate – mild to moderate limitation in activity, some assistance may be needed; 
no or minimal medical intervention/therapy required 
Grade 3 = Severe – marked limitation in activity, some assistance usually required; medical 
intervention/therapy required, hospitalization is possible 
Grade 4 = Life threatening – extreme limitation in activity, significant assistance required; 
significant medical intervention/therapy required, hospitalization or hospice care probable 


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Grade 5 = Death – the event results in death 
The term “severe” is often used to describe the intensity of a specific event (as in mild, 
moderate or severe myocardial infarction); the event itself, however, may be of relatively 
minor medical significance (such as severe headache). This criterion is not the same as 
“serious” which is based on patient/event outcome or action criteria associated with events 
that pose a threat to a patient’s life or functioning.   
Seriousness, not severity, serves as a guide for defining regulatory obligations. 
Note: 
 
Report the highest grade observed until resolution of the adverse event. In case the 
grade increases over time, update the grade only (Start date should remain the 
same).  
 
Baseline symptoms will be recorded on the Form 53-14 BAE and continuing events 
and worsening of grade during treatment have to be reported on the 53-14 AE Log. 
 
Any abnormal laboratory values grade ≥ 3 will have to be documented on the 53-14 
AE Log. 
 
AEs should not be reported in a narrative description, but rather by using the 
applicable CTCAE v4.0 term. 
12.1.2 Causality 
The Investigator must determine the relationship between the administration of trial drug(s) 
and the occurrence of an AE/SAE as Not Suspected or Suspected as defined below: 
Not suspected: 
The temporal relationship of the adverse event to trial drug(s) 
administration makes a causal relationship unlikely or remote, or 
other medications, therapeutic interventions, or underlying 
conditions provide a sufficient explanation for the observed 
event. 
Suspected: 
The temporal relationship of the adverse event to trial drug(s) 
administration makes a causal relationship possible, and other 
medications, therapeutic interventions, or underlying conditions 
do not provide a sufficient explanation for the observed event. 
12.1.3 Duration 
For both AEs and SAEs, the Investigator will provide a record of the start and stop dates of 
the event. 
12.1.4 Action taken 
The Investigator will report the action taken with trial drug(s) as a result of an AE or SAE, as 
applicable (e.g., discontinuation of trial drug(s)) and in case of an SAE report if concomitant 
and/or additional treatments were given for the event. 


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12.2 
Targeted adverse events 
The presence of the following AEs must be indicated on the Adverse Event Log (Form 53-
AE): 
 Neutrophil count decreased 
 Febrile neutropenia 
 Anemia 
 Platelet count decreased   
 Nausea  
 Diarrhea  
 Pulmonary embolism 
 Infections  
 Skin and subcutaneous tissue disorders 
12.3 
Adverse events of special interest (AESIs) 
Potential cases of drug-induced liver injury (see section 11.3) are considered AESIs and need 
to be notified to IBCSG immediately (i.e., within 24h), following the SAE reporting 
instructions described in Section 12.6, even if not fulfilling a seriousness criterion.  
12.4 
Otherwise reportable events 
Certain types of events, as identified below, are reportable to IBCSG under the reporting 
processes and requirements for SAEs, even if there is no associated adverse event. These are 
considered “otherwise reportable events” and generally reflect circumstances that could lead 
to an increased risk of an adverse event. Like an SAE, an otherwise reportable event is to be 
reported to IBCSG within 24 hours of awareness and followed up to determine outcome, 
including the later occurrence of an associated SAE. 
12.4.1 Pregnancy and lactation 
Pregnancy will be reported within 24h of awareness on the Pregnancy Form (53-PREG-
Initial) and on the Serious Adverse Event Form (53-SAE-A) in all cases.  
Exposure during lactation will be reported within 24h on the 53–SAE-A Form.  
In the event of pregnancy or lactation, all trial treatment must be discontinued.  
Follow-up of the pregnancy is mandatory until the outcome has been determined. Outcome 
will be reported on the Pregnancy Form (53-PREG-Outcome). 
12.4.2 Relevant overdose of IMP  
An overdose (accidental or intentional) with the IMP is an event suspected by the Investigator 
or spontaneously notified by the patient and defined as the intake of  
 more than 23 capsules during a cycle (e.g. two capsules on more than two days of a 
cycle, or more than two capsules during the week of break), or  
 more than 2 capsules on the same day. 
The overdose has to be reported within 24h on the 53–SAE–A Form. 


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12.5 
Serious adverse event (SAE)  
12.5.1 Definition 
An SAE is defined in general as any undesirable medical occurrence/adverse drug experience 
that occurs from signature of Informed Consent until 28 days after stopping all trial treatment 
that, at any dose, results in any of the following:  
 fatal (any cause) 
 life-threatening 
 requires or prolongs inpatient hospitalization 
 persistent or significant disability/incapacity 
 secondary (non-breast) malignancy 
 congenital anomaly or birth defect (including neonatal deaths) 
 constitutes an important medical event 
Important medical events are defined as those occurrences that may not be immediately life-
threatening or result in death, hospitalization, or disability, but may jeopardize the patient or 
require medical or surgical intervention to prevent one of the other outcomes listed above. 
Medical and scientific judgment should be exercised in deciding whether such an AE should 
be considered serious. 
After completion of trial treatments, report all SAEs beyond 28 days that are considered at 
least possibly related to previous trial treatment. Cases of second (non-breast) malignancies 
and congenital abnormalities are to be regarded as SAEs, regardless of whether they occur 
during or after trial treatment. These events should be reported during the whole trial duration 
on the Serious Adverse Event eCRFs (53–SAE–A and 53–SAE–B). 
A suspected unexpected serious adverse reaction (SUSAR) is an adverse event that is serious, 
related to the investigational drug and not listed as a known toxicity of the investigational 
drug in the Investigator’s brochure. All suspected unexpected serious adverse reactions 
judged by either the Investigator or IBCSG as the sponsor will be reported in accordance with 
applicable local regulations. 
12.5.2 Exceptions to the definition 
Hospitalizations occurring under the following circumstances are not considered to be serious 
adverse events:  
 elective surgery 
 occur on an outpatient basis and do not result in admission (hospitalization <24h) 
 are part of the normal treatment or monitoring of the studied treatment 
 progression of disease (by convention, clinical events related to the primary cancer 
being studied or to the primary cancer progression are not to be reported as SAEs, 
even if they meet any of the seriousness criteria from the standard SAE definition, 


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unless the event is more severe than expected and therefore the Investigator considers 
that their clinical significance deserves reporting). 
12.5.3 Causality assessment 
The Investigator needs to assess the relationship between protocol treatment and the 
occurrence of each SAE following the definitions in this table: 
 
Relationship to the 
protocol treatment  
Description 
suspected 
The possibility that the protocol treatment caused the event is 
deemed definite or probable or possible 
not suspected 
The possibility that the protocol treatment caused the event is 
deemed unlikely or unrelated 
 
The Investigator will use clinical judgment to determine the relationship. Alternative causes, 
such as natural history of the underlying diseases, medical history, concurrent conditions, 
concomitant therapy, other risk factors, and the temporal relationship of the event to the 
protocol treatment will be considered and investigated. 
The decision will be recorded on the SAE Form and if necessary the reason for the decision 
will also be recorded. 
12.5.4 Expectedness assessment / reference safety information 
The expectedness assessment is the responsibility of the sponsor of the trial. The expectedness 
assessment will be performed against the following reference documents: 
 For fulvestrant: Summary of Product Characteristics (SPC)  
 For palbociclib: latest version of Investigator’s Brochure 
12.6 
Reporting SAEs 
Any SAE and any AESI or other reportable event (sections 12.3 and 12.4) occurring in a 
patient after providing Informed Consent must be reported, including death due to any cause 
other than progression of breast cancer, which occurs within 28 days following cessation of 
treatment or the initiation of a new anticancer therapy, whichever is earlier, whether or not 
related to the investigational product. Information about all such events will be collected and 
recorded on the IBCSG Serious Adverse Event eCRFs (53–SAE–A and 53–SAE–B). 
To ensure patient safety, the IBCSG must be informed of each SAE using the procedures 
described below: 
 The Investigator/MD responsible for the patient must complete a Serious Adverse 
Event (SAE-A) eCRF in English within 24 hours of awareness via iDataFax. A copy 
is automatically forwarded to the IBCSG Safety Office for medical review. 
 Queries may be issued by the IBCSG Safety Office; a timely response by the 
Investigator to all SAE-related queries is crucial. 
 Follow-up information should be completed, via iDataFax, on the Serious Adverse 
Event (SAE-B) eCRF as early as possible but within 15 days of the initial report, even 


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if the event reported in the SAE-A eCRF is not yet resolved. If the event is not resolved 
within 15 days, revise the original Serious Adverse Event (SAE-B) eCRF in iDataFax 
to report the final resolution. 
 All SAEs that have not resolved upon discontinuation of the patient’s participation in 
the trial must be followed until recovered, recovered with sequelae, not recovered 
(death due to another cause) or death (due to the SAE). 
 If a non-serious adverse event becomes serious, this and other relevant follow-up 
information must also be provided within 24 hours. 
 Photocopies of all examinations carried out with the dates on which they were 
performed should be sent by fax or DFSend into the DataFax system. Care should be 
taken to ensure that the patient's identity is protected and the Patient ID number is 
properly included on ALL pages of any reports. For laboratory results, include the 
laboratory normal ranges. Please also note on each page that the information is “SAE 
related” so it can be properly categorized in iDF. 
 In the event the eCRF system is not working, the SAE Forms can be found in the trial 
site file or downloaded from the IBCSG trial webpage and sent via fax or DFSend into 
the DataFax system. 
If an SAE (SAE-A and SAE-B Forms) was submitted by fax or DFSend, the original forms 
and the fax confirmation sheet(s) must be kept at the Participating Center. 
The IBCSG will inform Pfizer Pharmacovigilance, AstraZeneca Pharmacovigilance and other 
appropriate persons about all SAEs within 24 hours of receipt at the IBCSG. 
The IBCSG will record the SAE and prepare a monthly SAE report. Principal Investigators 
will receive the summary report on a monthly basis, and these reports can be found on the 
IBCSG web site (www.ibcsg.org). 
12.7 
Occupational exposure 
If any patient-care or other personnel comes into contact with the content of palbociclib 
capsules then this needs to be reported to IBCSG via email to ibcsg53_pythia@fstrf.org.  
13 Disease assessment, response and progression (RECIST 1.1) 
13.1 
Introduction 
All enrolled patients will be assessed for disease response and progression according to the 
revised Response Evaluation Criteria in Solid Tumors (RECIST version 1.1)[57]. In this trial, 
patients may have measurable or non-measurable disease (see definitions below). Patients 
will be re-evaluated every 12 weeks until documented progression. 
Progression-free survival (primary endpoint) will be assessed using RECIST 1.1 criteria. 
13.2 
Methods of assessment 
The same method of assessment and the same technique should be used to characterize each 
identified and reported lesion at baseline and during treatment and follow-up. Imaging-based 


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evaluation is preferred to evaluation by clinical examination when both methods have been 
used to assess the antitumor effect of a treatment. 
CT scan is the best currently available and reproducible method to measure lesions selected 
for response assessment. CT scan should generally be performed using a ≤ 5 mm contiguous 
reconstruction algorithm. MRI is acceptable for certain situations (e.g., body scans). 
Clinical lesions will only be considered measurable when they are superficial (e.g., skin 
nodules) and ≥10 mm. In the case of skin lesions, documentation by color photography 
including a ruler to estimate the size of the lesion is recommended. 
Lesions on chest X-ray are acceptable as measurable lesions when they are clearly defined 
and surrounded by aerated lung. However, CT scan is preferable. 
Ultrasound is not useful in assessment of lesion size and is not accepted as a method of 
assessment. 
FDG-PET is not foreseen for regular response assessments. It may, however, be used to detect 
or confirm the appearance of new lesions. Attenuation correction CT scans performed as part 
of a PET/CT scan frequently show lower resolution; therefore, dedicated CT scans are 
preferred. However, if the site can demonstrate that the CT scan performed as part of a 
PET/CT is of the same diagnostic quality as a diagnostic CT scan (with i.v. and oral contrast), 
then the CT scan portion of the PET/CT can be used for RECIST measurements. 
13.3 
Measurability of tumor at baseline 
13.3.1 Measurable disease 
Measurable disease is defined as the presence of at least one measurable lesion. 
Measurable lesions: 
 Tumor lesions must be accurately measured in at least one dimension (longest 
diameter in the plane of measurement is to be recorded) with a minimum size of: 
o 10 mm by CT scan (CT scan slice thickness no greater than 5mm). 
o 10 mm caliper measurement by clinical exam (lesions which cannot be accurately 
measured with calipers should be recorded as non-measurable). 
o 20 mm by chest X-ray. 
Reminder: A lesion in a previously irradiated area is not eligible for measurable 
disease. 
 Malignant lymph nodes: To be considered pathologically enlarged and measurable, 
a lymph node must be ≥ 15 mm in short axis when assessed by CT scan, assuming the 
slice thickness is ≤ 5 mm. At baseline and in follow-up, only the short axis will be 
measured. 
13.3.2 Non-measurable disease 
Non-measurable disease is defined as lesions or sites of disease that cannot be measured.  
Non-measurable lesions/sites of disease and special considerations:  


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 Small non-nodal lesions (longest diameter < 10 mm in CT scan). 
 Small lymph nodes (short axis ≥ 10 and < 15 mm). Lymph nodes that have a short 
axis < 10 mm are considered non-pathological and should not be recorded or followed 
as measurable or non-measurable disease. 
 Bone lesions. Lytic bone lesions or mixed lytic-blastic lesions, with identifiable soft 
tissue components, can be considered as measurable lesions if the soft tissue 
component meets the definition of measurability described above. Blastic bone lesions 
are non-measurable. 
 Leptomeningeal disease 
 Ascites 
 Pleural or pericardial effusion 
 Inflammatory breast disease 
 Lymphangitic involvement of skin or lung 
 Cystic lesions. Cystic lesions thought to represent cystic metastases may be considered 
as measurable lesions. However, if non-cystic lesions are present, these are preferred 
as target lesions. 
 Tumor lesions situated in a previously irradiated area, or subjected to other 
locoregional therapy. Such lesions may be considered measurable if there has been 
demonstrated progression in the lesion. 
 Abdominal masses/abdominal organomegaly identified by physical exam that are not 
measurable by reproducible imaging techniques. 
13.4 
Selection of target lesions 
Target lesions should be identified, measured and recorded at baseline (Form 53-TEV-B). At 
baseline, there can be up to a maximum of 5 lesions representative of all involved organs, and 
up to 2 per organ. Target lesions should be selected on the basis of their size and their 
suitability for accurate repetitive measurements. A sum of diameters for all target lesions will 
be calculated and reported as the baseline sum of diameters. Lymph nodes selected as target 
lesions should always have the short axis recorded. All other lesions should always have their 
longest diameters recorded. The sum of diameters will be used as reference to further 
characterize the objective tumor response of the measurable dimension of the disease. 
13.5 
Selection of non-target lesions  
Non-target lesions should be identified. All other lesions (or sites of disease) not identified as 
target lesions should also be recorded as non-target lesions at baseline.  
For non-target lesions, measurements are not required, but the presence or absence of each 
should be noted throughout follow-up (Form 53-TEV). It is possible to record multiple non-
target lesions as a single item on the eCRF (e.g., "multiple liver metastases"). 


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13.6 
Evaluation of target lesions (measurable disease) 
All target lesions will be measured at each tumor assessment, and the sum of their diameters 
will be compared to previous assessments in order to assign the response status as specified 
below. 
 Complete Response (CR): Disappearance of all target lesions. Lymph nodes selected 
as target lesions must each have reduction in the short axis to <10 mm in order for the 
response to be considered complete. In this case, the sum of diameters may be >0. 
 Partial Response (PR): At least a 30% decrease in the sum of diameters of target 
lesions taking as reference the baseline sum of diameters. 
 Progression (PD): At least a 20% increase in the sum of diameters of target lesions, 
taking as reference the smallest sum recorded on trial. In addition to the relative 
increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. 
The appearance of one or more new lesions (see section 13.8) denotes disease 
progression. 
 Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient 
increase to qualify for PD taking as reference the smallest sum of diameters recorded 
on trial. 
Note: All target lesions, including lymph nodes, should have their actual measurements 
recorded at each subsequent evaluation, even when very small (e.g., 2 mm). If the radiologist 
does not feel comfortable assigning an exact measure and reports a lesion as "too small to 
measure", a default value of 5 mm should be recorded. If a target lesion is thought likely to 
have disappeared, use "0 mm." 
When no imaging/measurement is done at all at a particular time point, the patient is not 
evaluable (NE) at that time point. If only a subset of lesion measurements are made at an 
assessment, usually the case is also considered NE at that time point, unless a convincing 
argument can be made that the contribution of the individual missing lesion(s) would not 
change the assigned time point response. This would be most likely to happen in the case of 
PD. 
13.7 
Evaluation of non-target lesions  
All non-target lesions will be assessed at each tumor assessment, and compared to previous 
assessments in order to assign the response status as specified below. 
 Complete Response (CR): Disappearance of all non-target lesions; lymph nodes 
selected as non-target lesions must be non-pathological in size (< 10 mm). 
 Non-CR/non-PD: Persistence of one or more non-target lesions (non-CR). 
 Progression (PD): unequivocal progression of existing non-target lesions. 
Unequivocal means: comparable in magnitude to the increase that would be required 
to declare PD for measurable disease, or an overall substantial increase in tumor 
burden that merits treatment discontinuation. 


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When no imaging is done at all at a particular time point, the patient is not evaluable (NE) at 
that time point. If only a subset of lesions are evaluated at an assessment, usually the case is 
also considered NE at that time point, unless a convincing argument can be made that the 
contribution of the individual missing lesion(s) would not change the assigned time point 
response. This would be most likely to happen in the case of PD. 
13.8 
Determination of new lesions 
The appearance of any new malignant lesions denotes disease progression. The finding of a 
new lesion should be unequivocal (i.e., not attributable to differences in scanning technique 
or findings thought to represent something other than tumor). If a new lesion is equivocal, 
(e.g., because of its small size) the patient will stay on treatment (if the decision on PD is 
based on this lesion only). If the repeat scan documents that there is definitely a new lesion, 
then progression should be declared using the date of the previous scan when the lesion was 
discovered. 
Lesions or sites of disease found in a new location not included in the baseline scan (e.g., 
brain metastases) are considered new lesions. 
Note: The "re-appearance" of a previously "disappeared" target or non-target lesion does not 
in itself necessarily qualify as PD; this is the case only if the overall evaluation meets the PD 
criteria, or if the patient was previously in CR. 
13.9 
Additional considerations 
In some circumstances, it may be difficult to distinguish residual disease from normal tissue. 
When the evaluation of complete response depends upon this determination, it is 
recommended that the residual lesion be investigated (fine needle aspirate/biopsy) before 
confirming the complete response status. 
The cytological confirmation of the neoplastic origin of any effusion that appears or worsens 
during treatment when the measurable tumor has met criteria for response or stable disease is 
mandatory to differentiate between response or stable disease (an effusion may be a side effect 
of the treatment) and progressive disease. 
13.10 Determination of time point response 
Based on the responses of target lesions, non-target lesions, and the presence or absence of 
new lesions, the overall response will be determined at each tumor evaluation time point, 
according to Table 8 or Table 9 below. 
13.10.1 For patients with measurable disease 
Table 8. 
Measurable Disease - Overall Response 
Target lesions 
Non-target lesions 
New lesions 
Overall response 
CR 
CR 
No 
CR 
CR 
Non-CR / non-PD* 
No 
PR 
CR 
Not evaluated 
No 
PR 
PR 
Non-PD or not all evaluated 
No 
PR 
SD 
Non-PD or not all evaluated 
No 
SD 


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Target lesions 
Non-target lesions 
New lesions 
Overall response 
Not all evaluated 
Non-PD 
No 
NE 
PD 
Any  
Yes or no 
PD 
Any 
PD 
Yes or no 
PD 
Any 
Any 
Yes  
PD 
*Non-CR/non-PD should be used rather than SD for categorizing non-target lesions.  
13.10.2For patients with non-measurable disease only 
Table 9. 
 
Non-Measurable Disease - Overall Response 
Non-target lesions 
New lesions 
Overall response 
CR 
No 
CR 
Non-CR/non-PD* 
No 
Non-CR/non-PD* 
Not all evaluated 
No 
Not evaluated 
Unequivocal PD 
Yes or No 
PD 
Any 
Yes 
PD 
*Non-CR/non-PD should be used rather than SD for categorizing non-target lesions.  
 
13.11 Determination of best overall response  
Best overall response is defined as best response recorded from enrollment across all time 
points until disease progression. Confirmation of partial or complete response by an additional 
scan is not requested in this trial. Best overall response will be determined by the IBCSG 
Head of Medical Affairs, in consultation with the trial chair.  
Among patients with measurable disease, the duration of response is defined as the time 
from which the criteria for partial or complete response are first met until disease progression. 
Among patients with non-measurable disease, the duration of response is defined as the time 
from which the criteria for CR are first met until disease progression.   
13.12 Progression-free Survival 
The date of progression is the date that objective progression was first documented. 
Progression-free survival (PFS) is defined as time from treatment initiation until documented 
disease progression according to RECIST 1.1 criteria or death, whichever occurs first. For 
patients without progression, follow-up will be censored at the date of last disease assessment 
without progression, unless death occurs within a short period of time (12 weeks, 
corresponding to the interval of tumor re-evaluation) following the date last known 
progression-free, in which case the death will be counted as a PFS event.  
Patients who discontinue treatment prior to documented disease progression (see Section 
10.6), including those who initiate non-protocol therapy prior to progression, will be followed 
for disease progression, for a maximum of 12 months. A new (non-breast) cancer malignancy 
has to be reported on the 53-14 SAE Forms; such patients must continue to be followed for 
progression of the original breast cancer. 


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13.13 Storage of imaging 
CT/MRI images must be stored locally in electronic format for potential central review, please 
follow local guidelines and policies. 
14 Clinical and laboratory evaluations and follow-up 
14.1 
Screening  
Patients that have consented to enter the AURORA program  (NCT02102165) for metastatic 
breast cancer, conducted by BIG, will be candidates for enrollment in the current trial. Both 
primary breast tumor and one metastatic lesion (if available) will be subjected to next 
generation sequencing for an extended panel of cancer-related genes through the AURORA 
program, making available extensive molecular background information. The screening 
regarding the assessment of the eligibility of a candidate to be included in the trial will include 
securing biomaterial for the AURORA evaluation.  
The following examinations should be done within a maximum of 28 days before enrollment. 
If examinations were done prior to 28 days before enrollment (radiological tumor assessment: 
max 42 days), they have to be repeated. 
14.1.1 Make sure that the patient has consented to AURORA and that her breast cancer has 
been classified locally as ER-positive and HER2-negative. 
14.1.2 Confirmation of post-menopausal status (see 7.1.4). 
14.1.3 Obtain informed consent for screening evaluations and trial participation (Informed 
consent may be obtained earlier than within 28 days before enrollment). 
14.1.4 Medical history including details of malignancy: date of diagnosis, primary tumor type 
characteristics, metastatic tumor type characteristics, prior treatment. 
14.1.5 Clinical and radiological (by CT scan or MRI) tumor assessments of 
Chest/Abdomen/Pelvis should be done preferentially within 28 days prior to 
enrollment. If a biopsy of the metastatic disease is planned this should be done before 
treatment start, and the interval from radiological tumor assessment may be extended 
to max 42 days.  
14.1.6 Bone scan and FDG-PET if medically indicated. 
14.1.7 Cardiac evaluation: Electrocardiogram (ECG). 
14.1.8 Physical examination according to local standards, ECOG Performance Status, height, 
weight. 
14.1.9 Hematology: Hemoglobin, platelet count, white blood cell count including differential 
(absolute neutrophil count). 
14.1.10Coagulation function: PT, PTT and INR. 
14.1.11Biochemistry: serum potassium, HbA1c, glucose, sodium, calcium, LDH; 


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Liver function tests: albumin, total bilirubin, ALT, AST, ALP, GGT; 
Kidney function tests: urea, creatinine. 
14.1.12Baseline symptoms and adverse events graded according to CTCAE v4.0 (record on 
baseline adverse events form 53-BAE). Baseline symptoms and adverse events should 
be recorded from signature of informed consent to prior to start of treatment. 
14.1.13Record all concomitant medication from 28 days before enrollment to prior to start of 
treatment. 
14.2 
Day 1 of every treatment cycle 
Investigations marked with * need to be repeated on day 1 prior to start of treatment if not 
done within 3 days prior to this day. 
14.2.1 Physical examination according to local standards, ECOG Performance Status and 
weight. 
14.2.2 * Hematology: Hemoglobin, platelet count, white blood cell count including 
differential (absolute neutrophil count). 
14.2.3 * Biochemistry: serum potassium, HbA1c, glucose, sodium, calcium, LDH; 
Liver function tests: albumin, total bilirubin, ALT, AST, ALP, GGT; 
Kidney function tests: urea, creatinine. 
14.2.4 Cycle 1 only: Serum sample for TK1 assay 28 days after first dose (between day 26 
of cycle 1 and before start of cycle 2 treatment). 
14.2.5 Electrocardiogram if medically indicated. 
14.2.6 Coagulation function: PT, PTT and INR if medically indicated. 
14.2.7 Collection of any adverse event observed in the previous cycle and assignment of 
appropriate adverse events grade according to the NCI CTCAE Version 4.0. 
14.2.8 Record all concomitant medication. 
14.2.9 Compliance assessment (check patient diary; hand out new patient diary for next 
cycle). 
 
14.3 
Treatment day 15 (cycle 1) 
The Investigator needs to evaluate the status of the patient on day 15 of treatment (=day of 
second fulvestrant administration in cycle 1). Information to be collected during this visit 
includes the following: 
14.3.1 Physical examination according to local standards. 
14.3.2 Assessment of all AEs according to CTCAE v4.0 that have occurred since the day that 
treatment was initiated. 
14.3.3 Coagulation function: PT, PTT and INR if medically indicated. 


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14.3.4 Complete blood count, if medically indicated. 
14.3.5 Compliance assessment (check patient diary). 
14.3.6 Record all concomitant medication. 
14.3.7 Serum sample for TK1 assay 15 days (+/- 2 days) after first dose 
14.4 
Tumor assessments 
Tumor measurements according to RECIST 1.1 criteria (see section 13) have to be done at 
baseline, at weeks 12, 24, etc (every 12 weeks ± 2 weeks) until progression:  
14.4.1 Clinical and radiological (by CT scan or MRI) tumor assessments.  
14.4.2 Bone scan and FDG-PET will be done if clinically indicated at the same time points. 
14.5 
After discontinuation of trial treatment 
End of treatment visit within 30 days after stop of all trial treatment (or at the time of decision 
to stop the trial treatment if the decision is taken >30 days after last dose):  
14.5.1 Physical examination according to local standards, weight and ECOG Performance 
Status. 
14.5.2 Collection of any adverse event and assignment of appropriate adverse events grade 
according to the NCI CTCAE Version 4.0. 
14.5.3 Record all concomitant medication. 
14.5.4 Hematology: Hemoglobin, platelet count, white blood cell count including differential 
(absolute neutrophil count). 
14.5.5 Biochemistry: serum potassium, sodium, HbA1c, glucose, calcium, LDH; 
Liver function tests: albumin, total bilirubin, ALT, AST, ALP, GGT; 
Kidney function tests: urea, creatinine. 
14.5.6 Coagulation function: PT, PTT and INR if medically indicated. 
14.5.7 If not done in the 30 days prior to this visit, clinical and radiological tumor assessments 
by CT scan or MRI, and tumor measurements according to RECIST 1.1 criteria for 
determination of response (see section 13). 
14.5.8 Electrocardiogram 
 
14.6 
Follow-up prior to documented disease progression 
Every 12 weeks (± 2 weeks) in case trial treatment was stopped prior to documented objective 
disease progression, until progression is documented, or for a maximum of 12 months after 
treatment stop. The first visit should take place 12 weeks after the previous tumor evaluation: 
14.6.1 Physical examination according to local standards, weight and ECOG Performance 
Status. 


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14.6.2 Clinical and radiological (by CT scan or MRI) tumor assessments.   
14.6.3 Bone scan and FDG-PET if clinically indicated. 
14.6.4 Tumor measurements according to RECIST 1.1 for determination of disease 
progression. 
14.6.5 Serious adverse events up to 28 days after stop of all trial treatment. 
14.6.6 Any newly initiated anti-tumor therapy. 
15 Biological evaluations 
15.1 
Introduction and overview 
Biological material will be banked as foreseen in the AURORA study (NCT02102165). Two 
additional blood samples will be taken at two weeks and at the end of cycle 1 for the TK1 
assay (see section 15.2.3).  
Tumor tissue taken prior to start of treatment in the framework of AURORA will be used to 
define biomarkers that could identify patients with a high chance of responding to the 
combination therapy with fulvestrant and palbociclib. 
In the AURORA program, a whole blood sample for pharmacogenomics will be taken at 
inclusion into AURORA; plasma and serum samples will be taken every 6 months and at time 
of disease progression to monitor the disease and to better understand the contribution of 
ctDNA to the evolution of the disease. 
For successful inclusion in the AURORA program, the successful sequencing of at least 2 out 
of these 3 biospecimens is required. 
15.2 
Translational research 
The goals of the translational research will be to determine mechanisms and biomarkers for 
efficacy of and resistance to the combination of fulvestrant and palbociclib.  
15.2.1 Planned correlative studies: 
Within the AURORA program, next generation sequencing will be performed for a panel of 
411 cancer-related genes. Additionally, RNA sequencing data will become available at a later 
time point. Mutations will be assessed in a panel of genes on ctDNA. Based on this data, the 
following correlative studies will be performed:  
 gene mutations in a panel of genes assessed on ctDNA 
 The association of mutations and gene copy number aberrations of an extended panel 
of cancer related genes assessed in the primary tumor with the clinical outcome of the 
women enrolled in the present trial. 
 The association of mutations and gene copy number aberrations of the same panel of 
cancer related genes assessed in a metastatic lesion with the clinical outcome of the 
women enrolled in the present trial. 
 The association of mutations in a panel of genes assessed in ctDNA with the clinical 
outcome. 


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 The potential predictive value of gene signatures as inferred by RNA sequencing in 
regards to sensitivity towards the investigational palbociclib/fulvestrant treatment. 
15.2.2 Pharmacogenomics:  
The 9ml whole blood sample that is collected from each patient within the AURORA program 
will be analyzed in order to be able to differentiate germline from somatic genetic mutations 
during the analyses.  
15.2.3 Thymidine Kinase 1 assay  
Thymidine kinase-1 (TK1) is an enzyme that plays a critical role in the cell cycle and synthesis 
of DNA. All human cells express the TK1 enzyme during normal cell division through E2F-
dependent transcription, but only small amounts of TK1 are released into the serum. Due to 
uncontrolled cell growth and higher rates of replication compared to normal tissues, solid 
tumors can secrete pathological levels of TK1 that can be detected in serum with the 
DiviTum technology (Biovica International, Sweden). Given that TK1 expression is E2F-
dependent, there is a scientific rationale that TK1 will correlate well with CDK4 inhibitors 
like palbociclib. The CDK4 inhibitors prevent cellular DNA synthesis by prohibiting 
progression of the cell cycle from G1 into the S phase. The cell synthesizes TK1 during the S 
phase of cell division. If the CDK-inhibitor is active in a particular tumor, it is expected that 
this would be reflected by changes in serum TK1 levels and thereby TK1 can be used as a 
potential, non-invasive, efficacy biomarker for clinical benefit. 
The sampling requirements are described in the AURORA “Laboratory Manual: Procedures 
for tissue and blood samples collection, storage and shipment” and in the additional sheet 
entitled “IBCSG 53-14 / BIG 14-4 PYTHIA Serum Sample Instructions”. Besides the samples 
taken for AURORA, two serum samples specifically reserved for the TK1-assay should be 
taken on day 15 (+/- 2 days) of cycle 1 and between day 26 of cycle 1 and before any cycle 2 
treatment, at the latest on day 35. 
15.2.4 Not yet specified translational research 
The use of the biological material for future research not outlined in this protocol will be under 
the guardianship of BIG on behalf of the AURORA Steering Committee. As part of the 
AURORA Informed Consent process, patients are asked to indicate whether they agree to 
donate their sample for not yet specified future research. The patient’s decision is recorded 
on the AURORA Consent Form. 
Translational research proposals not outlined in this protocol will be assessed by the PYTHIA 
steering committee and the AURORA steering committee for merit and feasibility.  
Biomarkers that are published in the future and considered to be of relevance can then also be 
assessed in the context of this trial. 
16 FDG-PET substudy 
16.1 
Introduction 
Palbociclib combined with endocrine treatment has exhibited promising antitumor activity 
among patients with ER+, HER2- metastatic breast cancer. However, there is no predictive 


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biomarker associated with either sensitivity or resistance to this promising agent. Selecting 
patients who will achieve a benefit and thus justify the burden of treatment is of utmost 
importance.   
FDG-PET/CT offers a possible solution to early identification of patients who will not benefit 
from treatment with fulvestrant and palbociclib. Thus a substudy within the PYTHIA phase 
II trial evaluating the use of FDG uptake as a biomarker in patients with ER+/HER2- 
metastatic breast cancer treated with palbociclib plus fulvestrant will investigate the question 
of whether patients who will NOT derive benefit from this combination of therapy can be 
selected early in the therapeutic process using FDG-PET/CT metabolic response, thus 
minimizing unnecessary toxicities in these patients. 
16.2 
Study objectives 
16.2.1 Primary Objective 
To evaluate if early metabolic response (MR) using FDG-PET/CT is associated with 
progression free survival (PFS) in patients with ER+, HER2– metastatic breast cancer treated 
with fulvestrant plus palbociclib. 
16.2.2 Secondary Objective 
To evaluate if early metabolic response (MR) using FDG-PET is associated with disease 
control rate (DCR) in patients with endocrine resistant ER+, HER2– metastatic breast cancer 
treated with fulvestrant plus palbociclib. 
16.3 
Study design 
A subset of up to 45 patients enrolled by selected sites in Belgium into the PYTHIA trial will 
enter the FDG-PET/CT substudy to assess early FDG-PET/CT response as a predictor of lack 
of treatment response to fulvestrant and palbociclib. Because only patients with a 
metabolically measurable lesion at baseline will continue with the substudy and be 
statistically analyzed, it is expected that up to 45 patients will need to be enrolled in order to 
have the targeted number of 30 patients with both a baseline and a day 28 scan. Patients who 
do not have a metabolically measurable lesion at baseline will not have a day 28 scan.  
Screening, treatment and assessment of these patients, including tumor evaluations with CT 
or MRI as described in Section 14, will be performed as described in the current protocol for 
the PYTHIA trial population. In addition, for patients participating in the substudy: 
 FDG-PET/CT will be done at baseline (within 2 weeks prior to start of trial treatment) 
and on day (25-)28 (at end of first cycle of treatment, before start of cycle 2 treatment). 
The 53-PET form must be completed at Baseline and updated at day 28 with 
information regarding the FDG-PET/CT. 
 A blood sample (5mL) will be taken at the same time points for more accurate 
estimation of the blood glucose level. 
In order to ensure image quality and reproducibility, standardization and harmonization, the 
participating FDG-PET/CT centers are required to have FDG-PET/CT Accreditation through 
EARL (EANM Research Ltd.) and need to comply with the requirements of continued quality 
control (QC) of acquisition and reconstruction on a regular basis as outlined in EARL’s 


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accreditation manual. More information can be obtained via the EARL’s web site: 
http://earl.eanm.org. 
16.4 
Definitions 
This study is aimed at early identification of metabolic non-responders. FDG-PET/CT 
response will use the following methodology based on EORTC and PERCIST criteria. 
Response assessment will be performed centrally by 2 independent and blinded PET-CT 
experts. Consensus reading will be organized in case of discordant assessments. All analyses 
will be organized and coordinated by a central Imaging Core Lab (Orilab).  
Definition of metabolic target lesions on FDG PET/CT: 
Target lesions are defined on the baseline FDG-PET/CT based on all of the following criteria: 
a. Lesion with a very high probability of malignancy (all lesions potentially 
corresponding with a benign pathology should not be selected); AND 
b. Size ≥1.5 cm (preferably in anatomic images); AND  
c. FDG-PET avid lesion with uptake above the background liver uptake as follows: 
Metabolically measurable lesion, i.e. with a marked accumulation of FDG, at least 
1.5-fold greater than liver SUV mean + 2 SDs (in 3-cm spherical ROI in normal right 
lobe of liver). If liver is abnormal, target lesion should have uptake > 2.0 × SUV mean 
of blood pool in 1-cm-diameter ROI in descending thoracic aorta)  
Definition of FDG-PET-CT response: 
Both EORTC-based and PERCIST-based response classification methodologies will be 
performed: 
a. EORTC-based response assessment: Dominance Method [58]  
Patient-based classification of metabolic response within 4 classes: 
Class 1: 
All target lesions do respond (reduction of SUVmax of more than 25% 
seen in all lesions). 
Class 2: 
Mixed response with majority of whole body tumor load that responds. 
Class 3: 
Mixed response with majority of whole body tumor load that does not 
respond. 
Class 4: 
All target lesions do not respond OR presence of a progressive lesion 
(i.e., increase of SUVmax of more than 25% in a known lesion or 
appearance of a measurable (target) new PET lesion). 
b. PERCIST-based response assessment [59]  
A maximum of 10 target lesions (maximum of 2 per organ) will be identified. If more 
than 2 target lesions within one organ are available: only 2 lesions with the highest 
SUV will be defined as target lesions. 
SUVpeak at baseline and on day 28 of trial treatment will be calculated for all target 
lesions. 


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Significant lesion response is defined as a relative decrease of SUVpeak of more than 
30%, therefore patient will be classified as follows: 
 
Metabolic responder patient (MRP): the difference between the SUVpeak of the 
hottest lesion at baseline and during treatment is more than 30%; 
 
Metabolic NON responder patient (MNRP): patient that does not fulfil the 
definition of MRP. 
The primary method for defining response is the EORTC-based dominance method. 
16.5 
ORILAB – Imaging CoreLAB: 
FDG-PET images generated in this study are managed by the Oncology Related Imaging 
coreLAB (ORILAB) from the Institute Jules Bordet. 
16.5.1 Standardization 
To assure that centers performing FDG-PET for the PYTHIA PET sub-study follow the same 
acquisition procedures, ORILAB will provide them a Standard Procedures Imaging Manual 
(SPIM). The SPIM describes the procedures involved in patient preparation, data transfer, 
image analysis and storage.  
16.5.2 Data encoding 
The PYTHIA FDG-PET substudy imaging data will be encoded in an Acquisition electronic 
report form (AeCRF) and sent by the FDG-PET substudy centers through the electronic data 
capture tool (EDC) provided by ORILAB. The tool will also guarantee automatic study 
images anonymisation.  
16.5.3 Quality Control/Quality Assurance - QA/QC 
The encoded imaging data will be verified and study images will be visually checked to assure 
compliance with imaging protocol as described in SPIM and absence of artifacts. If non-
compliances or errors are detected queries will be sent to the participating center. 
16.5.4 Central Review 
Imaging data that successfully passed the QA/QC process will be reported and managed by 
ORILAB central review. To that, the predefined imaging analysis tools and a review 
electronic clinical report form (ReCRF) will be provided to the two PET experts to capture 
the required imaging information. The PYTHIA FDG-PET substudy images will be kept in 
ORILAB Picture Archiving and Communication System (PACS).  
17 Data submission 
We will conduct the trial according to the ICH Good Clinical Practice (GCP) guidelines.  
Keeping accurate and consistent records is essential to a cooperative trial. The following 
forms are to be submitted at the indicated times by the participating institutions for each 
patient: 


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17.1 Case report forms schedule 
Forms 
Description/Name 
Forms Submission  
ALL data should be completed in iDataFax 
(iDF) (unless otherwise specified) 
Informed 
Consent Form 
Consent to participation in clinical 
trial and biologic material 
submission 
Obtain before registration and keep with patient 
records as documentation (hard copy only). 
Registration and Enrollment 
53-A 
Confirmation of Registration Form 
Complete in iDF after you have registered the 
patient in the IBCSG Registration/ 
Randomization System and to confirm 
eligibility. Patient will be available in iDF 
within 24 hours of successful registration. 
53-AUR 
Confirmation of registration into 
AURORA Form 
Complete in iDF after you have enrolled the 
patient in the IBCSG Registration/ 
Randomization System.  
Baseline  
53-H 
History Form 
Complete in iDF within 1 week of enrollment. 
53-BAE 
Baseline Adverse Events and 
Symptoms Form 
Complete in iDF within 1 week of enrollment.   
53-CCT 
Concomitant Treatment Log  
Complete in iDF each time a treatment is 
started, amended, or ended, including treatments 
within 28 days prior to start of trial treatment 
(excluding prior treatment for 
primary/metastatic breast cancer). 
53-TEV-B 
Tumor Evaluation Baseline Form 
Complete in iDF within 1 week of enrollment.  
During trial treatment 
53-PT 
Protocol Therapy Form 
Complete in iDF at the end of each cycle until 
treatment stops. 
53-AE 
Adverse Events Log 
Complete/review in iDF at the end of each 
treatment cycle to report an event has started, 
grade has worsened, or event has resolved, 
including events observed up to 28 days after 
treatment stops. 
53-CCT 
Concomitant Treatment Log 
Review the original CCT Log in iDF at the end 
of each cycle.  Update any current medications 
if needed and add new medications taken, 
including medications taken up to 28 days after 
treatment stops. 
53-TEV 
Tumor Evaluation Form 
Complete in iDF at weeks 12, 24, then every 12 
weeks until progression.   
Follow Up (after discontinuation of trial treatment, up to documented progression) 
53-EoT 
End of Treatment Form 
Complete in iDF 30 days after ALL protocol 
treatment stops. 
53-AE 
Adverse Events Log 
Review the original AE Log in iDF 28 days after 
the last dose of trial treatment. Update any 
events if needed and add new adverse events . 
53-CCT 
Concomitant Treatment Log 
Review the original CCT Log entries in iDF 28 
days after the last dose of trial treatment.  
Update/add any treatments. 
53-TEV 
Tumor Evaluation Form 
If patient discontinued trial treatment prior to 
documented progression: every 12 weeks until 
progression, for a maximum of 12 months after 
all treatment stops. 
53-E 
Follow-up Form 
If patient discontinued trial treatment prior to 
documented progression: every 12 weeks until 
progression, for a maximum of 12 months after 
all treatment stops. Visit should be done in 
conjunction with tumor evaluation. 


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Forms 
Description/Name 
Forms Submission  
ALL data should be completed in iDataFax 
(iDF) (unless otherwise specified) 
Event Driven  
 
53-SAE-A 
Serious Adverse Event/Adverse 
Event of Special Interest Form A - 
Initial report 
Complete in iDF within 24 hours of the SAE 
awareness. If iDF is not available, fax the form 
within 24 hours to DataFax. 
53-SAE-B 
Serious Adverse Event/Adverse 
Event of Special Interest Form B - 
Follow-up report 
Complete in iDF within 15 days of the initial 
report (53-SAE-A). If event is not resolved in 15 
days, update 53-SAE-B again at the time of 
resolution. 
53-COC 
Change of Consent Form 
Complete in iDF if there is any change in 
patient's consent to participate in the trial, see 
Section 19.5.2. 
53-E-Death 
Death Form 
Complete in iDF if a patient dies, if patient is 
still in active follow-up. 
53-PREG (Initial) 
Pregnancy Form – Initial report 
Complete in iDF if a patient becomes pregnant 
while on trial treatment. 
53-PREG 
(Outcome) 
Pregnancy Form – Outcome report 
Complete in iDF at the outcome of pregnancy. 
The iDF User Manual and the Data Managers’ Manual for this trial contain instructions for 
completing and submitting forms using the iDataFax system.  
17.2 
Signing and submitting forms 
An Authorization Log (see section 17.5) should be completed at each Participating Center to 
identify the persons who are authorized to complete CRFs. 
CRFs should be completed on-line in iDataFax. Reports (lab, pathology, etc.) and any other 
non-CRF data will need to be sent to the DataFax system via fax or DFSend. Full instructions 
on submitting forms will be available on the IBCSG website (www.ibcsg.org). Also available 
on the website is a list of fax numbers that are available for faxing CRFs. 
17.3 
Data management 
Data collected in this trial will be submitted to the IBCSG Data Management Center in 
Amherst, NY, USA. The Data Management Center will process the data and will generate 
queries and forms requests. The Data Quality Control Office will oversee overall data 
submission and query resolution. The IBCSG Coordinating Center in Bern, Switzerland will 
provide medical review and summary of SAEs. The IBCSG Statistical Center in Boston, MA, 
USA will perform the data analysis. 
17.4 
Investigator Site File 
Each Participating Center should keep documentation about this trial in an Investigator Site 
File (ISF). Please arrange the documentation in the order foreseen in the ISF index which will 
be provided by IBCSG. The following documents should be included (list is not complete): 
 Protocol and appendices  
 Activation letter 
 Accrual reports 
 Amendments 


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 Copy of signed Protocol Signature Pages 
 Sample CRFs including blank SAE Forms 
 Data Managers’ Manual 
 Obvious Corrections Document and Signature Page 
 Registration/Randomization System Manual 
 iDataFax (iDF) Manual 
 Drug Supply Manual 
 SPIM (Belgian sites only) 
 Patient information and Informed Consent templates approved by Ethics Committee 
 Palbociclib Investigator's Brochure and updates 
 Fulvestrant SPC 
 Ethics Committee (and Health Authority, if applicable) approval of protocol, Patient 
Information Sheet and Informed Consent, amendments 
 Ethics Committee review of SAE, Investigators' alert, and other documents 
 Correspondence with Ethics Committee and Health Authority (if applicable) 
 Certificate of clinical trial insurance 
 Agreement with IBCSG 
 Center Activation email(s) from IBCSG Data Management Center (protocol and 
amendments, if any) 
 Correspondence with / Information issued by IBCSG Coordinating Center, Data 
Management Center 
 SAE Reports sent from IBCSG Data Management Center 
 Normal laboratory values/reference ranges 
 Laboratory Certifications 
 CV of Principal Investigator and Co-Investigators, GCP certificates  
 Trial Training Certificates issued by IBCSG Center Training Office 
 Documentation of any training done internally (e.g.,by use of IBCSG Training 
Confirmation Log) 
 Authorization Log 
 Center Information Sheet 
 Patient identification log (see section 17.6) 
 Drug shipment records 


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 Drug accountability log (including certificates of destruction if applicable)   
 Temperature logs 
 Weblink to ICH GCP guidelines/Declaration of Helsinki and updates 
 Audit certificates / monitoring follow-up letters 
17.5 
Authorization log 
The Principal Investigator (PI) should identify the other members of the Clinical Trial Team 
who are supervised by the PI and approved to provide information in CRFs, queries, etc. 
Instructions for completing the Authorization Log can be found in the Authorization Log 
Manual, posted on the IBCSG website.  All changes need to be communicated to IBCSG by 
updating and e-mailing the authorization log. 
17.6 
Patient identification log 
No patients’ names should be used in CRFs or any other documentation transmitted to IBCSG 
central offices. The only item used to identify a patient is the Patient ID (Registration 
Number). It is therefore imperative that the local data manager keep an identification log for 
all patients entered in this trial including: 
 Patient's name 
 Patient ID issued by the Registration/Randomization System 
 Date of birth 
 Date of enrollment 
18 Statistical considerations 
This is a single arm phase II trial for postmenopausal patients receiving fulvestrant and 
palbociclib for ER+ / HER2- metastatic or locally advanced breast cancer who have 
progressed under previous endocrine therapy. The primary objective is to interrogate in a 
prospective manner a series of potential biomarkers, which will be assessed for their 
association with PFS. A total of 120 patients will be enrolled. 
18.1 
Primary Objective 
A series of different molecular markers will be assessed for their association with the PFS of 
patients receiving fulvestrant plus palbociclib treatment. The primary efficacy endpoint of 
PFS is defined in Section 13.12. 
18.1.1 Design and Sample Size Determination 
Enrollment of 120 patients is expected to proceed over 12 months (approximately 10 patients 
per month), with an additional 12 months of follow-up after the last patient enrolled. The 
median PFS on palbociclib plus fulvestrant is expected to be in the range of 9 to 10.5 months, 
with about 67% of patients having documented PFS events (~80 events) at the time of 
analysis. For sample size / power considerations we conservatively assessed the simplified 


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situation wherein PFS is compared between groups of patients with and without a certain 
biomarker. This could be mutations or gene copy number aberrations in pre-treatment ctDNA 
or tumor-derived DNA as determined by the AURORA next generation sequencing platform, 
a gene signature as inferred by RNA sequencing, or a pre-treatment circulating marker level 
or change in that marker after 1 cycle of trial treatment. The assessment calculated the 
detectable hazard ratios with 80% power for alternative scenarios in which all 120 patients 
have biomarker data or conservatively in which ~80% (N=90) patients have biomarker data; 
and on the basis of nominal two-sided α=0.05 level test and α=0.001 level test as a rough 
consideration of multiple testing (Table X). For example, we expect approximately 40% of 
patients’ primary tumors to show cyclin D1 amplification and ER mutations in approximately 
15-20% of patients’ metastatic tumors. The statistical analysis plan will re-estimate power 
considerations based on the observed accrual pattern, overall treatment hazard ratio and 
overall average hazard, and with consideration of multiplicity of testing. The sample size of 
120 was selected to have 80% power to detect a HR of 2.0 for biomarker with 30-50% 
prevalence (two-sided α=0.05). 
Detectable hazard ratios (HR) with 80% power for the difference in PFS distributions between 
two biomarker groups (i.e., presence vs. absence), for a range of prevalence of the biomarker, 
two alpha levels and two analyzable sample sizes.  
Analyzable 
Sample Size 
2-sided 
α-level 
Biomarker Prevalence 
 
 
5% 
10% 
15% 
20% 
30% 
50% 
 
 
Detectable HR 
N=120 
0.05 
4.2 
2.85 
2.4 
2.2 
2.0 
1.9 
0.001 
8.3 
4.65 
3.65 
3.2 
2.75 
2.5 
N=90 
0.05 
5.25 
3.35 
2.75 
2.5 
2.2 
2.1 
0.001 
>10 
5.9 
4.5 
3.8 
3.2 
2.9 
Calculations based on George SL, Desu MM. Planning the size and duration of a clinical trial 
studying the time to some critical event. Stats Med 2000;19:441-452[60]. 
18.1.2 Analysis of Primary Endpoint 
On the basis of information that accumulates external to the trial during the years of 
enrollment and follow-up, the final statistical analysis plan will delineate hypothesis testing-
focused versus exploratory analyses, and the approaches to multiple testing and interpretation 
for each set of analyses. The Statistical Analysis Plan (SAP) will identify a few selected 
biomarkers as hypothesis testing-focused analysis to replicate or validate the clinical validity 
of the biomarker for its association with PFS, for future assessment as predictive biomarkers; 
in such analyses, the cutpoints or functional forms of the biomarker values would be pre-
specified.  


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As a general approach, PFS will be compared between biomarker groups or according to 
continuous biomarker values using by a stratified log-rank test using stratification factors 
defined for randomization. The hazard ratio with two-sided 90% CI will be estimated using a 
stratified Cox proportional hazards modeling, with estimation of hazard ratio with two-sided 
confidence intervals (CI). The distribution of PFS will be summarized for each treatment 
group using the method of Kaplan-Meier; median PFS with two-sided 95% confidence 
interval (CI) will be summarized, as well as other quartiles or t-month PFS times with CIs. 
18.2 
Secondary Objectives  
18.2.1 Safety and tolerability  
Adverse events (AE) will be collected using CTCAE v4.0. The maximum grade of each 
targeted AE while on treatment will be determined, and the frequencies summarized and 
tabulated according to grade and treatment assignment, with two-sided exact binomial 95% 
CIs. Table 10 shows the half-width of an asymptotic two-sided 95% CI for a range of possible 
true AE rates, with a sample size of 90 patients randomized to palbociclib with fulvestrant. 
Table 10. Confidence Intervals (CI) for AE Rates (%) in Experimental Group (N=90)  
True AE rate (%) 
5% 
10% 
20% 
30% 
40% 
50% 
95% CI width 
±4.5% 
±6.2% 
±8.3% 
±9.5% 
±10.1% 
±10.3% 
18.2.2 Disease Control 
Disease control is defined as best overall response of CR or PR, or SD (or non-CR/non-PD in 
the case of non-measurable disease only) lasting for at least 24 weeks, measured from 
enrollment until first documentation of progressive disease. Disease control will be 
summarized as proportion with two-sided exact binomial 95% CI. 
18.2.3 Overall survival 
Patients will not be followed for overall survival. However, at time of analysis, survival status 
of the patients will be available from AURORA and overall survival from time of treatment 
start will be summarized using the method of Kaplan-Meier.  
18.3 
Definitions of Trial Populations 
Included population: All enrolled patients. 
Treated population: All patients who initiate treatment. Safety population: All patients 
receiving at least one dose of trial treatment will be included in assessments of safety and 
tolerability. 
18.4 
Interim Analyses 
Interim analyses for efficacy or futility are not planned.  
18.5 
Accrual 
The overall accrual goal of this trial is 120 patients. We anticipate that the accrual rate will be 
approximately 10 patients per month over 12 months.  


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18.6 
Data and Safety Monitoring 
The trial will be presented for review to the IBCSG DSMC at each of their semi-annual 
meetings. Accrual, safety and accumulation of PFS events will be monitored. 
19 Ethical aspects, regulatory approval, and patient informed consent 
The Investigator will ensure that this trial is conducted in full conformance with the principles 
of the “Declaration of Helsinki” or with the laws and regulations of the country in which the 
research is conducted, whichever affords the greater protection to the individual. The trial 
must fully adhere to the principles outlined in “Guideline for Good Clinical Practice” ICH 
Tripartite Guideline (January 1997) or with local law if it affords greater protection to the 
patient. For trials conducted in the EU/EEA countries, the Investigator will ensure compliance 
with the EU Clinical Trial Directive (2001/20/EC). 
19.1 
Ethical Review Board/Ethics Committee 
All protocols and the patient Informed Consent forms must have the approval of a properly 
constituted committee or committees responsible for approving clinical trials. The Ethics 
Review Board (ERB) / Institution Review Board (IRB) written, signed approval letter/form 
must contain approval of the designated Investigator, the protocol (identifying protocol title 
and version number), and of the patient Informed Consent. Documentation of Ethics 
Committee approval(s) must be sent to the IBCSG Data Management Center prior to 
enrollment of the first patient. The IBCSG Ethics Committee also approves the protocol and 
reviews it annually. 
Any modifications made to the protocol will be reviewed by the IBCSG Ethics Committee 
and must also be submitted to the appropriate ERB/IRB for information or approval in 
accordance with local procedures and regulatory requirements and to Health Authorities if 
required.  
Once approved or acknowledged by the appropriate ERB/IRB and by the Health Authorities 
(if required), the Investigator shall implement the protocol modifications. Protocol 
modifications for urgent safety matters may be directly implemented following the 
instructions of IBCSG. 
19.2 
Regulatory approval procedures 
If applicable, in addition to the approval of the Ethics Committee according to national 
legislation, the protocol, other protocol-related documents including patient information and 
Informed Consent and other documents as required locally must be submitted to and be 
approved by the health authority. Documentation of health authority approval must be sent to 
the IBCSG Data Management Center prior to Participating Center activation. 
19.3 
Protection of human patients 
The IBCSG has an Office for Human Research Protection (OHRP) Federal Wide Assurance 
(FWA00009439) and follows all of the policies and procedures that are part of that assurance. 
All potential patients for this trial will receive a full explanation of the trial, its purpose, 


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treatments, risks, benefits, and all of the other items listed in section 19.4. Additional 
institution-specific sections should be added to Appendix I as needed. 
The medical record must be available for review by the IBCSG monitors and audit team and 
regulatory authorities as described in section 20.6. 
Serious Adverse Event (SAE) Reports are distributed monthly. In addition they are available 
on the IBCSG website (www.ibcsg.org) for participating Centers. 
19.4 
Informed Consent 
Informed Consent for each patient will be obtained prior to initiating any trial procedures in 
accordance with the “IBCSG Patient Information Sheet and Informed Consent" (See 
Appendix I). One signed and dated copy of the Informed Consent must be given to each 
patient and the original copy must be retained in the Investigator's trial records. The Informed 
Consent form must be available in the case of data audits. Verification of signed Informed 
Consent for both the PYTHIA trial and the AURORA program and the date signed are 
required for enrollment to this trial.   
The "Declaration of Helsinki" recommends that consent be obtained from each potential 
patient in biomedical research trials after the aims, methods, anticipated benefits, and potential 
hazards of the trial, and discomfort it may entail, are explained to the individual by the 
physician (http://www.wma.net/en/30publications/10policies/b3/index.html). The potential 
patient should also be informed of her right to not participate or to withdraw from the trial at 
any time. The patient should be told that material from her tumor will be stored and potentially 
used for additional studies not described in this protocol.  
If the patient is in a dependent relationship to the physician or gives consent under duress, the 
Informed Consent should be obtained by an independent physician. By signing this protocol, 
the Investigator agrees to conduct the trial in accordance with GCP and the "Declaration of 
Helsinki."  
The IBCSG recognizes that each institution has its own local, national, and international 
guidelines to follow with regard to Informed Consent. Therefore, we provide a template 
information sheet and Informed Consent form (Appendix I), which can be downloaded and 
edited to incorporate information specific to your institution (see www.ibcsg.org). The 
template Patient Information Sheet and Informed Consent (PIS/IC) has been written according 
to ICH guidelines which state the Informed Consent should adhere to GCP and to the ethical 
principles that have origin in the “Declaration of Helsinki”. The final version should receive 
the Institutional Review Board/ Local Ethics Committee approval in advance of its use. 
Centers should send their locally modified PIS/IC to the IBCSG Data Management Center for 
review and approval before submitting to their Ethics Committee.  
19.5 
Premature withdrawal 
19.5.1 Cessation of trial treatment 
Patients have the right to refuse further trial treatment at any time during the trial. Patients 
may also be withdrawn from trial treatment at any time at the discretion of the Investigator 


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due to an adverse event, or based on any other relevant medical condition. Such patients will 
remain in the trial. The patient will continue to be documented according to protocol. 
19.5.2 Withdrawal of consent 
Patients have the right to withdraw consent for further trial participation at any time without 
having to specify the reason. The data recorded up to the time point of withdrawal will 
continue to be evaluated in the trial.  
Withdrawal of consent should be documented in both the medical records and in the eCRF 
(Form 53-COC). For the patient’s safety, an end of treatment visit should be performed and 
documented in the eCRF. 
20 
Governance and Administrative Considerations 
20.1 
Insurance 
IBCSG will contract the appropriate liability insurance for this trial. Patients who suffer 
injuries due to the trial should report them immediately to their physician. The local Center 
should report all alleged claims immediately to the IBCSG. 
20.2 
Steering Committee 
A Steering Committee will be constituted for this trial. The primary responsibilities of the 
Steering Committee are twofold. First, the Steering Committee is responsible for maintaining 
the scientific integrity of the trial, for example, by recommending changes to the protocol in 
light of emerging clinical or scientific data from other trials. Second, the Steering Committee 
is responsible for the translation of recommendations of the IBCSG Data and Safety 
Monitoring Committee into decisions. Membership will include BIG and IBCSG officials, 
trial chair and co-chairs, trial statisticians, representatives from some Participating Centers, 
and one representative from Pfizer Inc. 
General partition of responsibilities: 
The Steering Committee has the authority to make and implement any final decisions, such 
as substudies of the trial or amendments to the trial protocol, and may recommend the 
termination/early termination of the trial. 
The IBCSG Foundation Council and the BIG Executive Board decide on the termination/early 
termination of the trial. 
20.3 
Data and Safety Monitoring Committee (DSMC) 
The trial will be presented for review to the IBCSG Data and Safety Monitoring Committee 
(DSMC) at each of their semi-annual meetings. Accrual, safety and accumulation of PFS 
events will be monitored.  
20.4 
Publication of trial results 
IBCSG and BIG will publish the results of the trial based on the final trial report. 


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20.5 
Premature discontinuation of the trial 
The trial may be discontinued early in parts or completely if the information on the trial 
treatment leads to doubt as to the benefit/risk ratio. 
The trial can be terminated at any time if the authorization and approval to conduct the Study 
is withdrawn by ethics committee or regulatory authority decision, insufficient accrual, 
emerging new data impacting the scientific value of the trial or ethical grounds. 
20.6 
Quality Assurance 
The IBCSG conducts trials according to the ICH GCP guidelines. The Trial IBCSG Data 
Manager reviews each CRF. In addition, the IBCSG Medical Reviewer reviews each case at 
specific timepoints. The IBCSG conducts periodic audit visits to ensure proper trial conduct, 
verify compliance with GCP, and perform source data verification. 
The Investigator should ensure that source documents are made available to appropriately 
qualified personnel from IBCSG or its designees, or to health authority inspectors after 
appropriate notification. 
At regular intervals during the clinical trial, the Center will be contacted, through monitoring 
visits, letters or telephone calls, by a representative of the Monitoring Team to review trial 
progress, Investigator and patient compliance with clinical trial protocol requirements and any 
emergent problems. These monitoring visits will include but not be limited to review of the 
following aspects: patient Informed Consent, patient recruitment and follow-up, SAE 
documentation and reporting, AEs with pre-specified monitoring documentation and 
reporting, AE documentation, trial treatment administration, patient compliance with the 
regimens, drug accountability, concomitant therapy use and quality of data. 
20.7 
Protocol adherence 
Investigators ascertain they will apply due diligence to avoid protocol deviations. Under no 
circumstances should the Investigator contact IBCSG or personnel monitoring the trial to 
request approval of a protocol deviation, as no deviations are permitted. The Investigator 
should document and explain any deviations from the approved protocol and promptly report 
them to IBCSG and to the EC concerned in accordance with the applicable EC policies and 
procedures. If the Investigator feels a protocol deviation would improve the conduct of the 
trial this must be considered a protocol amendment, and unless such an amendment is 
developed and activated by IBCSG and approved by the IRB/IEC/REB it cannot be 
implemented. All protocol deviations will be documented. 
20.8 
Data protection 
A unique Patient Identification (ID)/Registration Number will be assigned by the IBCSG 
Registration/ Randomization System to each patient registered into the trial. The names of the 
patients will not be disclosed to the IBCSG.  
Only the Patient ID will be used to identify a patient on the eCRF. Identification of patients 
must be guaranteed at the Participating Center. In order to avoid identification errors, Centers 
should keep a Patient Identification Log containing the patients’ name, year of birth, and the 
Patient ID allocated by IBCSG. 


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Regulatory authorities and the pertinent Ethics Committee (ERB/IRB) may have access to 
patient data on-site. IBCSG audit or monitoring personnel will also have access to such data 
on-site. 
20.9 
Record Retention 
The Center must retain all essential documents according to ICH GCP. This includes copies 
of the patient trial records, which are considered as source data, patient Informed Consent 
statement, laboratory printouts, drug inventory and destruction logs, and all other information 
collected during the trial. These documents are to be stored until at least 15 years after the 
termination of the trial. IBCSG guarantees access and availability of the data entered into 
iDataFax for at least 15 years after the termination of the trial. 
Longer retention may be required for Participating Centers according to national regulations. 
In the event that the Principal Investigator retires or changes employment, custody of the 
records may be transferred to another competent person who will accept responsibility for 
those records. Written notice of such transfer has to be given to IBCSG and the local Ethics 
Committee at least one month in advance. 
21 Confidentiality 
The protocol, CRFs and other protocol-related documents are confidential and are the 
property of the IBCSG. 
22 References 
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SAP_001.pdf: 
1 
 
 
 
 
 
 
 
 
 
 
International Breast Cancer Study Group Statistical Center 
 
 
TRIAL 53-14 / BIG 14-04 (PYTHIA) 
  
 
Statistical Analysis Plan for TK1 
 
Svitlana Tyekucheva 
Luca Malorni 
 
 
Version 7 January 2020 
 
 
 


2 
 
Introduction: 
The purpose of this analysis is to explore the prognostic value of  TK1 activity levels in serum 
samples of patients enrolled in the PYTHIA trial. 
 
Serum samples for TK1 analyses are taken at the following time points: 
1) Baseline 
2) D15C1 – day 15 of cycle 1 
3) D1C2 – day 1 of cycle 2 
4) EoT –  End of treatment  
 
The baseline and end of treatment samples were collected via AURORA; the D15 C1 and D1 C2 
samples were collected specifically for this TK1 analysis. 
 
Timepoints from 1) to 3) are defined “early timepoints” in the present document. 
 
TK1 activity is measured in DU/L (Divitum Unit/Liter) and the dynamic range is 20 DU/L to 
>10.000 DU/L. Coefficient of variation of the test is about 10%. 
 
Baseline TK1 activity is prognostic in patients treated with endocrine therapy alone (data from our 
group): patients with High TK1 baseline have an adverse outcome. This may not be true for patients 
treated with endocrine therapy + Palbociclib as proliferation markers do not seem to predict 
response to CDK4/6 inhibitors. Indeed, our data on the TREnd trial (McCartney et al, Clin Cancer 
Res 2020) show that baseline TK1 levels are not prognostic in patients treated with Palbociclib 
single agent. 
 
TK1 activity is expected to be reduced during treatment with Palbociclib and Fulvestrant as a 
pharmacodynamic effect of treatment, compared to baseline.  
Preliminary data from our group suggest that some patients (10-15%) do not experience a drop in 
TK1 during treatment , measured at day 1 cycle 2  (no data available for day 15 cycle 1 in our 
cohort). These patients show primary resistance to Palbociclib single agent (TREnd trial, submitted 
for AACR meeting 2019). These data may be confirmed in the PYTHIA cohort. 
 
As Palbociclib is administered with a 3 weeks on/one week off schedule, D15 C1 is “on treatment” 
while D1 C2 is “off treatment” (blood draw taken after one week rest). Based on published data in 
the neoadjuvant setting, it may be expected that TK1 levels have some rebound after the week off 
due to the absence of palbociclib treatment. The frequency and prognostic value of the “rebound” is 
non currently known. 
 
Our data suggest that TK1 levels rise significantly at time of disease progression. Patients with 
higher TK1 levels (based on the median value at progression) have an adverse outcome on 
subsequent treatment (post-palbociclib) compared with patients with lower TK1 levels. 
 
Hypotheses: 
 
- Baseline TK1 values are not prognostic 
 
- Patients who do not experience a drop in TK1 during treatment (measured either at D15 C1 or at 
D1 C2) have an adverse outcome (it may be hypothesized that the drug does not reach its target in 
such de novo resistant patients) 
 


3 
 
- Patients who experience a “rebound” between D15 C1 and D1 C2 have an adverse outcome as 
compared to those who have a drop at D15 C1 and stay low at D1 C2) 
(it may be hypothesized that those pts with a rebound have an incomplete suppression of cell cycle 
under treatment and have therefore a reduced benefit from treatment) 
 
- TK1 levels at time of disease progression to Palbociclib and Fulvestrant may inform prognosis on 
subsequent therapy post-study. 
 
- Dynamics of TK1 changes across the whole treatment history of the patient (from start to end of 
treatment) may identify predictive patterns. 
 
PRIMARY ANALYSIS 
 
The primary outcome measure for analysis is Progression Free Survival. 
Protocol definition: the date of progression is the date that objective progression was first 
documented. Progression-free survival (PFS) is defined as time from treatment initiation until 
documented disease progression according to RECIST 1.1 criteria or death, whichever occurs first. 
For patients without progression, follow-up will be censored at the date of last disease assessment 
without progression, unless death occurs within a short period of time (12 weeks, corresponding to 
the interval of tumor re-evaluation) following the date last known progression-free, in which case 
the death will be counted as a PFS event. Patients who discontinue treatment prior to documented 
disease progression, including those who initiate non-protocol therapy prior to progression, will be 
followed for disease progression, for a maximum of 12 months.  
TK1 values at different timepoints, as well as differences between each timepoint and baseline will 
be modelled as continuous variables. If appropriate, explanatory and TK1 variables will be log-
transformed. 
 
Note: EoT TK1 measurements will be done in a separate batch from early timepoints. According to 
Biovica DiviTum assay manual “Standard samples included in the kit are used to generate a 
standard curve by which the optical density readings from the patient samples are converted to TK 
activity expressed as DiviTum Units per liter (Du/L).”  Therefore, we do not expect issues with the 
batch effects when analyzing TK1 measurements from different  timepoints together (for e.g. when 
taking difference between BASELINE and  EoT). 
 
We will use Cox proportional hazards model to test if TK1 values have statistically significant 
effect on PFS.  We will report Hazard Ratios (HRs) relative to a one unit change in TK1 values (or 
differences from baseline) and their 95% CIs.  We will fit univariable models with TK1 values 
only, as well as multivariable models that include other prognostic factors, such as: (i) visceral vs 
non visceral disease; (ii)  number of disease sites (categorical: 1-2 vs 3+, or continuous); bone-only 
disease vs all other; (iv) PYTHIA therapy as first line treatment for MBC vs second line; (v) DFI; 
(vi) duration of prior ET from MBC; (vii) primary vs secondary ET resistance; (viii) ER+/PR+ vs 
ER+/PR-; (ix) de novo MBC vs not; (x) IHC LumA vs LumB.  
Models will also be assessed using c-index.  
 
For visualization we will consider discretization of TK1 values as described below. Survival curves 
will be estimated using Kaplan-Mayer method. If appropriate, we will also fit Cox models with 
TK1 groups as categorical predictors. HRs with 95% CIs will be reported, and  c-index will be used 
to further evaluate prognostic potential of the groups. Multivariate models with TK1 variable and 
known prognostic factors will be fitted as well.  


4 
 
 
Categories of interest for visualization using KM-curves:  
 
1) BASELINE time point: patients will be dichotomized in “HIGH TK1” or “LOW TK1” based 
on the median TK1 value  
2) Difference of BASELINE AND D15C1 time points: patients will be divided in three groups: 
“TK1 drop” (i.e. patients who have a significant decrease of TK1 levels at D15 compared to 
baseline);  “TK1 increase” (i.e. patients who have a significant increase of TK1 levels at D15 
compared to baseline); “TK1 no change” (i.e. patients who have no significant changes in 
TK1). Statistical significance of a change in a sample can be established by using a  z-statistic 
defined in following way:  
 
where δ is the desired threshold (for e.g. δ=0.1*TK1Baseline for a 10% change), and variances 
for TK1 values are derived from the coefficient of variation of the assay.  
 
 
3) Difference of BASELINE AND D1C2 – similarly to Difference between BASELINE and 
D15C1, as described in (2)  
4) Difference of BASELINE AND EoT – similarly to Difference between BASELINE and 
D15C1, as described in (2)  
5) Patterns across three early timepoints: the subgroup of patients with “TK1 drop” or “TK1 no 
change” based on difference of BASELINE AND D15 C1 will be divided in two groups:  
“rebound” if TK1 levels at D1 C2 are > D15 C1 (by 10%), “no rebound” if TK1 levels at D1 
C2 are < or = D15 C1.  
6) As an exploratory option we will consider unsupervised clustering of Baseline-C1D15-
C2D1-EoT trends using partitioning around medoids to identify data-driven patterns, similar 
to (4) but without thresholding.   
 
Note: if data prompts log transformation of TK1 values differences between time points of 
transformed variables will be interpretable as log fold changes.  
 
SECONDARY ANALYSES 
 
Exploratory analysis of TK1 distributions:  
 
We will perform exploratory data analysis (EDA) for TK1 values and examine distributions of TK1 
values at various time points between each other and with respect to other prognostic clinico-
pathologic variables. We will examine boxplots and/or density plots by categorical variables, and 
scatterplots for continuous ones. Distributions will described with 5-points summaries. If EDA 
suggests strong associations between clinico-pathologic variables and TK1, we will estimate 
variance explained in TK1 measurements by clinico-pathologic factors using linear regression. 
Explanatory and response variables might be transformed if appropriate. TK1 values for individual 
patients across all time points might be visualized using spaghetti plots. 
 
Analysis of TK1 relationship with secondary outcome measures:  
 
We will compare TK1 values at baseline as well as differences between baseline and C1D14 and 
C2D1 between “early progressors” (i.e. those patients who have disease progression at first 


5 
 
imaging) (expected 5-8% of all study population) and remaining patients. Parametric (t-test) or non-
parametric (Wilcoxon) tests will be used as appropriate.  
 
HANDLING OF MISSING DATA  
If up to 10% of  patients do not have TK1 Values for all timepoints – we will exclude patients with 
missing data from all of the analyses. If more than 10% of patients have incomplete data we will 
exclude patients only from the analyses corresponding to missing timepoints. 
 
TIMELINES: 
 
Serum samples will be analyzed in batches.  
 
- First batch analysis will be done after the collection of all “early time points” – projected in Q2 of 
2020. 
- Second batch analysis will be done when EoT samples assay data becomes available.