BREAst screening Tailored for HEr (BREATHE)-A study protocol on personalised risk-based breast cancer screening programme.

Routine mammography screening is currently the standard tool for finding cancers at an early stage, when treatment is most successful. Current breast screening programmes are one-size-fits-all which all women above a certain age threshold are encouraged to participate. However, breast cancer risk varies by individual. The BREAst screening Tailored for HEr (BREATHE) study aims to assess acceptability of a comprehensive risk-based personalised breast screening in Singapore. Advancing beyond the current age-based screening paradigm, BREATHE integrates both genetic and non-genetic breast cancer risk prediction tools to personalise screening recommendations. BREATHE is a cohort study targeting to recruit ~3,500 women. The first recruitment visit will include questionnaires and a buccal cheek swab. After receiving a tailored breast cancer risk report, participants will attend an in-person risk review, followed by a final session assessing the acceptability of our risk stratification programme. Risk prediction is based on: a) Gail model (non-genetic), b) mammographic density and recall, c) BOADICEA predictions (breast cancer predisposition genes), and d) breast cancer polygenic risk score. For national implementation of personalised risk-based breast screening, exploration of the acceptability within the target populace is critical, in addition to validated predication tools. To our knowledge, this is the first study to implement a comprehensive risk-based mammography screening programme in Asia. The BREATHE study will provide essential data for policy implementation which will transform the health system to deliver a better health and healthcare outcomes.

Introduction

Population-based mammography endeavours to reduce mortality via early detection and prompt treatment [1-3]. Despite growing evidence of high heterogeneity of breast cancer risk within populations, breast cancer screening programmes commonly recommend starting mammography screening at age 40 or 50 [4]. Furthermore, mammographic screening itself has many limitations–over-diagnosis and overtreatment being prime among them [5]. While substantially increasing the number of cases of early-stage breast cancer detected, it only marginally reduces the rate at which women present with advanced cancer, as illustrated in the Cochrane reviews [6], Canadian National Breast Screening Study [7] and other studies [8,9]. This has generated international interest in a more risk-stratified approach to the current “one-size-fits all” population screening programmes [10-15].

BreastScreen Singapore, a nation-wide mammography screening programme in Singapore established in 2002 by the Health Promotion Board, invites women aged 50 to 69 to participate in the early detection of breast cancer. However, only 66% of the target group have reported to ever had a mammogram, and half of them do not adhere to the recommended biennial screening guideline (<30% of the target group were reported to attend mammogram every 2 years) [16]. Lukewarm responses to these initiatives have been attributed to a low perception of risk and misperceptions of risk factors and knowledge of breast cancer by women [16-21]. A number of studies have since proposed that risk-based screening may improve timeliness of screening. Furthermore, under the current age-based screening paradigm, approximately 30% of diagnosed breast cancer cases in Singapore are women of a younger age than the recommended screening age by the national guidelines [22]. The striking difference of ~10 years in the peak age for breast cancer in between Asian (40 to 50 years) and Western (60 to 70 years) prompts the need to reconsider screening approaches adapted from Western studies in Asia [23]. The design and adoption of risk-stratified approach to screening is needful for timely identification and treatment of these high-risk individuals.

Personalised screening enhances an age-based screening paradigm by tailoring screening recommendations to the individual’s risk profile [24]. This reduces the rate of false positive results and over-diagnosis in lower risk individuals, thereby providing a more effective method to identify high risk individuals for intervention [25]. Currently, to identify high risk individuals, most screening programmes rely primarily on the evaluation of age, family history, clinical and lifestyle factors, and the testing of pathogenic variants in breast cancer predisposition genes [14,26].

Breast cancer is a multifactorial disease with both genetic and non-genetic risk factors. The Gail model (also known as the Breast Cancer Risk Assessment Tool) was first developed in 1989 for prediction using non-genetic risk factors in Whites, and has since been calibrated and validated for other ethnicities [27]. Furthermore, information from the first screen (i.e. mammographic density and false positive status) are indicators of elevated risk [28]. The validated Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) model is able to predict carriership of mutations in known breast cancer genes such as BRCA1 and BRCA2 [2,29-32].

Known pathogenic variants are rare. Due to cost issues, they are usually tested in only high risk individuals [33,34]. Common variants (i.e. single nucleotide polymorphism (SNP)) associated with breast cancer risk have been discovered through genome-wide association studies [35]. Individually, these SNPs have minimal effect on risk. However, Mavaddat et al. built a polygenic Risk Scores (PRS)–a tally of 313 SNPs–that emerged as a robust means to estimate an individual’s risk of breast cancer [36,37]. The PRS (313 SNPs) is able to reliably predict breast cancer risk, with those in the top centile having a lifetime absolute risk of 32.6% [38]. This PRS has been validated in women of Asian descent [38]. Despite a growing body of evidence illustrating the utility of PRS in population screening programmes, policy implementation has been low [3]. While the Gail model [27], mammographic density [39] and breast cancer predisposition genes [40] have been incorporated into prior risk stratification studies, implementation of PRS is less common [3].

BREATHE is a landmark study aiming to contextualise a personalised, risk-based screening approach to the Asian population (specific aims are listed in Table 1). The present study endeavours to explore the acceptability and potential impact on changes in screening behaviour of the BREATHE risk-stratified screening programme as the first step towards policy implementation. With the cost-effectiveness of similar approaches validated [41], it is hoped that BREATHE will greatly enhance resource allocation and patient outcomes in the era of precision medicine.

Table 1

Specific aims of BREAst screening Tailored for HEr (BREATHE).

The primary aims of our study are to:

1) Study the acceptability of risk stratification to aid women for decision making to attend regular screening2) Assess if risk-based screening will improve willingness to screen and recall rates3) Evaluate the cost-effectiveness of changing screening frequencies based on the risk-based BREATHE breast screening strategy over the current age-based paradigm.

The secondary aims of our study are:

1) Assess the current level of breast cancer awareness, given the increasing breast cancer education in the recent decade2) Study the association between breast cancer perceptions (e.g. family history, age, having children) and compliance to regular breast cancer screening3) Study changes in breast cancer risk factors (e.g. number of children, menopausal status/age)

Materials and methods

Study design and setting

BREATHE is a prospective multi-centre cohort to study a new modality of breast cancer screening in healthy Singaporean women aged between 35 and 59. Recruitment started in Oct 2021 and plans to recruit ~3,500 participants from two hospitals (Ng Teng Fong General Hospital and National University Hospital) and two polyclinics (Bukit Batok Polyclinic and Choa Chu Kang Polyclinic) over a period of two years. To achieve coverage of all age groups of interest, recruitment targets were allocated as such: 20% aged 35 to 39 years; 40% aged 40 to 49 years; and 40% aged 50 to 59 years. The proportion selected was based on the background population in the 2019 population report published on Singapore Department of Statistics. Participants will be on active follow-up for two years. In brief, enrolled participants will be asked (1) to provide a buccal swap for genotyping at study entry and (2) to answer various questionnaires and surveys at study entry and at the two follow-ups (at ~3 months and ~2 years after study entry) (Fig 1). All surveys are translated to the three major languages used in Singapore: Mandarin, Malay and Tamil.

Fig 1

Summary of the recruitment and follow-up process.

This study was approved by the National Healthcare Group Domain Specific Review. Board (reference no: 2020/01327). Written informed consent will be obtained from each participant.

Identification of eligible participants

The study team will identify potential participants (1) through the response to our advertisements on BREATHE (posters, flyers [see S1 Appendix], and blog.nus.edu.sg/BREATHE) or (2) by approaching them at the participating institutions. The locations include diagnostic departments, women’s clinics and waiting areas of the participating institutions. Responders to our advertisements can either call our hotline, email or fill up an online registration form (see S2 Appendix). They will be screened by study team members according to the eligibility criteria. Appointments will be scheduled for eligible participants to visit the participating hospitals or polyclinics for recruitment.

Eligibility criteria

Participants must be Singapore Citizens or Permanent Residents, female, aged 35–59 years old. Women who have histologically confirmed diagnosis of any cancer, cognitive impairment which prevent the participant from giving voluntary consent, or are pregnant during recruitment will be excluded. Informed consent will be sought by trained study coordinators in the participant’s language of choice (English, Chinese or Malay).

First visit

After providing informed consent, participants will be asked to complete a demographic and lifestyle questionnaire and provide a buccal swab sample. A brief education session on breast cancer knowledge and the importance of regular and timely breast self-examination/screening will be self-administered by participants. Depending on their age, the participant will be advised to attend mammography (aged 40 years and above) breast screening. The session will end with a recruitment experience survey.

First visit questionnaire

Participants will fill in a structured questionnaire detailing various factors associated with the development of breast cancer and related conditions at baseline and over time. These include non-genetic risk factors (demographic, lifestyle, reproductive), past treatments and other environmental factors.

Buccal swab, DNA extraction and genotyping

Buccal swab (DNA Genotek, ORAcollect-DNA kit) samples will be de-identified and sent in batches (weekly) for deoxyribonucleic acid (DNA) extraction (QIAamp DNA Blood Midi Kit, Part No. 51185). Genotyping will be done (Illumina Global Screening Array [GSA-MD v3.0]) as per manufacturers’ instructions.

DNA extracted from the bio-specimens will be stored in the freezer at -20 degrees Celsius for the duration of our research study. For participants who have agreed to the usage of their bio-specimens for future studies, DNA will be stored after study completion.

Breast cancer education session

A brief online education session will first assess the screening habits of the participants and their views about breast cancer (S3 Appendix). Various statements regarding breast cancer will be presented for participants to indicate their agreement. The correct answer and an accompanying explanation are given after every response submitted. The aim is to impart correct information about breast cancer and the importance of regular and timely breast self-examination/screening.

Experience survey

A short survey will be conducted to obtain feedback from the participants on their experience (including any discomfort) with the buccal swab and their initial views about risk-stratified breast cancer screening (S4 Appendix).

Mammography screening

Participants may choose to attend screening within the next few months. The study coordinator will assist with setting up appointments for mammography screening with the participating institutions if required. If the participant (aged 40 and above) chooses to attend mammography screen, the study coordinator will seek consent to extract the mammogram image from the service provider (National Healthcare Group Diagnostics). Participants who had a recent mammogram (within one year prior to recruitment) done with National Healthcare Group Diagnostics can choose to provide consent for the study to extract the mammogram image.

Risk stratification process and personal breast cancer risk report

Participants will be classified as above average, average or below average risk, based on (1) the Gail model; (2) information from the most recent mammography screening (mammography density and positive recall status); (3) BOADICEA; and (4) the PRS. Participants will first be considered average risk and reclassified as above average or below average based on the criteria in Table 2. A risk report will be produced and presented to the participant during the first follow-up session. All participants are recommended to follow current national guidelines (Table 3). In the BREATHE programme, women identified to be above average in breast cancer risk are referred to breast specialists at designated study sites, in addition to prevailing guidelines.

Table 2

Breast cancer risk reclassification criteria.

Individuals who met any one of the following criteria will be considered above average risk:

• Predicted to be carriers of BRCA1 or BRCA2 by BOADICEA    • Extremely dense breast, which is ascertained according to the breast composition categories of the Breast Imaging-Reporting and Data System (5th edition)    • Positive recall status    • Gail model five-year absolute risk above 1.3%a    • Polygenic Risk Score (PRS) five-year absolute risk above 3%b

Individuals who met all of the following criteria will be considered below average risk:

• Age <50 years    • Gail model five-year absolute risk below 1.3%a    • PRS five-year absolute risk below 1.3% a

a The threshold of 1.3% is equivalent to the five-year absolute risk of developing breast cancer for an average Caucasian woman aged 50 years [42]. b The risk of an average BRCA carrier [43].

Table 3

National guidelines for breast cancer screening in Singapore.

Age groups, years	National guidelines
35 to 39	No recommendation
40 to 49	Women are to attend yearly mammography screening, if recommended by their doctor.
50 to 59	Women are to attend mammography screening once every two years.

Gail model (non-genetic risk factors)

The Gail Model requires the following breast cancer risk factors from the questionnaire from the first visit: age, age at menarche, age at first live birth, number of previous benign breast biopsies, presence of atypical hyperplasia on biopsy, family history of breast cancer (mother, sisters or daughters), and ethnicity [44,45]. Weights (logistic regression coefficients derived from the Gail model) and attributable risks of Asian-Americans will be used in the calculation of five-year absolute risk based on the Gail model (“Asian.AABCS”, BCRA package in R) [45].

Information from most recent mammography screening

Mammographic density will be ascertained according to the breast composition categories of the Breast Imaging-Reporting and Data System (5th edition): almost entirely fatty, scattered areas of fibroglandular density, heterogeneously dense or extremely dense. A participant is considered recalled (i.e positive recall status) when she is asked to return for additional confirmatory examination or additional mammography views due to abnormal findings from initial screening.

Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) predictions for breast cancer predisposition genes

Carrier probabilities for breast cancer predisposition genes such as BRCA1 and BRCA2 will be predicted using the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA, web application v3, https://ccge.medschl.cam.ac.uk/boadicea/boadicea-web-application/, accessed Dec 28, 2021) [40]. Briefly, as described by Antoniou et al [40], the probability that an individual carries a mutation in BRCA1/BRCA2 or other breast cancer genes based on family history can be computed using Bayes theorem.

Breast cancer polygenic risk score

PRS is estimated as the weighted sum of effect alleles in 313 SNPs found to be associated with breast cancer; using PLINK (version 3) with the “scoresum” option [46]. where xk is the risk allele (0, 1, 2) for SNP k, βk is the corresponding weight. The weights of the 313 SNPs for overall breast cancer risk were obtained from are of the overall breast cancer risk published by Mavaddat et al [47].

First follow-up session

The first follow-up session occurs within three months of the recruitment date. This involves an in-person review of the risk reports and ends with a survey on their understanding of the risk report (S5 Appendix). Participants will be reimbursed S$10 for their time, inconvenience and transportation costs at the end of the first follow-up session.

Second follow-up session

This is the final in-person follow-up conducted for all participants and occurs approximately two years from date of recruitment. The study coordinator will administer a questionnaire on non-genetic risk factors to capture any changes in participant characteristics since the first visit. The session ends with a satisfaction survey to understand the acceptability of our proposed risk stratification screening programme (S6 Appendix). Participants will be reimbursed S$10 for their time, inconvenience and transportation costs at the end of the final study visit.

Passive follow-up

Mammogram images will be extracted if participants have undergone breast screening up to 31 March 2025. In addition, clinical information (e.g. radiology reports, medical conditions, medications and medical reports) related to this study will be retrieved from hospital/polyclinic medical records in accordance to the institutional guidelines, up to 31 December 2030. Clinical information may also be obtained through linkage to nation-wide health-related databases (Singapore Cancer Registry and the Registry of Births and Deaths), and may be done up to 31 December 2030.

Planned statistical analysis

To gauge the acceptability of risk stratification (Primary Aim 1) and the current level of breast cancer awareness (Secondary Aim 1), descriptive statistics will be performed. Chi-square test for categorical variables and Kruskal-Wallis test for continuous variables will be used for testing differences among risk groups. Post-hoc analysis may be applied for pairwise comparisons. Information will be obtained from the recruitment experience survey (e.g. “I will recommend doing a breast cancer risk classification to others [options: strongly agree, agree, neither agree nor disagree, disagree, strongly disagree]”), report feedback survey (e.g. “I am confident that my personalized breast cancer risk profiles are reliable [options: strongly agree, agree, neither agree nor disagree, disagree, strongly disagree]”), and satisfaction survey (e.g. “What do you like about your experience in this study?”) (Primary Aim 1); and from the breast cancer education questionnaire (e.g. “I am still young, therefore I do not need to screen for breast cancer [options: agree, disagree]”) (Secondary Aim 1).

Logistic regression will be used to study the association between risk perceptions (i.e. risk categories and perceived risk) and follow-up events, which includes actual attendance of breast cancer screening and recall rates (Primary Aim 2 and Secondary Aim 2). Other modelling techniques will be employed dependent on event rates. Adjusted analysis may be done if variability in demographic variables are significant (e.g. conditional logistic models).

To understand potential short-term changes in breast cancer risk factors, paired analysis (e.g. paired-t-test, rank-sum test) between information from the first visit and follow-up will be performed (Secondary Aim 3).

Taking the healthcare system perspective, a cost-utility analysis will be conducted to compare BREATHE’s recommendation with the prevailing breast cancer screening guidelines using a Markov model (Aim 3). Utility weights and various unit costs will be sourced from existing literature. Additional costs associated with breast cancer risk profiling, and changes in healthcare expenditure and health outcomes for different risk groups will be examined. The incremental cost-effectiveness ratio (incremental cost/incremental quality-adjusted life years) will be calculated to understand the cost-effectiveness of BREATH recommendations. The resulting model code and parameters will be made publicly available in an independent study.

Discussion

Many previous works have evaluated the validity and discriminatory power of breast cancer risk calculators, alone or in combination [27,28,48]. In spite of the advances in breast cancer risk prediction, screening recommendations in practice have remained largely unchanged for the past few decades [23]. Several large-scale studies conducted in populations of European ancestry, such as KARMA—KARolinska MAmmography Project for Risk Prediction of Breast Cancer [49], PROCAS—Predicting the Risk of Cancer at Screening [50], WISDOM—Women Informed to Screen Depending On Measures of risk [43], are already underway to evaluate the feasibility of implementing risk stratification in breast screening programmes. However, prediction tools should be validated and calibrated to the target population [51]. To our knowledge, BREATHE is the first initiative to incorporate risk stratification approaches to enhance the efficacy of existing breast screening protocols in Asia.

Our study leverages on the existing national breast cancer screening programme (BreastScreen Singapore) [21]. Hence mammography service is consistent across all participants. The setup is scalable to include additional hospitals and polyclinics in the future. Singapore is geographically small and convenient for participants to visit breast clinics for recommendations to manage their breast cancer risk. While potential participants can visit multiple hospitals or polyclinics throughout our recruitment period, each individual’s unique National Registration Identity Card number allows us to track them for follow-up. Loss to passive follow-up due to emigration is expected to be minimal for the duration of the study. The BREATHE risk classification is adapted from the established WISDOM Personalized Breast Cancer Screening Trial [43]. WISDOM uses a five-year absolute risk threshold of 6% (risk of an average BRCA carrier) for stratification [43]. However, it is known that the incidence of breast cancer among Asian women is lower [38,43]. Hence, the BREATHE study uses five-year absolute risk above 3% as a threshold (equivalent to women aged 50 years at the top risk percentile based on PRS in Singapore, S7 Appendix).

In this study, only one risk report is generated per participant based on information at recruitment. Over time, the participant’s risk will change. An updated risk report is recommended if the programme were to be implemented in a nation-wide screening programme. As our risk estimates reflect a five-year absolute risk by the Gail model and PRS, a review of breast cancer risk should be done at a minimum frequency of five years.

Personal risk of breast cancer is a difficult concept to grasp. Breast cancer risk prediction tools are mainly designed for providers, hence the participant may have a limited understanding of the results [52]. We deliberated over the readiness of the Singapore’s population to receive personalised disease risk results with various stakeholders (representatives of the hospitals and clinics, ethics representatives, public health experts, and researchers), and concluded that only the general risk classification (below-average, average and above-average risk) is to be conveyed to the participants as part of this research study.

Details on the risk classification categories are communicated and explained to the participants by healthcare professionals to ensure that the results are interpreted accurately and meaningfully. At recruitment, participants are briefed about the risk stratification tools used in this study. We understand that in this current information age, it is likely that there will be participants especially those in the above-average risk group will want details about their risk levels. Our main concern is in the participants’ understanding of genetic risk, in particular the genetic risk component. As we want to create an inclusive risk-based screening program, we are not performing clinical genetic testing as part of this screening design. Unlike the better known and more expensive clinical genetic testing, PRS is a new method of breast cancer risk assessment that is not familiar to our study population. To avoid the situation where participants reject screening due to any misconception about risk (i.e. the idea that if I am genetically above-average risk there is nothing I can do to avoid getting breast cancer), we will only indicate the general risk level in the risk report. Based on the risk assessment by our ethics board, it is prudent that the follow-up of the above-average risk group and conveying of the details of the risk classification is done based on current clinical practice by clinicians and not part of the research study. Participants who are predicted carriers of BRCA1/2 by BOADICEA will be recommended to see a breast specialist and referred to genetic counselling and subsequently genetic testing if appropriate.

We expect challenges in attaining our target recruitment of 3500 healthy women aged between 35 and 59. As this is a hospital led research study, publicity is limited to the hospitals and affiliated polyclinics. BREATHE may be extended to other sites (e.g. Alexandra Hospital and Jurong Medical Centre) to increase recruitment numbers. Participants are encouraged to refer family and friends to the program; they are given flyers with the sign-up link for enrolment. In addition, given the small geographic size of Singapore, redeployment of recruiters from sites with low numbers of potential participants to sites with higher numbers of potential participants is in our strategy to reach out to as many women as possible.

BREATHE has some limitations worth noting. Selection bias may arise due to systematic differences between baseline characteristics of responders and non-responders to BREATHE’s advertisements. BREATHE participants may be more health conscious or are already attending breast screening. Such a bias may affect sample representativeness and generalizability of findings. However, the BREATHE study collects information on the study participants (e.g. profession, socio-economic status, highest education attained) and how they found out about the study. This information will allow us to assess the implementation of a risk-based screening approach in this population first, before rolling out the initiative on a larger scale. The BREATHE risk report is based on information available from each participant. For example, if the participant does not participate in or is ineligible for mammography screening, information from first screen will not be in the risk report (participant is assumed to be of average risk). When information is incomplete, breast cancer risk will be underestimated. Barriers to active follow-up two years later are expected. However, the study coordinators will be actively contacting the participants to remind them about the follow-up visit. In addition, the questionnaire is designed such that the participant does not need to be present in-person (conducted electronically or over a phone call).

Conclusions

The aims of BREATHE are aligned with efforts to use personalized health for tailored interventions. For breast cancer screening, multiple studies have supported a risk-stratified approach over the current age-based paradigm due to potentially higher cost-effectiveness and reduced over-diagnosis [13,24,53,54]. If BREATHE is successful, women will gain a realistic understanding of their personal risk of breast cancer as well as strategies to reduce their risk, and fewer women will suffer from the anxiety of false positive mammograms and unnecessary biopsies. This work puts Singapore on the world map as a pioneer in integrating state-of-the-art breast cancer risk prediction tools, in particular, breast cancer PRS, in breast cancer screening. This study has real potential to transform breast cancer screening in Singapore.

BREATHE has assembled a multidisciplinary team to build on best practices and emerging data from other risk-based breast cancer screening studies elsewhere. Data-driven and patient-centric value-based care will benefit the healthcare system in many aspects. At the personal level: Women will gain a realistic understanding of their personal breast cancer risk and be empowered to make informed decisions together with their physicians on strategies to manage their risk. At the clinic: The comprehensive risk classification will aid physicians in the conversation on the need for further genetic testing as well as screening and risk reduction strategies. At the population-level: BREATHE generates real-world evidence on how to change the breast cancer screening paradigm to recognize the different needs of individuals. This includes assessment of the organizational readiness, effectiveness, efficiency, resources, costs and cost-effectiveness of implementing a risk-based breast cancer screening approach in Singapore. BREATHE puts Singapore on the world map as one of the pioneers in integrating state-of-the-art risk prediction tools in breast cancer screening, with a real potential to transform the health system to deliver better health and healthcare outcomes.

Data availability

No datasets were generated or analysed as part of this submission. All relevant data from this study will be made available upon study completion. The data generated by this study is owned by the providing institutions (e.g. NTFGH, NUH, NUP). Data may be obtained with reasonable request to the main Principal Investigator Mikael Hartman (mikael_hartman@nuhs.edu.sg). The data is not publicly available due to privacy or ethical restrictions. Legal agreements will need to be drawn up between data requesters and providers for access to the de-identified data. The proposed studies will need to be in compliance with Singapore’s laws and regulations with regards to human biomedical research and clinical investigation including The Declaration of Helsinki, International Good Clinical Practice Guidelines, Good Clinical Practice guidelines by Singapore’s Health Science Authority and the Ministry of Health.

Advertisement for BREATHE.

(PDF)

Click here for additional data file.

Online registration form for potential participants who are interested in BREATHE study.

(PDF)

Click here for additional data file.

Breast cancer education survey.

(PDF)

Click here for additional data file.

Experience survey.

(PDF)

Click here for additional data file.

Risk report feedback survey.

(PDF)

Click here for additional data file.

Satisfaction survey.

(PDF)

Click here for additional data file.

Obtaining appropriate threshold for the five-year absolute risk by polygenic risk score (PRS).

(PDF)

Click here for additional data file.

23 Dec 2021

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This study protocol reports on an important research endeavor. It starts by reiterating the clear case for risk stratification for breast cancer screening. It further notes that there is a lack of evidence on this topic for Asian populations.

The study has multiple aims. Its foundation is the recruitment of a cohort of 3,500 women aged 35 to 59 for whom a range of risk factors will be collected, including the latest in genetic markers. Based on these risk factors, the women in the cohort are being triaged into high, average, and low risk groups. They will be actively followed for 2 years, and passively followed via health care records until 2030. All women in the study will be asked to complete several questionnaires, and recommended to follow the current national guidelines (which are screening starting at age 40). Further, based on their assessed risk, those classified as high risk will be referred to a “breast specialist”.

The definition of high risk is cautious, in that any one of five criteria will be sufficient for a woman to be classified as high risk. However, there are two important questions about these criteria:

-- The BOADICEA model is being used to estimate the likelihood that the woman is a carrier of the BRCA1/2 mutations. However, recent versions of BOADICEA incorporate the likelihoods of 5 pathogenic variants plus the Mavaddat PRS plus a number of non-genetic risk factors, though this newer version of BOADICEA has not been validated in Asian populations. At the least, some discussion of the choice of the version of the BOADICEA model is needed.

-- The Gail model and PRS risk thresholds are expressed in terms of fixed absolute risk levels, independently of each woman’s age. This is a serious concern, as absolute breast cancer risk increases very rapidly with age. These thresholds will therefore over-estimate women at high risk at younger ages, and under-estimate those at higher ages.

The project has many and diverse aims, so there is a question of practical feasibility. The aims range from using questionnaires to assess women’s knowledge of and interests in risk stratified screening, to their actual responses (and implicitly those of their health care providers) to information about their assessed breast cancer risks, to a Markov model-based cost-utility analysis. Some indications of the scale of funding and scope of activities would therefore be helpful. For example, where will the utility weights and various unit costs for this latter analysis be sourced?

The paper notes the risks of sample selection bias in the recruitment of women for the cohort study, but makes no mention of the challenges of actually attaining the target sample size. Experience has shown that this can be a major problem. It is also not clear that the sample size will be sufficient to generate a large enough sample of high-risk women to provide adequate power to address the various aims of the study.

Regarding the PLOS questions, my reasoning is as follows:

2. The protocol will likely work, but the responses to some eventualities, like low sample recruitment rates or a seriously biased sample, are not described in sufficient detail. Similarly, the level of detail on the Markov cost-utility modeling is not adequate, though this could be remedied by undertaking to make the resulting model code and parameters fully public.

3. The methodology is likely feasible, but not described in sufficient detail to allow replicbility. Still, in such a large and complex study, this may not be a reasonable expectation. A very real challenge is the confidentiality of the cohort members’ health care records, so the data used for the final analysis will likely not be publicly available. This is not the fault of the authors, but rather a larger problem with this kind of research.

4. As just noted, it will likely be impossible for all the data to be made public, for reasons of patient confidentiality. However, it would be acceptable if the Singaporean authorities had a means whereby duly authorized independent researchers could access the full data under appropriate controls, as is possible in other countries.

Reviewer #2: The authors introduced the study protocol of personalized breast cancer screening programs. Various breast cancer risk prediction models were developed, but they have not been implemented in clinical practice. The study is essential for the implementation of personalized breast cancer screening.

1. According to the personalized risk evaluation, the participants will be classified into three risk levels. Can the participants know their reasons for risk classification? If participants have a high probability of familial breast cancer, they need genetic counseling and close follow-ups. Is there any rule for the potential familial breast cancer variant carriers?

2. The authors defined several primary and secondary aims in the study; however, the measures of the aims were not clear. How did the authors evaluate the acceptability of risk stratification (primary aim 1)? What is the measure for breast cancer awareness (secondary aim 1)?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Michael Wolfson

Reviewer #2: Yes: Isao Oze

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

13 Jan 2022

Below are the responses to editor and reviewers. A same copy of Word document has been uploaded as "Response to Reviewers".

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2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

Response: We would like to update the ‘Financial Disclosure’ section to the following: “This study is funded by the JurongHealth Fund (reference number JHF-20-RE-003) and the Precision Health Research Singapore Clinical Implementation Pilot (PRECISE CIP) Fund. M.H. is supported by the JurongHealth Fund, PRECISE CIP Fund, the Breast Cancer Prevention Programme under Saw Swee Hock School of Public Health Programme of Research Seed Funding (SSHSPH-Res-Prog-BCPP), Breast Cancer Screening Prevention Programme under Yong Loo Lin School of Medicine (NUHSRO/2020/121/BCSPP/LOA), National Medical Research Council Clinician Scientist Award (Senior Investigator Category, NMRC/CSA-SI/0015/2017), the National University Cancer Institute Singapore Centre Grant Programme (CGAug16M005), and Asian Breast Cancer Research Fund. J.Li is supported by the National Research Foundation Singapore (NRF-NRFF2017-02) and BMRC Central Research Fund (Applied Translational Research). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” In addition, we have revised the grant information to ensure the information provided in the ‘Funding Information’ matches that in the ‘Financial Disclosure’ section.

3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

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We will update your Data Availability statement to reflect the information you provide in your cover letter.

Response: We have updated the data availability statement to state that there is no data generated for this submission and future data generated from this study will be available upon request. No datasets were generated or analysed as part of this submission. All relevant data from this study will be made available upon study completion. The data generated by this study is owned by the providing institutions (e.g. NTFGH, NUH, NUP). Data may be obtained with reasonable request to the main Principal Investigator Mikael Hartman (mikael_hartman@nuhs.edu.sg). The data is not publicly available due to privacy or ethical restrictions. Legal agreements will need to be drawn up between data requesters and providers for access to the de-identified data. The proposed studies will need to be in compliance with Singapore’s laws and regulations with regards to human biomedical research and clinical investigation including The Declaration of Helsinki, International Good Clinical Practice Guidelines, Good Clinical Practice guidelines by Singapore’s Health Science Authority and the Ministry of Health.

4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Response: We have included the information as a supplementary document (S7 Appendix).

5. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well.

Response: We have included the ethic statement in the methods section (page 8, line 155-157 in tracked changes version).

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Response: All the figure files have been uploaded to PACE and passed the PLOS requirements.

Review Comments to the Author:

Reviewer #1: This study protocol reports on an important research endeavor. It starts by reiterating the clear case for risk stratification for breast cancer screening. It further notes that there is a lack of evidence on this topic for Asian populations.

The study has multiple aims. Its foundation is the recruitment of a cohort of 3,500 women aged 35 to 59 for whom a range of risk factors will be collected, including the latest in genetic markers. Based on these risk factors, the women in the cohort are being triaged into high, average, and low risk groups. They will be actively followed for 2 years, and passively followed via health care records until 2030. All women in the study will be asked to complete several questionnaires, and recommended to follow the current national guidelines (which are screening starting at age 40). Further, based on their assessed risk, those classified as high risk will be referred to a “breast specialist”.

The definition of high risk is cautious, in that any one of five criteria will be sufficient for a woman to be classified as high risk. However, there are two important questions about these criteria:

-- The BOADICEA model is being used to estimate the likelihood that the woman is a carrier of the BRCA1/2 mutations. However, recent versions of BOADICEA incorporate the likelihoods of 5 pathogenic variants plus the Mavaddat PRS plus a number of non-genetic risk factors, though this newer version of BOADICEA has not been validated in Asian populations. At the least, some discussion of the choice of the version of the BOADICEA model is needed.

Response: BOADICEA Web Application (BWA) v3 (https://ccge.medschl.cam.ac.uk/boadicea/boadicea-web-application/, accessed Dec 28, 2021) will be used to compute BRCA mutation carrier probabilities. Prediction of other breast cancer predisposition genes is not available using the BWA. Gail model risk estimates and PRS are computed separately based on our understanding of Asian breast cancer risk based on Singaporean breast cancer incidence and mortality rates.

We have included this in (page 14, line 261-265 in tracked changes version): “Carrier probabilities for breast cancer predisposition genes such as BRCA1 and BRCA2 will be predicted using the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA, web application v3, https://ccge.medschl.cam.ac.uk/boadicea/boadicea-web-application/, accessed Dec 28, 2021) (40).”

-- The Gail model and PRS risk thresholds are expressed in terms of fixed absolute risk levels, independently of each woman’s age. This is a serious concern, as absolute breast cancer risk increases very rapidly with age. These thresholds will therefore over-estimate women at high risk at younger ages, and under-estimate those at higher ages.

Response: Our study looks at the short-term risk, i.e. the risk of breast cancer in the next five years from screening. As rightfully pointed out by the Reviewer, using the fixed threshold for absolute risk does not convey the risk of the woman throughout her lifetime. Re-assessment and conveying of changes in risk level should be done at five-years intervals.

We have included additional information (page 19, line 369-376 in tracked changes version): “In this study, only one risk report is generated per participant based on information at recruitment. Over time, the participant’s risk will change. An updated risk report is recommended if the programme were to be implemented in a nation-wide screening programme. As our risk estimates reflect a five-year absolute risk by the Gail model and PRS, a review of breast cancer risk should be done at a minimum frequency of five years.”

The project has many and diverse aims, so there is a question of practical feasibility. The aims range from using questionnaires to assess women’s knowledge of and interests in risk stratified screening, to their actual responses (and implicitly those of their health care providers) to information about their assessed breast cancer risks, to a Markov model-based cost-utility analysis. Some indications of the scale of funding and scope of activities would therefore be helpful. For example, where will the utility weights and various unit costs for this latter analysis be sourced?

Response: Utility weights and various unit costs for the cost-effectiveness analysis will be sourced from existing literature.

We have included this in (page 17, line 331-332 in tracked changes version): “Utility weights and various unit costs will be sourced from existing literature. “

The paper notes the risks of sample selection bias in the recruitment of women for the cohort study, but makes no mention of the challenges of actually attaining the target sample size. Experience has shown that this can be a major problem. It is also not clear that the sample size will be sufficient to generate a large enough sample of high-risk women to provide adequate power to address the various aims of the study.

Response: BREATHE will be expanded to cover two more sites with additional funding. Staggered opening of sites was done to ensure sufficient effort is provided to smoothen out any logistics issues. We have recruited 286 women after two months from the opening of the first recruitment site. Additional funding has been secured to expand BREATHE to two additional sites. Within the first 100 participants, 20% were identified as at above-average risk. This is higher than expected; in existing data sources we are expecting ~12% of women to be at above-average risk. Selection bias is evident and the recruiters have been briefed to not only target the subpopulations of women who are at the mammogram clinics but also at other waiting areas (e.g. for other purposes than health screening) of the polyclinics.

We have included this in (page 20-21, line 407-415 in tracked changes version): “We expect challenges in attaining our target recruitment of 3500 healthy women aged between 35 and 59. As this is a hospital led research study, publicity is limited to the hospitals and affiliated polyclinics. BREATHE may be extended to other sites (e.g. Alexandra Hospital and Jurong Medical Centre) to increase recruitment numbers. Participants are encouraged to refer family and friends to the program; they are given flyers with the sign-up link for enrolment. In addition, given the small geographic size of Singapore, redeployment of recruiters from sites with low numbers of potential participants to sites with higher numbers of potential participants is in our strategy to reach out to as many women as possible.”

Regarding the PLOS questions, my reasoning is as follows:

2. The protocol will likely work, but the responses to some eventualities, like low sample recruitment rates or a seriously biased sample, are not described in sufficient detail. Similarly, the level of detail on the Markov cost-utility modeling is not adequate, though this could be remedied by undertaking to make the resulting model code and parameters fully public.

Response:

We have included this in (page 17, line 336-337 in tracked changes version): “The resulting model code and parameters will be made publicly available in an independent study.”

3. The methodology is likely feasible, but not described in sufficient detail to allow replicbility. Still, in such a large and complex study, this may not be a reasonable expectation. A very real challenge is the confidentiality of the cohort members’ health care records, so the data used for the final analysis will likely not be publicly available. This is not the fault of the authors, but rather a larger problem with this kind of research.

Response: "The data generated by this study is owned by the providing institutions (e.g. NTFGH, NUH, NUP). Data may be obtained with reasonable request to the main Principal Investigator Mikael Hartman (mikael_hartman@nuhs.edu.sg). The data is not publicly available due to privacy or ethical restrictions. Legal agreements will need to be drawn up between data requesters and providers for access to the de-identified data. The proposed studies will need to be in compliance with Singapore’s laws and regulations with regards to human biomedical research and clinical investigation including The Declaration of Helsinki, International Good Clinical Practice Guidelines, Good Clinical Practice guidelines by Singapore’s Health Science Authority and the Ministry of Health."

We have included the above text in the “Data availability” section.

4. As just noted, it will likely be impossible for all the data to be made public, for reasons of patient confidentiality. However, it would be acceptable if the Singaporean authorities had a means whereby duly authorized independent researchers could access the full data under appropriate controls, as is possible in other countries.

Response: The data generated by this study is owned by the providing institutions (NTFGH, NUH, NUP). Legal agreements may be drawn up between data requesters and providers for access to the de-identified data. For example, in this current study, the collaborator GIS has a legal agreement with the providers to use the data generated from this study for analysis. The proposed study will need to be in compliance with Singapore’s laws and regulations with regards to human biomedical research and clinical investigation including The Declaration of Helsinki, International Good Clinical Practice Guidelines, Good Clinical Practice guidelines by Singapore’s Health Science Authority and the Ministry of Health.

Reviewer #2: The authors introduced the study protocol of personalized breast cancer screening programs. Various breast cancer risk prediction models were developed, but they have not been implemented in clinical practice. The study is essential for the implementation of personalized breast cancer screening.

1. According to the personalized risk evaluation, the participants will be classified into three risk levels. Can the participants know their reasons for risk classification? If participants have a high probability of familial breast cancer, they need genetic counseling and close follow-ups. Is there any rule for the potential familial breast cancer variant carriers?

Response: Participants are briefed about the process of risk classification, which includes the tools used (i.e. the Gail model, Polygenic Risk Scores, BOADICEA, mammogram density and recall status). Participants in the below-average and average risk category will know they are below-average or average risk based on all available information they provided. Participants in the above-average risk category will know their reasons for being at elevated risk if they attend the recommended clinic visit with a breast specialist. Based on risk assessment by our ethics board, it is prudent that the follow-up of the above-average risk group and conveying of why the participant is this risk level is done based on clinical practice by clinicians and not part of the research study. The research study will have access to the medical records to follow up on the recommendations by the breast specialist. Only participants who chose to attend the clinic incurring out-of-pocket expenses will know their risk, the reason is to simulate a nationwide screening scenario.

We have included this in (page 19-20, line 378-405 in tracked changes version): “Personal risk of breast cancer is a difficult concept to grasp. Breast cancer risk prediction tools are mainly designed for providers, hence the participant may have a limited understanding of the results (52). We deliberated over the readiness of the Singapore’s population to receive personalised disease risk results with various stakeholders (representatives of the hospitals and clinics, ethics representatives, public health experts, and researchers), and concluded that only the general risk classification (below-average, average and above-average risk) is to be conveyed to the participants as part of this research study. Details on the risk classification categories are communicated and explained to the participants by healthcare professionals to ensure that the results are interpreted accurately and meaningfully. At recruitment, participants are briefed about the risk stratification tools used in this study. We understand that in this current information age, it is likely that there will be participants especially those in the above-average risk group will want details about their risk levels. Our main concern is in the participants’ understanding of genetic risk, in particular the genetic risk component. As we want to create an inclusive risk-based screening program, we are not performing clinical genetic testing as part of this screening design. Unlike the better known and more expensive clinical genetic testing, PRS is a new method of breast cancer risk assessment that is not familiar to our study population. To avoid the situation where participants reject screening due to any misconception about risk (i.e. the idea that if I am genetically above-average risk there is nothing I can do to avoid getting breast cancer), we will only indicate the general risk level in the risk report. Based on the risk assessment by our ethics board, it is prudent that the follow-up of the above-average risk group and conveying of the details of the risk classification is done based on current clinical practice by clinicians and not part of the research study. Participants who are predicted carriers of BRCA1/2 by BOADICEA will be recommended to see a breast specialist and referred to genetic counselling and subsequently genetic testing if appropriate.”

2. The authors defined several primary and secondary aims in the study; however, the measures of the aims were not clear. How did the authors evaluate the acceptability of risk stratification (primary aim 1)? What is the measure for breast cancer awareness (secondary aim 1)?

Response: We are using surveys tailored to Singapore’s population. The questionnaires are not validated, however, they are designed with reference to various publicity materials on breast cancer available in Singapore. We have included these surveys as supplementary materials (S3 Appendix – S6 Appendix).

The acceptability of risk stratification is measured by our recruitment experience survey (done at the end of recruitment, S4 Appendix), report feedback survey (done after the return of risk report ~3 months after recruitment, S5 Appendix), and the satisfaction survey (done at the end of the study participation, ~2 years after recruitment, S6 Appendix). The three surveys ask questions about their physical and emotional experience 1) during recruitment, 2) while waiting for the risk report, 3) their understanding of the risk report, and 4) their thoughts about risk stratification in terms of inclusion of the PRS. Some questions include: “Since receiving my personalized breast cancer risk profile I feel_________”, “I will follow the recommended breast cancer screening frequency (options: strongly agree, agree, neither agree nor disagree, disagree, strongly disagree)”. “I am confident that my personalized breast cancer risk profiles are reliable (options: strongly agree, agree, neither agree nor disagree, disagree, strongly disagree)”.

We will be using a descriptive method to understand the current breast cancer awareness in Singapore. At recruitment, we will administer a breast cancer education questionnaire (S3 Appendix) including questions “I am still young, therefore I do not need to screen for breast cancer [options: agree, disagree]” and “If breast cancer is detected early, chances of surviving is high [options: agree, disagree]”.

We have included this in (page 16, line 308-316 in tracked changes version): “Information will be obtained from the recruitment experience survey (e.g. “I will recommend doing a breast cancer risk classification to others [options: strongly agree, agree, neither agree nor disagree, disagree, strongly disagree]”), report feedback survey (e.g. “I am confident that my personalized breast cancer risk profiles are reliable [options: strongly agree, agree, neither agree nor disagree, disagree, strongly disagree]”), and satisfaction survey (e.g. “What do you like about your experience in this study?”) (Primary Aim 1); and from the breast cancer education questionnaire (e.g. “I am still young, therefore I do not need to screen for breast cancer [options: agree, disagree]”) (Secondary Aim 1).”

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

11 Mar 2022

BREAst screening Tailored for HEr (BREATHE) - A study protocol on personalised risk-based breast cancer screening programme

PONE-D-21-33800R1

Dear Dr. Li,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Yonglan Zheng, Ph.D.

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #2: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #2: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #2: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors clearly answered all comments from the reviewer 2.

I have no more comments to the article.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

23 Mar 2022

PONE-D-21-33800R1

BREAst screening Tailored for HEr (BREATHE) - A study protocol on personalised risk-based breast cancer screening programme

Dear Dr. Li:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Dr. Yonglan Zheng

Academic Editor

PLOS ONE