A Multicenter Retrospective Study on the Prognostic Impact of Pregnancy in Women With History of BRCA Mutated Breast Cancer
Conditions: Breast Cancer
Sponsor: Jules Bordet Institute

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
1 
                        
 
                    
 
 
 
 
 
A multicenter retrospective study on the 
prognostic impact of pregnancy in women with 
history of BRCA mutated breast cancer 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
2 
STUDY CHAIRS 
 
 
 
 
 
 
 
STUDY SECRETARIAT 
 
 
STUDY STATISTICIANS 
 
Matteo Lambertini, MD PhD 
Medical Oncology Department 
IRCCS Ospedale Policlinico San Martino – University of 
Genova 
Genova, Italy 
Email: matteo.lambertini@unige.it 
 
Hatem A. Azim Jr., MD PhD 
Breast Cancer Center 
Hospital Zambrano Hellion Tecnolgico de 
MonterreySan Pedro Garza Garcia, Mexico 
Email: hatem.azim@gmail.com 
 
Ameye Lieveke, MSc PhD & Marianne Paesmans  
Data Centre 
Institut Jules Bordet 
Brussels, Belgium 
Email: ameye.lieveke@bordet.be 
Email: marianne.paesmans@bordet.be 
Marco Bruzzone, MSc & Marcello Ceppi, 
MSc 
Clinical Epidemiology Unit 
IRCCS Ospedale Policlinico San 
MartinoGenova, Italy 
Email: marco.bruzzone@hsanmartino.it 
Email: marcello.ceppi@hsanmartino.it 
Eva Blondeaux, MD  
Clinical Epidemiology Unit  
IRCCS Ospedale Policlinico San Martino  
Genova, ItalyEmail: evablx@libero.it 
 
Elisa Agostinetto, MD 
Medical Oncology Department 
Institut Jules Bordet 
Brussels, Belgium 
Email: elisa.agostinetto@bordet.be 
 
Michail Ignatiadis, MD PhD 
Medical Oncology Department 
Academic Trials Promoting Team 
Institut Jules Bordet, Université Libre De Bruxelles 
Brussels, Belgium  
Email: michail.ignatiadis@bordet.be 
 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
3 
SPONSOR 
 
 
Institut Jules Bordet 
Boulevard de Waterloo, 121 
1000 Brussels, Belgium 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
4 
SIGNATURE PAGE 
 
Name and Surname 
Date 
Signature 
 
Matteo Lambertini, MD PhD 
 
January 3, 2022 
 
 
Michail Ignatiadis, MD PhD 
 
January 10, 2022 
 
 
Hatem A. Azim Jr., MD PhD 
 
January 4, 2022 
 
 
 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
5 
DOCUMENT HISTORY OF REVISIONS  
 
Type of 
amendment 
Protocol 
version 
number 
Final version 
date 
Nature of amendments 
Not 
applicable 
1.0 
20 December 
2016 
Not applicable 
Amendment 
1 
2.0 
10 
January 
2022 
1) Update of the study team and its affiliations with the 
addition of Elisa Agostinetto and Eva Blondeaux as study 
secretariat, as well as Marco Bruzzone and Marcello Ceppi 
among study statisticians. 
2) Among primary objectives, revision of the definition of 
“incidence of pregnancy” and the way to analyze it; current 
definition is “rate of pregnancy” to be analyzed according 
to the Kaplan-Meier method.  
3) Addition of the three following secondary objectives 
(and respective statistical analyses): 
- To describe baseline, tumor and treatment characteristics 
as well as patterns of care including access to risk-reducing 
surgeries in BRCA mutated patients. 
- To evaluate the prognostic impact of baseline, tumor and 
treatment characteristics as well as of risk-reducing 
surgeries in BRCA mutated patients. 
- To evaluate tumor infiltrating lymphocytes (TILs) in 
BRCA mutated patients. 
4) Clarification and update of study eligibility criteria: 
accrual is allowed for patients diagnosed up to December 
2020, while patients with invasive breast cancer and 
germline BRCA variants of unknown significance are 
excluded. 
5) Inclusion of the following information for data 
collection: cause of death. 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
6 
6) Clarification of the priority for the analysis of survival 
endpoints: DFS is the primary endpoint and BCSS/OS are 
secondary endpoints. 
7) Clarifications of the type of survival analyses to be 
conducted for reducing the impact of selection bias (i.e. 
guaranteed time bias). Two analyses are expected: the 
extended Cox model analysis with occurrence of pregnancy 
as a time-varying covariate and a “case-control” analysis. 
Type of matching for the “case-control” analysis has been 
clarified and will be performed according to the following 
factors: disease-free interval, year at diagnosis, nodal status, 
hormone receptor status and type of BRCA mutation are the 
matching criteria. 
8) Addition of the ClinicalTrials.gov identifier of this study: 
NCT02308085. 
9) Corrections of typos and inconsistencies within the text. 
10) Addition of 2 columns in the excel file for data 
collection 
11) Updated list of participating centers. 
 
 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
7 
Contents 
SYNOPSIS ........................................................................................................................................... 8 
1. BACKGROUND AND SCIENTIFIC RATIONALE ................................................................... 11 
2. STUDY OBJECTIVES .................................................................................................................. 13 
3. METHODOLOGY......................................................................................................................... 14 
3.1. Eligibility criteria ................................................................................................................ 14 
3.2. Contact with Investigators .................................................................................................. 15 
3.3. Data collection .................................................................................................................... 15 
4. STATISTICS ................................................................................................................................. 16 
5. ETHICAL ASPECTS .................................................................................................................... 20 
6. DATA HANDLING AND RECORD KEEPING ......................................................................... 21 
7. DATA CONFIDENTIALITY AND TIMELINE .......................................................................... 21 
8. PUBLICATION POLICY.............................................................................................................. 21 
9. FUNDING ...................................................................................................................................... 22 
10. REFERENCES............................................................................................................................. 22 
 
 
 
 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
8 
SYNOPSIS 
 
Title of the study 
A multicenter retrospective study on the prognostic impact of 
pregnancy in women with history of BRCA mutated breast cancer 
Protocol version 
2.0 – 10 January 2022 
Sponsor 
Institut Jules Bordet, Brussels, Belgium 
Study chairs 
 Matteo Lambertini, MD PhD, IRCCS Ospedale Policlinico San 
Martino – University of Genova 
 Michail Ignatiadis, MD PhD, Institut Jules Bordet 
 Hatem A. Azim Jr., MD PhD, Hospital Zambrano Hellion 
Tecnolgico de Monterrey 
Study Objectives 
Primary objectives: 
- To evaluate the prognostic impact of pregnancy following breast 
cancer diagnosis in BRCA mutated patients. 
- To evaluate the rate of pregnancy following breast cancer diagnosis 
in BRCA mutated patients. 
 
Secondary objectives: 
- To evaluate the prognostic impact of pregnancy following breast 
cancer diagnosis in various subgroups of BRCA mutated patients 
according to: 
 Type of BRCA mutation; 
 Hormone receptor status; 
 HER2 status; 
 Exposure to chemotherapy;  
 Exposure to endocrine therapy; 
 Interval between diagnosis and pregnancy;  
 Outcome of pregnancy; 
 Breastfeeding status. 
- To evaluate the pregnancy, fetal and obstetrical outcomes of the 
pregnancies following breast cancer diagnosis in BRCA mutated 
patients. 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
9 
- To evaluate the safety of assisted reproductive technology (ART) 
procedures following breast cancer diagnosis in BRCA mutated 
patients. 
- To evaluate the impact of anticancer therapies on patients’ ovarian 
function. 
- To describe baseline, tumor and treatment characteristics as well as 
patterns of care including access to risk-reducing surgeries in BRCA 
mutated patients. 
- To evaluate the prognostic impact of baseline, tumor and treatment 
characteristics as well as of risk-reducing surgeries in BRCA mutated 
patients. 
- To evaluate tumor infiltrating lymphocytes (TILs) in BRCA mutated 
patients. 
Number of patients 
More than 2000 non-pregnant BRCA mutated breast cancer patients and 
more than 200 pregnant BRCA mutated breast cancer patients 
(estimated numbers, considering a rate of pregnancy of approximately 
10%). 
Eligibility criteria 
Inclusion criteria: 
- 
Diagnosis of invasive breast cancer between January 2000 and 
December 2020; 
- 
Breast cancer diagnosis at the age of ≤ 40 years; 
- 
Known presence of germline BRCA pathogenic variant. 
Exclusion criteria: 
- 
Known BRCA mutation with no diagnosis of invasive breast 
cancer; 
- 
Diagnosis of ovarian cancer or other malignancies with no 
history of invasive breast cancer; 
- 
Diagnosis of hereditary or familiar invasive breast cancer 
without BRCA mutation or with BRCA genes not tested; 
- 
Diagnosis of invasive breast cancer with germline BRCA 
variants of unknown significance. 
Design of the trial 
Retrospective multicenter hospital-based study 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
10
Statistics 
The achievement of pregnancy after diagnosis of breast cancer will be 
the criteria used to distinguish between two cohorts of patients: women 
with no subsequent pregnancies after breast cancer diagnosis (“non-
pregnant cohort”), and women with one or more pregnancies any time 
after breast cancer diagnosis (“pregnant cohort”). To evaluate the 
prognostic impact of pregnancy following breast cancer diagnosis in 
BRCA mutated survivors, three survival endpoints will be considered 
in the two cohorts: disease-free survival (DFS, as primary endpoint) 
breast cancer specific survival (BCSS, as secondary endpoint) and 
overall survival (OS, as secondary endpoint). To reduce the impact of 
selection bias (i.e. guaranteed time bias), two analyses will be 
performed. In the extended Cox model analysis with occurrence of 
pregnancy as a time-varying covariate, all eligible patients included in 
the study will be considered. In the “case-control” analysis, each patient 
in the pregnant cohort (case) will be matched with 3 patients from the 
non-pregnant cohort (controls). Each nonpregnant control should have 
a disease-free interval equal to or longer than the time elapsing between 
breast cancer diagnosis and date of pregnancy of the matched pregnant 
case. Patients who became pregnant after the date of the DFS event will 
be dropped from this analysis. The other matching factors will be year 
at diagnosis (± 2.5 years), nodal status (negative vs. positive), hormone 
receptor status (positive vs. negative), and type of BRCA mutation 
(BRCA1 vs. BRCA2). Because all included patients are aged ≤ 40 years 
at the time of diagnosis, matching according to age will not be 
performed. The matching will be done centrally at the coordinating 
center. The prognostic impact (in terms of DFS, BCSS and OS) of 
pregnancy following breast cancer diagnosis will be then evaluated in 
various subgroups of BRCA mutated survivors. Pregnancy rate will be 
computed according to the Kaplan-Meier method.  
 
 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
11
1. BACKGROUND AND SCIENTIFIC RATIONALE 
 
Breast cancer is the most common tumor type diagnosed in women of reproductive age [1]. The 
inheritance of a mutation in one of the breast cancer susceptibility genes (i.e. BRCA1 or BRCA2) 
generates the hereditary breast and ovarian cancer syndrome [2]. For healthy women with BRCA1 
mutated gene (i.e. BRCA1 carriers), the average lifetime risk of developing breast and ovarian cancer 
is approximately 67% and 45%, respectively; for BRCA2 carriers, these average cumulative risks are 
66% and 12%, respectively [3]. 
 
Since young women with breast cancer, including those with BRCA mutation, are more likely to 
develop biologically aggressive tumors [4], the majority of them are candidates to receive 
antineoplastic treatments that include chemotherapy. The use of cytotoxic therapy in premenopausal 
patients may result in the occurrence of treatment-related premature ovarian failure (POF) [5]. BRCA 
mutations seem to negatively affect ovarian reserve with consequent accelerated ovarian aging [6]. 
Although very limited data are available on this topic, it has been suggested that BRCA carriers may 
be more sensitive to the gonadotoxic effect of chemotherapy as compared to patients without 
mutations with subsequent higher risk of developing this side effect [6]. The impairment in ovarian 
function negatively impacts on global health of young breast cancer survivors being associated with 
several side effects including infertility [7].  
 
Concerns about fertility preservation and future chance of achieving a pregnancy are prevalent issues 
affecting young breast cancer patients [8]. Hence, accordingly, major international guidelines 
recommend physicians to address with patients as part of informed consent before anticancer 
therapies the possibility of developing POF and infertility, and then to discuss fertility preservation 
options in interested patients [9–11]. Specific issues on this regard should be considered in patients 
with BRCA mutation in the context of their unique concerns [12]. For carriers, given the increasing 
risk of ovarian cancer with age, bilateral salpingo-oophorectomy is generally recommended for both 
BRCA1 and BRCA2 carriers between the ages of 35 and 40 years and upon completion of childbearing 
[13]. Moreover, some carriers wish to eliminate the mutation from future offspring through pre-
implantation genetic diagnosis [14]. Finally, given the increasing risk of ovarian and breast cancer 
and the risk of developing long-term treatment-related POF [15], the window for fertility and 
pregnancy may be narrow in such young women. Of note, although, the last years have brought many 
safety and efficacy data on the available options for fertility preservation increasing the chances of 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
12
future offspring in cancer survivors, numerous challenges remain for young breast cancer patients 
considering fertility preservation and to have a pregnancy after the end of treatments, especially for 
those with BRCA mutations. Thus, further research is needed in the field; particularly, reproduction 
studies to address the specific issues of women with BRCA mutations are lacking and should be 
considered a research priority. 
 
Approximately 40% to 50% of women in whom breast cancer is diagnosed during childbearing age 
wish to have a subsequent pregnancy [16]. However, the percentage of breast cancer patients reported 
in the literature that has at least one full-term pregnancy after breast cancer diagnosis is very low. Of 
note, among cancer survivors, breast cancer patients have the lowest pregnancy rate with an overall 
67% reduction in the chance of giving birth after cancer treatment as compared to the general 
population [17]. This observation reflects not only the damage to ovarian reserve due to the 
gonadotoxic treatments required, but also patient and provider concerns related to a possible negative 
impact of pregnancy on the evolution of breast cancer [18].  
 
Recent data support the statement that pregnancy in cancer survivors after adequate treatment and 
follow-up should not be discouraged including among patients with breast cancer [19]. As shown in 
a meta-analysis of 14 retrospective control-matched studies, breast cancer patients who became 
pregnant following diagnosis and treatment had a 41% reduced risk of death compared to women 
who did not get pregnant (pooled relative risk [PRR], 0.59; 95% confidence intervals [CI], 0.50-0.70) 
[20]. Furthermore, a more recent multicenter retrospective cohort study confirmed the safety of 
pregnancy after breast cancer even in patients with endocrine-sensitive disease [21]. Importantly, no 
impact of abortion on patient outcome was observed either [21]. Nevertheless, in the specific 
subgroup of patients with BRCA mutations very limited data consisting in one single study are 
available on the impact of pregnancy on breast cancer survival [22]. In this study, out of the 128 
pregnant cases included, only 53 had a pregnancy following breast cancer; no difference in breast 
cancer specific mortality was observed between pregnant cases and matched non-pregnant control 
(adjusted hazard ratio [HR], 0.73; 95% CI, 0.21-2.68) [22]. However, due to the limited number of 
patients included in the analysis, no solid conclusion can be drawn from this study to counsel BRCA 
mutated survivors on the safety of having a pregnancy after breast cancer diagnosis. Furthermore, no 
information is available on the impact of breastfeeding in these patients, and it remains not clear yet 
the ideal interval to wait between the end of anticancer treatments and conception. Moreover, other 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
13
highly relevant issues remain elusive pertaining on subsequent pregnancy in BRCA mutated breast 
cancer patients, including the safety of assisted reproductive technology (ART), pregnancy outcomes, 
risk of congenital malformations, breastfeeding patterns and many more.  
 
Thus, due to the lack of data on several issues on this regard, the present study aims at refining the 
understanding of the effect of pregnancy on breast cancer outcomes in the specific population of 
BRCA mutated patients with known history of breast cancer.  
 
2. STUDY OBJECTIVES 
 
Primary objectives: 
- To evaluate the prognostic impact of pregnancy following breast cancer diagnosis in BRCA mutated 
patients. The following survival endpoints will be considered: disease-free survival (DFS, as primary 
endpoint), breast cancer specific survival (BCSS, as secondary endpoint) and overall survival (OS, 
as secondary endpoint). 
- To evaluate the rate of pregnancy following breast cancer diagnosis in BRCA mutated patients. 
 
Secondary objectives: 
- To evaluate the prognostic impact (in terms of DFS, BCSS and OS) of pregnancy following breast 
cancer diagnosis in various subgroups of BRCA mutated patients according to: 
 Type of BRCA mutation: BRCA1 vs. BRCA2; 
 Hormone receptor status: positive vs. negative; 
 HER2 status: positive vs. negative; 
 Exposure to chemotherapy: prior exposure vs. no prior exposure; 
 Exposure to endocrine therapy: prior exposure vs. no prior exposure; 
 Interval between diagnosis and pregnancy: ≤ 2 years vs. > 2 years after diagnosis; 
 Outcome of pregnancy: abortion vs. completed pregnancy; 
 Breastfeeding status: breastfeeding vs. no breastfeeding. 
- To evaluate the pregnancy, fetal and obstetrical outcomes of the pregnancies following breast cancer 
diagnosis in BRCA mutated patients. 
- To evaluate the safety of ART procedures following breast cancer diagnosis in BRCA mutated 
patients.  

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
14
- To evaluate the impact of anticancer therapies on patients’ ovarian function, in terms of incidence 
of treatment-induced POF and age at menopause. 
- To describe baseline, tumor and treatment characteristics as well as patterns of care including access 
to risk-reducing surgeries in BRCA mutated patients. 
- To evaluate the prognostic impact of baseline, tumor and treatment characteristics as well as of risk-
reducing surgeries in BRCA mutated patients. 
- To evaluate tumor infiltrating lymphocytes (TILs) in BRCA mutated patients. 
 
3. METHODOLOGY  
 
This is a multicenter hospital-based retrospective cohort study (ClinicalTrials.gov identifier: 
NCT02308085) conducted across several Institutions managing patients with hereditary breast 
cancer. The study aims to refine the understanding of the effect of pregnancy on breast cancer 
outcomes in BRCA mutated patients with known history of breast cancer. 
 
3.1. Eligibility criteria 
 
The eligibility criteria are intentionally broad for trying to exclude as few patients as possible so that 
true population-based data can be obtained. To be eligible for the present study, patients should fulfill 
the following inclusion and exclusion criteria. 
 
Inclusion criteria: 
- 
Diagnosis of invasive breast cancer between January 2000 and December 2020; 
- 
Breast cancer diagnosis at the age of ≤ 40 years; 
- 
Known presence of germline BRCA pathogenic variant. 
 
Exclusion criteria: 
- 
Known BRCA mutation with no diagnosis of invasive breast cancer; 
- 
Diagnosis of ovarian cancer or other malignancies with no history of invasive breast cancer; 
- 
Diagnosis of hereditary or familiar invasive breast cancer without BRCA mutation or with 
BRCA genes not tested; 
- 
Diagnosis of invasive breast cancer with germline BRCA variants of unknown significance. 
 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
15
3.2. Contact with Investigators 
 
The study chairs and secretariat of this project will invite investigators from different institutions 
worldwide to participate in this project.  
Upon agreement, investigators will be requested to provide anonymous individual patient data. A 
principal contact will be requested per Institution in case of data clarification needs. A timeframe of 
4 months is given for data collection from each Institution after a positive opinion is granted by the 
respective Independent Ethical Committee / Institutional Review Board. 
 
3.3. Data collection 
 
For all consecutive eligible patients included in the study from each participating Institution, their 
medical records will be retrieved and anonymized data entered into a database. The following 
information will be collected, if available: 
 
1) Baseline characteristics: body mass index; smoking habit; age at menarche; age at menopause; 
menopausal status at the time of breast cancer diagnosis; use of birth control pills; prior children 
before breast cancer diagnosis; history of spontaneous abortion; history of induced abortion; prior 
treatment for infertility; prior gynecological surgery and/or medical history with possible impact on 
fertility.  
2) Breast cancer history: date of diagnosis; age at diagnosis; disease stage; laterality of the primary 
tumor; type of breast and axillary surgery; use of breast reconstruction surgery; grade; histology; 
tumor size and nodal status (for patients who underwent primary systemic therapy, only clinical tumor 
size and nodal status will be collected); hormone receptor status (estrogen and progesterone 
receptors); HER2 status. 
3) Treatment of the primary tumor: timing of systemic therapy administration; use of chemotherapy 
(type and number of cycles); use of endocrine therapy (type and duration); use of anti-HER2 targeted 
therapy (type and duration); use of radiotherapy. 
4) Survival status: any recurrence of invasive breast cancer including date of the event; any second 
primary breast cancer including date of the event; any second primary malignancy including date of 
the event; date of last follow-up and/or death; cause of death (breast cancer-related or not). 
5) Ovarian function after chemotherapy: treatment-induced POF; age at menopause. 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
16
6) Pregnancy after breast cancer: any pregnancy including date of the event; pregnancy outcome; 
gestational age at delivery; any pregnancy/fetal/birth complications; breastfeeding and its duration; 
use and type of ART. 
7) BRCA mutation: date of BRCA testing; type of BRCA mutation; use and date of breast prophylactic 
surgery; use and date of gynecological prophylactic surgery. 
8) Additional information: diagnosis of breast cancer during pregnancy, availability of digital scanned 
images for TILs analysis 
 
Data can be sent in confidence to Matteo Lambertini (matteo.lambertini@unige.it) and Elisa 
Agostinetto (elisa.agostinetto@bordet.be) where they will be held secure and only used for the 
purposes of this study.Analyses will only be undertaken under the supervision of the study 
statisticians. 
 
4. STATISTICS 
 
The achievement of pregnancy after diagnosis of breast cancer will be the criteria used to distinguish 
between two cohorts of patients: women with no subsequent pregnancies after breast cancer diagnosis 
(“non-pregnant cohort”), and women with one or more pregnancies any time after breast cancer 
diagnosis (“pregnant cohort”). Pregnancies could have resulted in live births, therapeutic or 
spontaneous abortions. 
 
More than 2000 non-pregnant BRCA mutated breast cancer patients and more than 200 pregnant 
BRCA mutated breast cancer patients are expected to be included in the present study (estimated 
numbers, considering an incidence of pregnancy of approximately 10%). 
 
To evaluate the prognostic impact of pregnancy following breast cancer diagnosis in BRCA mutated 
survivors, three survival endpoints will be considered in the two cohorts: DFS (as primary endpoint) 
BCSS (as secondary endpoint) and OS (as secondary endpoint). DFS event is defined by the 
occurrence of one of the following invasive events: local recurrence, distant metastases, contralateral 
or ipsilateral breast tumor, second primary malignancy, or death from any cause. BCSS event is 
defined as death from breast cancer. OS event is defined as death from any cause. For DFS, 
observation times of patients without the event will be censored on the date of their last contact. Using 
individual patient data from all patients included in the study, survival outcomes of patients in the 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
17
pregnant cohort will be compared to those of patients in the non-pregnant cohort. To reduce the 
impact of selection bias (i.e. guaranteed time bias), two analyses will be performed. In the extended 
Cox model analysis with occurrence of pregnancy as a time-varying covariate, all eligible patients 
included in the study will be considered.  In the “case-control” analysis, each patient in the pregnant 
cohort (case) will be matched with 3 patients from the non-pregnant cohort (controls). To adjust for 
guaranteed time bias, each patient in the non-pregnant cohort should have a disease-free interval equal 
to or longer than the time elapsing between breast cancer diagnosis and date of pregnancy of the 
matched patient in the pregnant cohort. Patients who became pregnant after the date of the DFS event 
will be dropped from this analysis. Patients will be also matched according to several variables 
including type of BRCA mutation (BRCA1 vs. BRCA2), hormone receptor status (positive vs. 
negative), nodal status (negative vs. positive), and year at diagnosis (± 2.5 years). Because all 
included patients are aged ≤ 40 years at the time of diagnosis, matching according to age will not be 
performed. The matching will be done centrally at the coordinating center. Two data sets will be 
constituted: one for the cases and one for the potential controls. Cases will be sorted randomly and 
matching will be done starting for the first case until the last one. For each case, all possible controls 
will be extracted from the data set of potential controls. If they are only 2 controls available, matching 
criteria will be relaxed one by one (starting from year of diagnosis, etc) until at least a third control 
is found. It is anticipated, given the expected low rate of cases in the full database, to reach matching 
for almost each case. In these analyses, all survival endpoints will be calculated from the date of 
pregnancy until the date of the event. In the non-pregnant cohort, survival endpoints will be calculated 
from the date of diagnosis, adding the time elapsing between diagnosis and pregnancy of the matched 
pregnant case. 
The prognostic impact (in terms of DFS, BCSS and OS) of pregnancy following breast cancer 
diagnosis will be then evaluated in various subgroups of BRCA mutated survivors according to: type 
of BRCA mutation (BRCA1 vs. BRCA2), hormone receptor status (positive vs. negative), HER2 status 
(positive vs. negative), exposure to chemotherapy (prior exposure vs. no prior exposure), exposure to 
endocrine therapy (prior exposure vs. no prior exposure), interval between diagnosis and pregnancy 
(≤ 2 years vs. > 2 years after diagnosis), outcome of pregnancy (abortion vs. completed pregnancy), 
and breastfeeding status (breastfeeding vs. no breastfeeding). 
Considering a rate of pregnancy of approximately 10%, with 2000 non-pregnant and 200 pregnant 
BRCA mutated breast cancer patients, assuming a DFS rate of 65% at 5 years in the non-pregnant 
cohort [21], the study will have a power of 0.83 to detect a hazard ratio of 0.75 and a power of 0.62 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
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to detect a hazard ratio of 0.80 in favor of the pregnant cohort, at a two-sided significance level 0.05 
(accrual duration 2000-2020, total study duration 2000-2030). 
 
The rate of pregnancy following breast cancer diagnosis will be computed according to the Kaplan-
Meier method. A possible increasing or decreasing trend in rate of pregnancy following breast cancer 
over the years will be also assessed. 
 
The statistical analysis to evaluate the pregnancy, fetal and obstetrical outcomes of the pregnancies 
following breast cancer diagnosis in BRCA mutated survivors will be mainly descriptive and will 
include the following parameters: 
1. Gestational age at delivery; 
2. Incidence of early pre-term (< 34 weeks), late-preterm (34-36 weeks) and full-term (≥ 
37 weeks) pregnancies; 
3. Number of pregnancies resulting in live birth and number of children born; 
4. Incidence of induced and spontaneous abortion; 
5. Incidence and nature of pregnancy complications (if any); 
6. Incidence and nature of fetal complication and/or congenital malformations (if any); 
7. Incidence and nature of obstetrical complications (if any). 
 
To evaluate the safety of ART procedures following breast cancer diagnosis in BRCA mutated 
survivors, two groups of patients within the pregnant cohort will be considered: patients who achieved 
pregnancy with the use of any ART procedure (i.e. “ART group”) and patients who achieved 
pregnancy without the use of any ART procedure (i.e. “no ART group”). A subgroup analysis will 
be performed to evaluate the safety of controlled ovarian stimulation as part of ART within the ART 
group. 
 
In patients who underwent chemotherapy as part of (neo)adjuvant systemic therapy, the impact of 
anticancer therapies (i.e. chemotherapy) on patients’ ovarian function, in terms of incidence of 
treatment-induced POF and age at menopause will be evaluated, if available. According to the World 
Health Organization definition of postmenopausal status [23], treatment-induced amenorrhea will be 
defined by the absence of menses for ≥ 12 months after the end of chemotherapy. Age at menopause 
will be defined as age in which patients had absence of menses for ≥ 12 months. For patients who 

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Protocol version 2.0: 10 January 2022 
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19
developed treatment-induced amenorrhea but then had a recovery of menses more than 12 months 
after the end of chemotherapy and with active menstrual function within 12 months prior to the last 
follow-up visit, they will not be considered as having developed menopause. Subgroup analyses will 
be performed according to age at diagnosis, type of chemotherapy used (anthracycline- and taxane-
based regimens vs. anthracycline-based regimens vs. other regimens), and use of endocrine therapy 
(yes vs. no). These results will be compared to historical data in the BRCA wild-type breast cancer 
population. 
 
Considering the sample size and the uniqueness of the young cohort of BRCA mutated patients 
expected to be included in the study, additional analyses will be conducted to describe baseline, tumor 
and treatment characteristics as well as patterns of care including access to risk-reducing surgeries. 
Possible changes over time will be also assessed. Moreover, similarly, the prognostic impact of 
baseline, tumor and treatment characteristics as well as of risk-reducing surgeries in BRCA mutated 
patients will be also evaluated. 
In order to conduct the evaluation of TILs in this cohort, there will be no need for new 
cutting/preparation of tumor slides. The evaluation will be conducted on one archived diagnostic 
tumor-containing slide stained with hematoxylin and eosin representative of the primary tumor. The 
TILs scoring will be performed centrally. If available, anonymized digitally scanned images of the 
diagnostic hematoxylin and eosin-stained slide should be sent via email to Maria Vittoria Dieci 
(mariavittoria.dieci@unipd.it) at University of Padova-Istituto Oncologico Veneto (Padova, Italy). 
The University of Padova-Istituto Oncologico Veneto will take care of costs related to TILs scoring 
and storage support (hard disk). The evaluation will be performed according to the TILs working 
group recommendations [24]. 
 
Patients demographic, clinical and pathologic baseline characteristics will be compared by pregnancy 
status using chi-square test or Fisher Exact test for categorical variables and Wilcoxon rank-sum test 
or t-test for continuous variables, as appropriate. 
Dichotomous outcomes will be compared using univariate and multivariate logistic regression models. 
Time-to-event outcomes will be estimated and plotted using Kaplan-Meier methods. Survival rates 
will be compared using log-rank test and Cox proportional hazards models for univariate and 
multivariate analysis, respectively.   
All tests will be two-sided and p-values of < 0.05 will be considered statistically significant. All 

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20
analyses will be performed using standard available statistical programs.  
 
5. ETHICAL ASPECTS 
 
The study will be submitted by the investigator(s) or the Sponsor (or its legal representative) in 
accordance with local regulations to and approved by an appropriate Independent Ethical Review 
Committee / Institutional Review Board and a Regulatory Authority if required by the national laws 
of the countries where the study will be conducted. 
 
The investigators and the Sponsor will ensure that the study is conducted in full conformance with 
the principles of the “Declaration of Helsinki” 1964, as revised from time to time or with the laws 
and regulations of the country in which the research is conducted, whichever affords the greater 
protection to the individual. For the study, the principal investigators will ensure compliance with the 
latest European Union (EU) Data Protection Directive or with local law especially if it affords greater 
protection to the patient. 
 
If required according to the national regulations and laws, it is the responsibility of the Investigator 
or a person designed by the Investigator (if acceptable by local regulations) to obtain written informed 
consent from each potential subject prior any study data collection being carried out. However, when 
allowed by the national regulations and laws, the investigators of the present study will apply to the 
Independent Ethical Review Committee / Institutional Review Board for a waiver of the need to 
obtain informed consent. The reasons are: 
(i) it is a retrospective study that involves no interventions with human subjects. Medical records will 
be reviewed, but none of the patients will be contacted; 
(ii) the research involves no more than minimal risk to the subjects;  
(iii) the waiver will not adversely affect the rights and welfare of the subjects; moreover, the research 
could not practicably be carried out without the waiver; 
(iv) the information collected does not include information that may be damaging to the individual 
should it be wrongfully disclosed; patients’ privacy will be protected throughout the study; 
(v) data will be anonymized and stored electronically in a restricted access network folder and will 
only be accessible to the research team. 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
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Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
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21
 
6. DATA HANDLING AND RECORD KEEPING 
 
The Patient’s right to confidentiality is paramount. Collected data will be anonymized. All data will 
be kept confidential and will be used only for the purpose of this study. Local data privacy laws, rules 
and guidelines must be respected. 
 
7. DATA CONFIDENTIALITY AND TIMELINE 
 
Data will be provided by investigators of the participating Institutions in confidence to Institut Jules 
Bordet, where they will be kept secure. The data collection will be performed by Matteo Lambertini 
and Elisa Agostinetto, whereas the database set up, data cleaning and the statistical analysis will be 
performed by the study secretariat and statisticians in charge of the overall analysis once the database 
has been cleaned and locked.  
The proposed timeline for project from the receipt of all data, database assembly and cleaning, 
statistical analysis to first results is 6 months. 
 
8. PUBLICATION POLICY 
 
A manuscript summarizing the results of the main analysis will be prepared for submission to and 
publication in a peer-reviewed scientific journal. Regular emails, teleconferences and/or face-to-face 
meetings will be organized to discuss progress of the data analysis. The manuscript will be prepared 
by one or more of the study chairs along with the study secretariat and statistical team and will be 
submitted to the all collaborators for review. Authorship will be based on number of included patients: 
the 3 Institutions with the highest number of included patients will be granted to up to two authors, 
while each of the collaborating Institutions will allocate one or no author.   
 
Any other publication arising from this project will be made on behalf of the study chairs directly 
involved in that specific analysis and the principal investigators from the participating Institutions 
that actively contributed to that specific analysis.  
 

A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
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Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
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22
All authors will be given sufficient time to provide comments and attempts will be made to come to 
mutual agreement. No financial benefits will be pursued or derived by the study and the 
corresponding results. 
 
9. FUNDING 
 
This project will be supported by Institut Jules Bordet. Unfortunately, it cannot be offered any 
financial reimbursement for involved investigators. 
 
10. REFERENCES 
 
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A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
23
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A multicenter retrospective study on the prognostic impact of pregnancy in women with history of BRCA mutated breast cancer 
 
Protocol version 2.0: 10 January 2022 
CONFIDENTIAL 
Information and data included in this protocol contain trade secrets and privileged or confidential information which is the property of the Institut 
Jules Bordet. No person is authorised to make it public without the written permission of the Institut Jules Bordet 
 
24
(TILs) in breast cancer: recommendations by an International TILs Working Group 2014. Ann Oncol. 
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