BACKGROUND: Polygenic scores (PGSs) are weighted sum scores of trait-associated alleles from up to millions of SNPs. As PGS research pivots to translation into health care settings a key issue for laboratories providing PGS is demonstration of analytical validity of PGS. METHODS: We report data from 6 individuals who have been genotyped multiple times using the same and different technologies. These data were generated as part of standard experimental design for quality control purposes in two research settings over many studies and over many years. Using this opportunistic design of technical variability, we provide an empirical evaluation of technical reproducibility of PGS from 115 traits of different genetic architectures. RESULTS: Given a predefined set of SNP weights variability in PGS can reflect only SNP missingness or incorrect genotype call. We find very high reproducibility of SNP genotypes. In particular, the technical reproducibility of PGS generated from the same array technology and processed through the same quality control and imputation pipeline is very high. However, impact of missing SNPs varies between traits depending on the SNP’s weight for a trait. We provide a PGS quality score statistic (PGS:QS) that can be reported for each trait-specific score for an individual, to provide a quantitative assessment of the proportion of variation of the score that is captured by the SNPs genotyped/imputed for the individual. We provide an algorithm (PGS-impute) that updates the SNP weights of the scoring algorithm to the SNPs available for an individual, improving PGS accuracy. CONCLUSIONS: While validity of directly measured genotypes (whether from microarray or whole genome sequencing) is well-established, objective approaches to evaluate analytical reproducibility of PGS post-genotyping pipeline have been lacking. Here, we provide empirical data and an analysis framework which can be used by PGS providers to support understanding of analytical reproducibility and robustness.

Background Polygenic risk scores are increasingly being used in cancer care to inform population screening programs, personalise risk estimates in familial cancer clinics, and inform therapeutic decision-making. However, lack of clear guidelines for PRS laboratory accreditation restricts the ability of researchers, clinicians and regulatory bodies to evaluate test quality. Via stakeholder consultation, this study aimed to describe the unique aspects of PRS laboratory accreditation to support safe test implementation in cancer care. Methods Semi-structured interviews explored the unique challenges and considerations of PRS testing. Eligible participants were stakeholders with experience in ordering, performing, reporting, and accrediting PRS. Interviews were transcribed verbatim, and deductive content analysis conducted. Results Participants were clinicians, laboratory scientists, researchers, and industry experts with experience in PRS laboratory accreditation (n = 13). Five themes were developed covering the entire PRS testing pipeline: i) laboratory methods, ii) PRS algorithm, iii) clinical context, and iv) reporting results, and a fifth theme, impact of ancestry that affected all aspects of the PRS pipeline. Laboratory methods for sample collection, genotyping, and quality control were identified as well-established and transferable to PRS testing. Use of imputation was identified as a unique consideration for PRS accreditation, which impacts transparency and quality assessments of the result. Imputation is particularly relevant in cancer applications where accuracy may affect risk classification and subsequent clinical management decisions. PRS algorithm updates were considered manageable within existing accreditation frameworks for test improvements. Additional considerations included the need to develop quality standards for the management of missing data, defined validation processes, protocols for collecting additional risk factors if delivering integrated risk (e.g. breast density and family history), and education of laboratory pathologists. Finally, the importance of clear reporting of risk assessment was explored. Based on findings, a conceptual overview and checklists were developed to aid providers and accreditation bodies alike assessing PRS testing. Conclusions Findings highlight the unique considerations for PRS laboratory accreditation and offer a practical framework to guide accreditation guidelines. These considerations are particularly urgent in the cancer setting, where PRS is being most rapidly implemented, and where robust accreditation frameworks are essential to ensure accurate and equitable risk assessments.

Background Pathogenic germline variants in certain genes are associated with somatic tumour mutation signatures. The use of somatic tumour mutation data has the potential to improve the identification of true pathogenic variants but remains underexplored. We investigated the integration of tumour homologous recombination (HR) deficiency status as a predictor of pathogenicity for germline BRCA1 and BRCA2 variants, building on the established link between HR deficiency and germline pathogenic variants in these genes. Methods We analysed breast tumour whole-genome sequence and matching germline data from 350 patients across four datasets: Familial Breast Cancer (N = 77), The Cancer Genome Atlas (TCGA-BRCA, N = 96), the MAGIC study (N = 136), and Q-IMPROvE (N = 41). A total of 15,156 germline variants (including structural variations) in BRCA1, BRCA2, and other cancer genes (ATM, BARD1, BRIP1, CHEK2, PALB2, PTEN, RAD51C, RAD51D, TP53) underwent variant curation. Patients were categorised based on germline classification as BRCA1 positive (N = 27), BRCA2 positive (N = 21), and BRCA1/2 negative (N = 232), excluding those with BRCA1/2 variants of uncertain significance (N = 8) and pathogenic or only uncertain variants in other cancer genes (N = 62). Somatic HR status (deficient or proficient) was predicted using three algorithms: HRDetect, CHORD, and HRDsum. HR-deficient and HR-proficient status were significant predictors of germline BRCA1/2 pathogenic variant status (positive and negative directions). Findings The CHORD algorithm, which estimates BRCA1 and BRCA2 subtype specifically, added precision contributing evidence towards pathogenicity for the corresponding gene, reaching pathogenic moderate strength for the relevant gene-subtype. Finally, we assessed CHORD HR predictions for variants of uncertain significance in BRCA1 and BRCA2, and reported their tumour HR status for potential use as additional evidence in variant curation. Interpretation Analysis across multiple tumour whole-genome sequencing datasets has shown that HR status prediction algorithms can separate profiles for BRCA1 and BRCA2 pathogenic variants and provide further evidence at increased weight to aid in the classification of germline BRCA1 and BRCA2 variants. Tumour sequencing offers a promising strategy for reducing the uncertainty in germline variant interpretation. Funding This work was funded by the National Breast Cancer Foundation.

Abstract Breast cancer in women with germline BRCA1/2 pathogenic variants (gBRCA1/2) are generally treated with platinum-based therapies and PARP inhibitors (PARPi) with resistance commonly emerging. As the tumor microenvironment (TME) in gBRCA1 triple-negative breast cancer (TNBC) is enriched with tumor-infiltrating lymphocytes (TILs) and CD8 T cells, treatment trials have been done combining PARPi and immune checkpoint inhibitors (ICIs) in BRCA1 TNBC. This combination has not been shown to be more effective than PARPi alone. Evaluating the TME in gBRCA1/2 TNBC may help identify tumors most likely to benefit from PARPi/ICI therapy. We performed a detailed spatial proteomic analysis to characterize tumor-immune cell interactions in patients with gBRCA1/2 and wild-type (WT) TNBC with spatial tissue multiplexing (PhenoCycler) in 101 gBRCA1, 24 gBRCA2, and 30 WT TNBCs with matched RNAseq for 34 gBRCA1, 8 gBRCA2, and 16 WT TNBCs. A 43-plex antibody panel was developed featuring markers of DNA damage and repair, immune subtypes and exhaustion. We detected single tumor cells (PANCK+) in S/G2 phase (Geminin+) with double-stranded DNA breaks (yH2AX+) and DNA repair capacity (RAD51+) across all three cohorts. gBRCA1/2 TNBC patients exhibited a significantly lower proportion of tumor cells with homologous recombination proficiency (HRP) (gBRCA1 p = 0.006; gBRCA2 p = 0.007) compared to WT TNBC. CD4 & CD8 T cells, and CD20 B cells had intact DNA repair in WT and gBRCA1/2 TNBC. The frequency of CD8+ T (p=0.016) and CD20 B (p=0.003) cells was significantly higher in gBRCA1 compared to WT TNBC; BRCA2 and WT TNBC showed no differences. A detailed characterization of CD8 T cells revealed significantly increased numbers of potentially dysfunctional CD8 T cells in BRCA1 (TOX, p<0.0001; LAG-3, p=0.028; PD-1, p=0.033) and BRCA2 (LAG-3, p=0.033) compared to WT TNBC. We observed two types of TMEs in gBRCA1 TNBC: 1) CD8 low (mean<9.38%) with 1.4-fold increased immune checkpoint (PD-1) expression (mean: 15.4%) and high DNA damage in tumor cells; and 2) CD8 high (>9.38%) with reduced PD-1 and low DNA damage in tumor cells. Our findings suggest that although gBRCA1/2 variants lead to DNA damage and impaired repair in tumor cells, T cells (CD4, CD8) and B cells (CD20) retain intact DNA repair mechanisms. We also found that gBRCA1/2 TNBCs exhibit higher levels of immune checkpoint proteins LAG-3 and PD-1 on CD8 T cells compared to WT TNBC. This finding suggests the potential utility of additional ICI (LAG-3, PD-1) beyond PD-L1 blockade. Importantly, patients with gBRCA1-associated TNBC exhibit two different TMEs, suggesting that the response to ICI- and DNA-damaging-based therapies may differ between tumors, and anticipated prior to treatment. Defining treatment-naïve TME is crucial for designing personalized, targeted ICI strategies for individuals with BRCA-mutated TNBC. Citation Format: Dana Pueschl, Danielle Bragen, Jia-Ren Lin, Anupma Nayak, Derek A. Oldridge, Kate Bennett, Victoria Fang, kConFab Investigators, Kenneth Offit, Andrew K. Godwin, Paul A. James, Phuong L. Mai, Soo Hwang Teo, Antonis Antoniou, Georgia Chenevix-Trench, E. John Wherry, Susan M. Domchek, Katherine L. Nathanson. A single-cell spatial proteomic analysis of the TNBC microenvironment defines genotype-specific features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4966.

Abstract Pathogenic coding variants in BRCA1, BRCA2 and PALB2 confer hereditary breast/ovarian cancer risk, yet these regions comprise less than 10% of the genomic footprint of these genes, leaving most sequence unexplored. We investigated the contribution of non-coding variation to hereditary breast cancer by analyzing intronic variants and 5′ upstream regions of BRCA1, BRCA2 and PALB2 in the BEACCON case–control study of over 11,000 participants. Full-gene sequencing showed that 46.3% of cases carried at least one rare non-coding variant. This was associated with a modest increase in breast cancer risk (OR = 1.2, p < 0.0001), most likely reflecting the presence of a small proportion of pathogenic variants within a larger background of predominantly neutral variation. Stronger enrichment was observed for triple-negative disease, particularly for BRCA1 (OR = 1.5, p = 0.0001). Tumor sequencing of 42 high-priority variants identified 11 (26.2%) with wild-type allele loss and high homologous recombination deficiency. Functional CRISPR/Cas9 knock-in assays in MCF10A cells confirmed that two deep intronic variants created aberrant splice sites, disrupted splicing and impacted transcript expression.

Objectives Polygenic risk scores are increasingly available to consumers to provide an estimate of the genetic contribution to health conditions. However, healthcare providers report limited knowledge and confidence using polygenic risk scores. Clinical implementation necessitates educational programmes to support clinicians to integrate this new test into practice. This study aimed to identify healthcare providers’ learning needs and preferences for polygenic risk education to inform the design of tailored education initiatives.Design, setting and participants This pragmatic qualitative study used focus groups to capture healthcare providers’ perspectives. To ensure informed responses, genetic healthcare providers with prior experience using polygenic risk scores, and/or who had completed polygenic risk education were recruited to participate in focus groups or interviews (n=30). There were no exclusions based on country of practice. Recordings were transcribed and content analysis conducted to identify learning needs. Themes related to education engagement were mapped to the capability, opportunity and motivation model for behaviour change.Results Among this cohort of experienced providers, residual gaps existed in polygenic risk-related knowledge, skills and local guidelines to inform practice. Learning needs encompassed: (i) polygenic risk-specific knowledge, and (ii) communication skills needed to discuss results and facilitate risk management. Themes related to engaging with polygenic risk education mapped to capability included awareness of, and access to educational resources and initiatives, including practice resources and position statements from professional bodies. Time-poorness was a primary barrier to accessing education. Opportunities comprised of building on existing workplace training and activities such as multidisciplinary team meetings and journal clubs. All participants noted that motivation for completing polygenic risk training was primarily driven by a desire to improve patient-centred care and clinical outcomes.Conclusion This study highlights priority learning areas to inform the development of tailored polygenic risk education initiatives, and resources and delivery strategies that meet the identified needs. Participants’ expert insights reveal potential barriers as well as solutions to engaging healthcare providers with polygenic risk score education to ultimately facilitate implementation into clinical practice.

BackgroundAs demand for breast cancer germline testing has increased, mainstream genetic testing models of care have been implemented to increase access to genetic testing. This study examined patients' experiences of mainstream genetic testing and compared outcomes between patients seen only by their breast specialist and those who also received genetic counseling.Patients and methodsA mixed methods approach using a survey and qualitative interviews examined decision-making, adaption to result, and family communication. Outcomes were compared by receipt of genetic counseling or not. Qualitative data were analyzed deductively.ResultsSixty-eight participants completed the survey and 20 were interviewed. Most appreciated the mainstream pathway due to an existing relationship with their cancer specialist and avoidance of extra appointments. There were no differences between those who received genetic counseling compared to those who did not regarding decision regret, cancer risk perception, or adaptation to their genetic test result. Participants who had genetic counseling had higher knowledge scores likely due to the lasting impact of their positive genetic test result.ConclusionPatients preferred the mainstream model of care, however, genetic counselor involvement was valued by those who received a pathogenic variant due to receiving counseling about the familial implications of their genetic diagnosis.

Background:Pharmacogenomics (PGx) is increasingly recognised as a cornerstone of personalised medicine, with established recommendations for many commonly prescribed medicines. Despite this, translation into real-world clinical practice remains inconsistent, reflecting persistent implementation and health-system gaps. Objective:To provide a multidisciplinary perspective on the system-level conditions required to normalise PGx within contemporary hospital care, drawing on structured knowledge exchange and priority-setting across clinical, digital and governance domains. Methods:A multidisciplinary knowledge exchange program convened clinicians, pharmacists, genetic services, informatics teams, researchers and health-service leaders across oncology, transplantation, peri-operative care and palliative care. Knowledge was exchanged through presentations, informing the generation and ranking of shared priorities in groups and individually using the Nominal Group Technique. Aggregate scores were calculated, and priorities further refined. Presentation insights were synthesised using a narrative, interpretive approach, to identify cross-cutting translational themes. Results:Eleven shared priorities were identified across four broad critical levers for sustainable integration: (1) computable, reusable PGx data embedded within electronic medical records (EMR); (2) coordinated and equitable testing and prescribing pathways; (3) workforce capability and stewardship; and (4) governance. Equity, access and data sovereignty were highlighted as foundational to responsible scale-up, particularly for First Nations peoples and under-represented communities. Conclusion:Insights from this knowledge-exchange program highlight the feasibility and clinical relevance of PGx, and identify workforce capability, EMR integration, and system-level governance as critical implementation levers of adoption. Coordinated national investment and policy alignment will be central to realising the full value of PGx.

TP53 germline pathogenic variants are among the most significant genetic causes of cancer across all age groups. Current TP53 variant classification guidelines are designed to identify high penetrance TP53 variants that lead to a phenotype called Li-Fraumeni syndrome. However, they are insufficient to accurately classify variants conferring atypical penetrance. These atypical penetrance variants are disease-causing, but the phenotype is often attenuated. Using current TP53 variant classification guidelines, atypical penetrance variants are not properly recognized leading to sub-optimal management of individuals carrying such variants. We highlight the need to develop strategies to consistently identify atypical penetrance disease-causing germline TP53 variants including development of variant classification specifications tailored to distinguish such variants, and to define the associated cancer spectrum and age-related risks. These studies will inform modifications to the existing standard risk management recommendations.

The clinical implementation of polygenic risk scores (PGS) in comprehensive cancer risk assessments is imminent. The successful implementation relies on clear communication to consumers to facilitate understanding of personalised risk information and motivate cancer prevention behaviours. Development of provider- and consumer-friendly resources to aid communication and understanding of PGS results is a critical implementation task. This study aimed to produce an acceptable report for delivering comprehensive, PGS-informed breast cancer risk assessments in research and clinical settings. A two-phase, multi-methods research program was conducted, focusing on co-design with a broad group of interest holders to gather preferences and feedback on the content, layout and visual tools of a risk report. Phase 1 involved a literature review, a quantitative survey with consumers and qualitative interviews with consumers and medical practitioners to generate an initial prototype report. Phase 2 involved qualitative interviews with genetic counsellors to seek feedback about the prototype and refine it into a final version. The report was operationalised as a web-based application that generates personalised versions in PDF. The evidence-based, interest-holder-informed comprehensive breast cancer risk report produced is highly usable, able to support ongoing clinical implementation research on PGS across cancer use-cases and contexts. Future work should explore consumer evaluation of the report and the feasibility of interactive and digital delivery mechanisms.

Background: Germline genetic testing is an increasingly important component of treatment decision-making for clinicians and patients with breast cancer. To address increased demand and expedite access to genetic testing for these patients, the Parkville Familial Cancer Centre (PFCC) in Victoria, Australia, implemented a breast mainstream genetic testing program. The program educates and supports breast cancer specialists to provide eligible patients with pre-test information, gain consent, and arrange genetic testing during their routine cancer appointments. This study aimed to explore breast cancer specialists' experiences and opinions of the education program and of facilitating mainstream genetic testing for their patients. Methods: Specialists who had attended the mainstream genetic testing education were invited to complete an online survey about the training provided through the education program and their experience of deploying mainstream genetic testing in their practice. Descriptive statistics were compiled, and content analysis used for open text responses. Results: Forty-five breast cancer specialists (breast surgeons, medical oncologists, radiation oncologists and breast care nurses) responded (45% response rate). Most respondents had discussed (87%) and consented (80%) patients for mainstream genetic testing. Most specialists (81%) rated their confidence levels as high or very high for consenting patients to mainstream genetic testing. The majority (89%) indicated that they believed mainstream genetic testing should be part of their role and felt well supported by the PFCC (90%). Conclusion: Breast cancer specialists used the education they received in the mainstream education program and were supported to deliver mainstream breast cancer genetic testing to their patients.

The experiences and outcomes for women identified with a BRCA1/2 pathogenic variant during young adulthood are qualitatively described but not well quantified. This study investigated the impact of BRCA1/2 status on women’s reproduction, intimate partner relationships, and sexual functioning. Australian women aged 18–40 years who had predictive BRCA1/2 testing, received either a positive or negative result, and had no personal cancer history, completed an online survey that used a case-control design. Outcome measures included childbearing, use of reproductive technologies, relationship status, and sexual functioning. 579 women participated (62.0

BACKGROUND:Ovarian cancer is characterised by high mortality and lacks effective screening, making prevention critical. Polygenic risk scores (PRS), which aggregate the effects of multiple common alleles, may capture a proportion of currently unexplained genetic risk. While PRS have been evaluated for risk prediction, their association with treatment response and survival remains unclear. This study assessed the utility of a PRS for predicting high-grade serous ovarian cancer (HGSOC) risk in an Australian population and its association with chemotherapy response and outcomes. METHODS:PRS were calculated for 1,097 HGSOC, and 812 controls using data from Australian research programs. Associations between PRS, OC risk, chemotherapy response, and survival were analysed. RESULTS:Each standard-deviation increase in PRS was associated with a 40% increase in HGSOC risk (OR 1.40, p < 0.001). Women in the top 1% of the PRS distribution had a lifetime OC risk approaching 3%. Higher PRS values showed a trend toward poorer outcomes, however these associations were not consistent across analyses. CONCLUSIONS:PRS were not clearly associated with chemotherapy response or survival but represent a significant risk factor for the development of HGSOC. Incorporating PRS into clinical models may improve risk stratification and support targeted prevention. IMPACT:As the first study to evaluate how PRS relate to both HGSOC risk and chemotherapy response and outcomes, we show that PRS are unlikely to serve as therapeutic biomarkers but support their use for enhanced risk-stratified prevention.

PURPOSE:The return of polygenic risk scores (PGS) is currently being assessed in research settings for clinical utility and validity, and it is anticipated that PGS will soon be implemented in a clinical setting. There are limited guidelines regarding PGS communication and reporting; thus, there is a need to identify and analyze the current research to determine the most acceptable means of presenting PGS results through reports. The aim of this review is to examine the literature regarding the development and evaluation of PGS communication tools, including risk reports, visual aids, and online tools. METHODS:Research studies that evaluated preferences, understanding or interpretation of PGS through a report, visual aid, or tool were included. The search strategy was applied to MEDLINE (via Ovid) and American Psychological Association PsychInfo. RESULTS:Thirteen studies met the inclusion criteria. The presentation of PGS differed across studies, including icon arrays and bell curves for visual presentation and absolute risk, relative risk, and genetic risk score for numerical presentation. Participants' understanding of PGS differed between studies. Studies supported using absolute risk and avoiding stigmatizing colors to communicate results. CONCLUSION:To support PGS clinical implementation, the development of an evidence-based PGS report evaluated by consumers and various health care professionals is needed.

BACKGROUND:With widening therapeutic indications, germline genetic testing is offered to an increasing proportion of patients with breast cancer (BC) via mainstream oncology services. However, the gene set tested varies widely from just BRCA1/BRCA2 through to 'pan-cancer' panels of nearly 100 genes. If a germline pathogenic variant (GPV) is detected, the BC proband and other family GPV-carriers may be offered interventions such as risk-reducing surgery and intensive surveillance over decades for the various cancers linked to that gene. METHODS:The European Society for Medical Oncology (ESMO) Precision Oncology Working Group established an international expert working group (EWG) in BC germline genetics. This EWG firstly established a framework of criteria by which to evaluate each breast cancer susceptibility gene (BCSG) for potential inclusion on a breast cancer multigene panel test (BC-MGPT) for universal mainstream testing for BC cases. Next, the EWG scored BCSGs for impact regarding (i) BC risk estimation, (ii) clinical actionability and (iii) cancer-related mortality. RESULTS:The group agreed that they would constitute a BC-MGPT based on net clinical-public health utility, as quantified by likelihood of impact on cancer-related mortality. Judged as of high or moderate impact on this basis were six BCSGs: BRCA1, BRCA2, PALB2, RAD51C, RAD51D and TP53 (for BC diagnosed <40 years of age), with possible addition of BRIP1. While potentially informative for BC risk estimation, CHEK2 and ATM were judged to offer insufficient evidence for improving cancer-related mortality. The EWG recommended strongly against inclusion of 'syndromic' genes such as STK11, PTEN, NF1 and CDH1. CONCLUSIONS:With expanded germline testing in patients with BC (and cascade testing into families), the number and nature of resultant GPV-carriers identified will be dictated by the genes included on the upfront BC-MGPT. The potential harms, opportunity and economic costs of decades of surveillance of multiple organs and risk-reducing surgeries for GPV-carriers should be justified by strong evidence of meaningful improvement in cancer-related mortality (or health-related quality of life).

Frequencies of all reported germline CHEK2 variant carriers and carriers of variants concordantly categorized by functional our kinase assays in breast cancer patients and controls in 12 analyzed population datasets.

Associations between gene expression and stage group and OS in MOC.

Disease-causing variants with penetrance that is lower than the average expected for a given gene complicate classification, even when using gene-specific guidelines. For TP53, a gene associated with some of the highest cancer risks, even reduced penetrance disease-predisposition variants remain clinically actionable. We conducted a review of ClinVar submissions to identify TP53 variants flagged as having reduced penetrance by genetic testing laboratories and analyzed functional, bioinformatic, immunogenicity, frequency, and clinical features of these variants compared with standard pathogenic and benign variants. Our findings show that reported reduced penetrance TP53 variants are more likely to exhibit intermediate functional activity in multiple assays and are predicted as deleterious with bioinformatic tools, though with lower scores than pathogenic variants. These variants also have a higher population frequency than pathogenic variants, and heterozygotes tend to manifest disease later in life, suggesting a need for refined clinical criteria to better capture attenuated Li-Fraumeni syndrome phenotypes. Finally, by applying a random forest prediction model to all TP53 uncertain or conflicting variants in ClinVar, we identified 106 additional variants with potential reduced penetrance.

The breast cancer risk conferred by germline protein truncating variants (PTVs) in known and putative breast cancer genes has been extensively investigated. However, the effect of FANCM PTVs on breast cancer risk remains unclear. Our previous clinical, genetic and functional results on the N-terminal p.Arg658∗ and the two C-terminal p.Gln1701∗ and p.Gly1906Alafs∗12 variants suggested that FANCM PTVs may confer different risks for ER-negative (ER-neg) and triple-negative (TN) breast cancer subtypes. Here, we performed meta-analyses of seven studies totaling 144 681 breast cancer cases and 123 632 controls. FANCM PTVs were tested for association with breast cancer risk overall and the disease clinical subtypes by single variant and burden analyses. Two CRISPR-Cas9-based functional assays were also conducted to test the fitness of cells after knock-in of the p.Arg658∗, p.Gln1701∗ and p.Gly1906Alafs∗12 PTVs and the sensitivity of different FANCM regions to genome editing. Our results suggest that the N-terminal FANCM region upstream of p.Tyr725 harbors essential functions, whereas downstream regions appear dispensable. This is supported by our genetic data which indicate that all FANCM PTVs, excluding the two C-terminal p.Gln1701∗ and p.Gly1906Alafs∗12, are associated with an increased risk of ER-neg (OR = 1.41, P = 0.023) and TN (OR = 1.64, P = 0.0023). Notably, PTVs upstream of AA position 670 are associated with a moderate risk of developing TN breast cancer, and that even when the p.Arg658∗ carriers were excluded from the analysis. Importantly, our results confirm previous data indicating that p.Arg658∗ carriers are at moderate risk of developing ER-neg (OR = 2.08, P = 0.030) and TN (OR = 3.26; P = 0.0034), whereas carriers of p.Gln1701∗ and p.Gly1906Alafs∗12 should not be considered at increased risk. Our data are useful for counseling carriers of FANCM PTVs, but further analyses are warranted to obtain more precise risk estimates.