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BackgroundPALB2is the most important contributor to familial breast cancer afterBRCA1andBRCA2. Large genomic rearrangements (LGRs) inBRCA1andBRCA2are routinely assessed in clinical testing and are a significant contributor to the yield of actionable findings. In contrast, the contribution of LGRs inPALB2has not been systematically studied.MethodsWe performed targeted sequencing and real-time qPCR validation to identify LGRs inPALB2in 5770 unrelated patients with familial breast cancer and 5741 cancer-free control women from the same Australian population.ResultsSeven large deletions ranging in size from 0.96 kbp to 18.07 kbp involvingPALB2were identified in seven cases, while no LGRs were identified in any of the controls. Six LGRs were considered pathogenic as they included one or more exons ofPALB2and disrupted the WD40 domain at the C terminal end of the PALB2 protein while one LGR only involved a partial region of intron 10 and was considered a variant of unknown significance. Altogether, pathogenic LGRs identified in this study accounted for 10.3% (6 of 58) of the pathogenicPALB2variants detected among the 5770 families with familial breast cancer.ConclusionsOur data show that a clinically important proportion ofPALB2pathogenic mutations in Australian patients with familial breast cancer are LGRs. Such observations have provided strong support for inclusion ofPALB2LGRs in routine clinical genetic testing.

Supplementary Table 1 from Gene Methylation in Breast Ductal Fluid from BRCA1 and BRCA2 Mutation Carriers

Purpose: Genomic alterations (including gene hypermethylation) are likely to precede the phenotypic changes associated with breast tumorigenesis. From a prospective collection of ductal lavage (DL) samples from women with a known mutation in BRCA1 or BRCA2, we have assessed promoter methylation with a comparison of results with several variables, including breast cancer (BC) outcome.Experimental Design: Hypermethylation of p16, RASSF1A, twist, and RARβ was assessed using a qualitative, real-time, nested PCR assay. Associations between methylation status and variables were tested using Fisher's exact test or logistic regression. Analyses were done at three levels: a single breast, a single duct (both over time), and each DL sample in isolation.Results: A total of 168 samples from 93 ducts in 54 breasts have been analyzed in 34 women (16 BRCA1 and 18 BRCA2 mutation carriers). A median of 2 DL was done (range, 1–5), with 7 women developing BC on study, 1 bilateral. Methylation of p16 was associated with a known BRCA1 mutation (P = 0.001, P < 0.001, and P < 0.001 for breast, duct, and sample levels, respectively) and women with a history of contralateral BC (P = 0.001 and P < 0.001 for duct and sample levels, respectively). An association was seen for women who developed BC on study and RASSF1A methylation (P = 0.001 for sample level).Conclusions: Genetic methylation patterns could potentially be used to predict future BC risk. In addition, p16 methylation may be a predictor of BRCA1 mutation status. Further research is required to corroborate these findings. Cancer Epidemiol Biomarkers Prev; 19(1); 265–74

Background :For patients with newly diagnosed breast cancer, real-time identification of pathogenic germline mutations in hereditary breast cancer (HBC) genes can provide important information to inform decisions regarding surgery, medical oncology, radiation oncology and to enable activate risk mitigation strategies for unaffected relatives. The potential to dramatically improve outcomes by identifying HBC gene mutation carriers at diagnosis has been demonstrated in the OlympiA. The current strategy of offering germline testing based on NCCN guidelines or validated scores inherently fails to identify all patients with germline HBC gene mutations with adverse consequences for patients and their families. Also, in about 15% of patients without HBC gene mutations, the breast tumors have characteristics suggestive of a more aggressive course. Although universal HBC gene testing has been reported for sub-types of breast cancer or at specialist centers, the MAGIC study is the first prospective trial of unselected invasive breast cancers in general practice combining upfront germline and somatic sequencing with well-validated methods to assess the acceptability of universal testing to patients and clinicians. Methods :This is an Australian multi-center prospective study with 150 consecutive consented patients who are newly diagnosed with nonmetastatic breast cancers. High-grade ductal carcinoma in-situ and pleomorphic lobular cancers were included. Germline testing was performed by whole genome sequencing on DNA from blood or saliva and the data analyzed for actionable HBC gene mutations, including large genomic rearrangements. Whole genome Tumor sequencing was performed on DNA extracted from formalin fixed paraffin-embedded diagnostic tumor and the data analyzed for actionable somatic mutations as well as scoring for homologous recombination deficiency using HRDetect and mutational signatures. The frequency of actionable HBC gene mutations and the number of additional carriers identified compared to standard referral guidelines was the primary objective. For additional analysis, a 3-generation pedigree was done and NCCN, MANCHESTER, and BOADICEA scores were calculated to see whether they would qualify for germline testing according to American and Australian guidelines. Well validated questionnaires were given pre and post-testing for all patients to assess the favorability of universal testing. Health economic analysis will be performed to see the cost vs benefit of offering germline testing. Results :A total of 12 carriers of actionable germline mutations were identified (8.0%) in BRCA1 (n=2), BRCA2 (n=1), PALB2 (n=3), CHEK2 (n=2), ATM (n=2) and PMS2 (n=2). No actionable HBC germline mutations were identified in the 14 cases diagnosed with DCIS only. Only 3 of the 12 mutation positive cases (25%) were referred by the treating clinician for germline testing and including BOADICEA and MANCHESTER scores, would only have identified 6 cases (50%) eligible for germline testing. 9/12 (75%) cases were ER positive including 4 carriers of BRCA1, BRCA2 or PALB2 mutations. Preliminary ascertainment of patient acceptance demonstrates >90% were in favor of universal testing. Among the 12 HBOC gene mutation carriers, the study changed the surgical recommendation for 7 patients (58%) which included the recommendation of B/L salpingo-oophorectomy and radiation management for 3 (25%) patients. Conclusion :Universal germline HBC gene testing is the best method for detecting carriers as over 50% are missed using current. Identifying an actionable mutation in real-time can inform the decision in all specialties involved in the treatment and has a high rate of impacting the clinical decision-making process. This approach was favored by the patients and the clinicians and provided a pathway forward for breast cancer management. Citation Format: Dilanka L De Silva, Anita R Skandarajah, Michelle Sinclair, Maira Kentwell, Lisa Devereux, Magnus Zeethoven, Kirsten Hogg, Luxi Lal, Lesley Stafford, Paul A James, Geoffrey J Lindeman, Gregory B Mann, Ian G Campbell. Mutational assessment of newly diagnosed breast cancer using Germline and tumor genomICs [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-02.

Background: While protein truncating variants in RAD51C have been shown to predispose to triple negative breast cancer (TNBC) and ovarian cancer, little is known about the pathogenicity of missense (MS) variants. Methods: The frequency of rare RAD51C MS variants were assessed in the BEACCON study of 5,734 familial breast cancer cases and 14,382 population controls, and integrated with tumour sequencing data from 21 cases carrying a candidate variant to assess bi-allelic inactivation. Results: Collectively, a significant enrichment of rare missense variants was detected in cases (MAF < 0.001, OR 1.57, 95%CI 1.00 - 2.44, p = 0.05), particularly for variants with a REVEL score > 0.5 (OR 3.95, 95%CI 1.40 - 12.01, p = 0.006). Despite the large sample size, the majority of variants detected were very rare, precluding definitive conclusions about pathogenicity based solely on the case-control data. Sequencing of 21 tumours from carriers of one of eight candidate MS variants, identified four cases with bi-allelic inactivation through loss of the wild-type allele, while six lost the variant allele and ten remained heterozygous. Loss of the wild-type alleles corresponded strongly with ER- and triple-negative breast tumours and a high homologous recombination deficiency score. Conclusions: Using this approach, the p.Gly264Ser variant, which was previously suspected to be pathogenic based on small case-control analyses and loss of activity in in vitro functional assays, was shown to be benign with similar prevalence in cases and controls, and eight out of nine tumours showing loss of the variant allele or retention of heterozygosity. Conversely, the combined case-control and tumour sequencing data identified p.Ile144Thr, p.Arg212His, p.Gln143Arg and p.Gly114Arg as variants warranting further investigation.

Purpose: Risk-stratified screening has potential to improve the cost effectiveness of national breast cancer screening programs. This study aimed to inform a socially acceptable and equitable implementation framework by determining what influences a woman's decision to accept a personalized breast cancer risk assessment and what the relative impact of these key determinants is. Methods: Multicriteria decision analysis was used to elicit the relative weights for 8 criteria that women reported influenced their decision. Preference heterogeneity was explored through cluster analysis. Results: The 2 criteria valued most by the 347 participants related to program access, “Mode of invitation” and “Testing process”. Both criteria significantly influenced participation (P

ImmunoPET is a multicentre, single arm, phase 0-1 study that aims to establish ifThe phase 0 study recruited 5 PD-L1+ patients with metastatic non-small cell lung cancer (NSCLC). Patients received 60MBq/70 kgThis phase 0 study has ethics approval (HREC/65450/PMCC 20/100) and is registered on the Australian Clinical Trials Network (ACTRN12621000171819). The protocol, technical and clinical data will be disseminated by conference presentations and publications. Any modifications to the protocol will be formally documented by administrative letters and must be submitted to the approving HREC for review and approval.Australian Clinical Trials Network ACTRN12621000171819.

Preclinical testing is a crucial step in evaluating cancer therapeutics. We aimed to establish a significant resource of patient-derived xenografts (PDXs) of prostate cancer for rapid and systematic evaluation of candidate therapies. The PDX collection comprises 59 tumors collected from 30 patients between 2012–2020, coinciding with availability of abiraterone and enzalutamide. The PDXs represent the clinico-pathological and genomic spectrum of prostate cancer, from treatment-naïve primary tumors to castration-resistant metastases. Inter- and intra-tumor heterogeneity in adenocarcinoma and neuroendocrine phenotypes is evident from bulk and single-cell RNA sequencing data. Organoids can be cultured from PDXs, providing further capabilities for preclinical studies. Using a 1 x 1 x 1 design, we rapidly identify tumors with exceptional responses to combination treatments. To govern the distribution of PDXs, we formed the Melbourne Urological Research Alliance (MURAL). This PDX collection is a substantial resource, expanding the capacity to test and prioritize effective treatments for prospective clinical trials in prostate cancer., The prognosis of castration-resistant prostate cancers remains dismal, but accurate preclinical models can lead to effective therapies. Here the Melbourne Urological Research Alliance establish prostate cancer patient-derived xenografts, use the tumors for organoids and single-cell RNA-seq, and show the efficacy of PARP inhibitor combination treatments.

Various forms of private investment are considered necessary for the sustainability of biobanks, yet pose significant challenges to public trust. To manage this tension, it is vital to identify the concerns of relevant stakeholders to ensure effective and acceptable policy and practice. This research examines the aspects of commercialisation that are of most concern to the Australian public (n = 800) and patients who had donated their tissue to two large disease specific (cancer) public biobanks (n = 564). Overall, we found a commercialisation effect (higher support for public relative to private) in relation to funding, research location and access to stored biospecimens. The effect was strongest for research locations and access compared to funding. A latent class analysis revealed the pattern of concern differed, with the majority (34.1%) opposing all aspects of commercialisation, a minority supporting all (15.7%), one quarter (26.8%) opposing some (sharing and selling tissue) but not others (research locations and funding), and a group who were unsure about most aspects but opposed selling tissue (23.5%). Patient donors were found to be more accepting of and unsure about most aspects of commercialisation. Members of the (general) public who were motivated to participate in biobanking were more likely to oppose some aspects while supporting others, while those who indicated they would not donate to a biobank were more likely to oppose all aspects of commercialisation. The results suggest that approaches to policy, engagement and awareness raising need to be tailored for different publics and patient groups to increase participation.

Background Research identifying and returning clinically actionable germline variants offer a new avenue of access to genetic information. The psychosocial and clinical outcomes for women who have received this ‘genome-first care’ delivering hereditary breast and ovarian cancer risk information outside of clinical genetics services are unknown. Methods: An exploratory sequential mixed-methods case-control study compared outcomes between women who did (cases; group 1) and did not (controls; group 2) receive clinically actionable genetic information from a research cohort in Victoria, Australia. Participants completed an online survey examining cancer risk perception and worry, and group 1 also completed distress and adaptation measures. Group 1 participants subsequently completed a semi structured interview. Results: Forty-five participants (group 1) and 96 (group 2) completed the online survey, and 31 group 1 participants were interviewed. There were no demographic differences between groups 1 and 2, although more of group 1 participants had children (p = 0.03). Group 1 reported significantly higher breast cancer risk perception (p < 0.001) compared to group 2, and higher cancer worry than group 2 (p < 0.001). Some group 1 participants described how receiving their genetic information heightened their cancer risk perception and exacerbated their cancer worry while waiting for risk-reducing surgery. Group 1 participants reported a MICRA mean score of 27.4 (SD 11.8, range 9–56; possible range 0–95), and an adaptation score of 2.9 (SD = 1.1). Conclusion: There were no adverse psychological outcomes amongst women who received clinically actionable germline information through a model of ‘genome-first’ care compared to those who did not. These findings support the return of clinically actionable research results to research participants.

Background Breast cancers (BCs) that arise in individuals heterozygous for a germline pathogenic variant in a susceptibility gene, such as BRCA1 and BRCA2, PALB2, and RAD51C, have been shown to exhibit biallelic loss in the respective genes and be associated with triple-negative breast cancer (TNBC) and distinctive somatic mutational signatures. Tumor sequencing thus presents an orthogonal approach to assess the role of candidate genes in BC development. Methods Exome sequencing was performed on paired normal-breast tumor DNA from 124 carriers of germline loss-of-function (LoF) or missense variant carriers in 15 known and candidate BC predisposition genes identified in the BEACCON case-control study. Biallelic inactivation and association with tumor genome features including mutational signatures and homologous recombination deficiency (HRD) score were investigated. Results BARD1-carrying TNBC (4 of 5) displayed biallelic loss and associated high HRD scores and mutational signature 3, as did a RAD51D-carrying TNBC and ovarian cancer. Biallelic loss was less frequent in BRIP1 BCs (4 of 13) and had low HRD scores. In contrast to other established BC genes, BCs from carriers of CHEK2 LoF (6 of 17) or missense (2 of 20) variant had low rates of biallelic loss. Exploratory analysis of BC from carriers of LoF variants in candidate genes such as BLM, FANCM, PARP2, and RAD50 found little evidence of biallelic inactivation. Conclusions BARD1 and RAD51D behave as classic BRCA-like predisposition genes with biallelic inactivation, but this was not observed for any of the candidate genes. However, as demonstrated for CHEK2, the absence of biallelic inactivation does not provide definitive evidence against the gene’s involvement in BC predisposition.

We performed targeted sequencing and real-time qPCR validation to identify LGRs inSeven large deletions ranging in size from 0.96 kbp to 18.07 kbp involvingOur data show that a clinically important proportion of

Personal Breast Cancer (BC) Risk Assessments (PBCRA) have potential to stratify women into clinically-actionable BC risk categories. As this could involve population-wide genomic testing, women’s attitudes to PBCRA and views on acceptable implementation platforms must be considered to ensure optimal population participation. We explored these issues with 31 women with different BC risk profiles through semi-structured focus group discussions or interviews. Inductive thematic coding of transcripts was performed. Subsequently, women listed factors that would impact on their decision to participate. Participants’ attitudes to PBCRA were positive. Identified themes included that PBCRA acceptance hinges on result actionability. Women value the ability to inform decision-making. Participants reported anxiety, stress, and genetic discrimination as potential barriers. The age at which PBCRA was offered, ease of access, and how results are returned held importance. Most women value the opportunity for PBCRA to inform increased surveillance, while highlighting hesitance to accept reduced surveillance as they find reassurance in regular screening. Women with BRCA pathogenic variants value the potential for PBCRA to identify a lower cancer risk and potentially inform delayed prophylactic surgery. This study highlights complexities in adopting advances in BC early detection, especially for current users who value existing processes as a social good.

Background There is significant interest in personalised, risk-based breast cancer screening. This requires high quality risk assessment. The ‘Gail model’ risk assessment tool has been validated on over 40,000 BreastScreen Australia participants. We assess whether adding mammographic density (MD) information improves risk stratification on that cohort. Methods We used questionnaire data, baseline MD readings (using AutoDensity) and linked screening and cancer registry records from 40,158 BreastScreen Australia participants aged 50–69 years (via the lifepool cohort). We investigated incident invasive breast cancer rates by quintiles of Gail model scores, MD, and combinations of Gail and MD. Results Gail scores and MD values were weakly correlated (r≤0.02). Gail and MD were each strong predictors of incident breast cancer, but stronger predictors when used in combination. For example, the odds ratio for incident invasive breast cancer was 3.6 (95%CI 2.5-6.3) for the 17% of women in the upper two quintiles of both Gail and MD scores compared to the 17% of women in the lower two quintiles of both scores. In comparison, the odds ratio for breast cancer between same-size (each 17%) upper and lower groups for Gail score alone was 2.5 (95%CI 1.8-3.4), and for MD 1.9 (95%CI 1.2-2.9). Conclusions Combining Gail and MD categories improves risk stratification on BreastScreen Australia participants, compared to using Gail or MD alone. Key messages While questionnaire data and MD measures are each strong predictors of future invasive breast cancer among BreastScreen Australia participants, risk prediction is stronger when questionnaire and MD measures are combined.

Background Loss-of-function variants in RAD51C are associated with familial ovarian cancer, but its role in hereditary breast cancer remains unclear. The aim of this study was to couple breast tumor sequencing with case-control data to clarify the contribution of RAD51C to hereditary breast cancer. Methods RAD51C was sequenced in 3080 breast cancer index cases that were negative in BRCA1/2 clinical tests and 4840 population-matched cancer-free controls. Pedigree and pathology data were analyzed. Nine breast cancers and one ovarian cancer from RAD51C variant carriers were sequenced to identify biallelic inactivation of RAD51C, copy number variation, mutational signatures, and the spectrum of somatic mutations in breast cancer driver genes. The promoter of RAD51C was analyzed for DNA methylation. Results A statistically significant excess of loss-of-function variants was identified in 3080 cases (0.4%) compared with 2 among 4840 controls (0.04%; odds ratio = 8.67, 95% confidence interval = 1.89 to 80.52, P< .001), with more than half of the carriers having no personal or family history of ovarian cancer. In addition, the association was highly statistically significant among cases with estrogen-negative (P Conclusions This study provides evidence that germline loss-of-function variants of RAD51C are associated with hereditary breast cancer, particularly triple-negative type. RAD51C-null breast cancers possess similar genomic and clinical features to BRCA1-null cancers and may also be vulnerable to DNA double-strand break inducing chemotherapies and poly ADP-ribose polymerase inhibitors.

Breast cancer (BC) diagnosed after a negative mammogram but prior to the next screening episode is termed an 'interval BC' (IBC). Understanding the molecular differences between IBC and screen-detected BCs (SDBC) could improve mammographic screening and management options. Therefore, we assessed both germline and somatic genomic aberrations in a prospective cohort. Utilising the Lifepool cohort of >54 000 women attending mammographic screening programs, 930 BC cases with screening status were identified (726 SDBC and 204 IBC). Clinico-pathological and family history information were recorded. Germline and tumour DNA were collected where available and sequenced for BC predisposition and driver gene mutations. Compared to SDBC, IBCs were significantly associated with a younger age at diagnosis and tumour characteristics associated with worse prognosis. Germline DNA assessment of BC cases that developed post-enrolment (276 SDBCs and 77 IBCs) for pathogenic mutations in 12 hereditary BC predisposition genes identified 8 carriers (2.27%). The germline mutation frequency was higher in IBC versus SDBC, although not statistically significant (3.90% versus 1.81%, p = 0.174). Comparing somatic genetic features of IBC and SDBC matched for grade, histological subtype and hormone receptor revealed no significant differences, with the exception of higher homologous recombination deficiency scores in IBC, and copy number changes on chromosome Xq in triple negative SDBCs. Our data demonstrates that while IBCs are clinically more aggressive than SDBC, when matched for confounding clinico-pathological features they do not represent a unique molecular class of invasive BC, but could be a consequence of timing of tumour initiation and mammographic screening. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Bi-allelic loss-of-function (LoF) variants in the base excision repair (BER) gene NTHL1 cause a high-risk hereditary multi-tumor syndrome that includes breast cancer, but the contribution of heterozygous variants to hereditary breast cancer is unknown. An analysis of 4985 women with breast cancer, enriched for familial features, and 4786 cancer-free women revealed significant enrichment for NTHL1 LoF variants. Immunohistochemistry confirmed reduced NTHL1 expression in tumors from heterozygous carriers but the NTHL1 bi-allelic loss characteristic mutational signature (SBS 30) was not present. The analysis was extended to 27,421 breast cancer cases and 19,759 controls from 10 international studies revealing 138 cases and 93 controls with a heterozygous LoF variant (OR 1.06, 95% CI: 0.82–1.39) and 316 cases and 179 controls with a missense variant (OR 1.31, 95% CI: 1.09–1.57). Missense variants selected for deleterious features by a number of in silico bioinformatic prediction tools or located within the endonuclease III functional domain showed a stronger association with breast cancer. Somatic sequencing of breast cancers from carriers indicated that the risk associated with NTHL1 appears to operate through haploinsufficiency, consistent with other described low-penetrance breast cancer genes. Data from this very large international multicenter study suggests that heterozygous pathogenic germline coding variants in NTHL1 may be associated with low- to moderate- increased risk of breast cancer.

Introduction: Up to 25% of cases of ductal carcinoma in situ (DCIS) recur, and half of these recur as invasive breast cancer (IBC). There is no biomarker to predict which DCIS cases will recur but it has been suggested that a biomarker would allow de-escalation of the current paradigm of treating most patients with surgery and/or radiotherapy. However, we hypothesize that not all recurrences are genetically similar to the primary DCIS (i.e. non-clonal) and therefore arise de novo. To test this we assembled a large recurrence cohort to explore the clonal relatedness of primary-recurrence tumor pairs prior to proposing a predictive biomarker (Gorringe et al Mod Pathol 2015). Method: We microdissected and extracted DNA from 65 pairs of primary DCIS and recurrences. Half of these recurrences were IBC. We analyzed 21 pairs by targeted sequencing or low-coverage whole-genome sequencing (LCWGS, 2x depth) and 44 pairs with Whole Exome Sequencing (WES, average depth 95x). We similarly analyzed a set of non-recurrent DCIS cases (n=29) treated with wide local excision and with a minimum of 7 years follow-up. No matched normal samples were available. Several approaches were utilized to investigate clonal relatedness using copy number alterations and mutations (when detected), including the Clonality package (Mauguen et al Biometrics 2018), manual breakpoint inspection (Bollet et al JNCI 2008), and clonality indexes (Schultheis et al JNCI 2016). Phylogenetic analyses were carried out by MEDICC2 (Petkovic et al bioRxiv 2021) on WES samples. Results: 62% of cases were clonal (40/65), 28% were non-clonal. There were 7/65 that were equivocal, although further validation will be performed. There was no significant difference in clonal relatedness whether the recurrence was IBC or DCIS. IBC recurrent cases showed a significantly higher ploidy than the DCIS recurrences. The final phylogenetic analysis with MEDICC2 will be presented, which will take into account any subclonal whole-genome doubling events. Furthermore, clonal primary DCIS showed a significantly higher number of TP53 mutations compared to non-recurrent and non-clonal primaries (p Conclusion: Detailed molecular analysis of a large cohort of matched DCIS primary and their recurrences using high sequencing resolution showed a substantial proportion of recurrences were not genetically related to the primary DCIS. Our observations raise the question of whether a tumor intrinsic biomarker alone would be sufficient to predict DCIS recurrences. Citation Format: Tanjina Kader, Sakshi Mahale, Magnus Zethoven, Hugo Saunders, Dorothea Lesche, David Byrne, Siqi Lai, Lauren Tjoeka, Claire Candido, Maree Pechlevanis, Olivia Craig, Jia-Min Pang, Lisa Devereux, Shona Hendry, Eloise House, Sureshni Jayasinghe, Michael Toss, Islam M. Miligy, Emad Rakha, Stephen Fox, Bruce Mann, Ian Campbell, Kylie Gorringe. Predictive biomarkers of recurrence may not be useful for deescalating treatment of breast ductal carcinoma in situ due to de novo ipsilateral breast carcinoma development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 43.

Mammographic density (MD) influences breast cancer risk, but how this is mediated is unknown. Molecular differences between breast cancers arising in the context of the lowest and highest quintiles of mammographic density may identify the mechanism through which MD drives breast cancer development. Women diagnosed with invasive or in situ breast cancer where MD measurement was also available (n = 842) were identified from the Lifepool cohort of >54,000 women participating in population-based mammographic screening. This group included 142 carcinomas in the lowest quintile of MD and 119 carcinomas in the highest quintile. Clinico-pathological and family history information were recorded. Tumor DNA was collected where available (n = 56) and sequenced for breast cancer predisposition and driver gene mutations, including copy number alterations. Compared to carcinomas from low-MD breasts, those from high-MD breasts were significantly associated with a younger age at diagnosis and features associated with poor prognosis. Low- and high-MD carcinomas matched for grade, histological subtype, and hormone receptor status were compared for somatic genetic features. Low-MD carcinomas had a significantly increased frequency of TP53 mutations, higher homologous recombination deficiency, higher fraction of the genome altered, and more copy number gains on chromosome 1q and losses on 17p. While high-MD carcinomas showed enrichment of tumor-infiltrating lymphocytes in the stroma. The data demonstrate that when tumors were matched for confounding clinico-pathological features, a proportion in the lowest quintile of MD appear biologically distinct, reflective of microenvironment differences between the lowest and highest quintiles of MD.

Background. Germline mutations in certain genes account for a large proportion of inherited risk for breast and ovarian cancer. The identification of asymptomatic mutation carriers could significantly reduce the incidence of these diseases as active risk management can dramatically reduce the risk of developing cancer. In most countries, identifying high-risk individuals is based on their family history. In general, a family is first identified because one family member develops cancer and, because of high-risk indicators is referred to a familial cancer centre (FCC). However, current data suggests that many BRCA1 or BRCA2 mutation carriers do not have a remarkable history of cancer in a close relative. Population-based genetic testing would be a far more effective strategy for identification of at-risk individuals. To test the feasibility of such a strategy we are conducting a population genetic testing trial for actionable mutations in 11 breast/ovarian cancer predisposition genes (BRCA1, BRCA2, PALB2, ATM, CDH1, PTEN, STK11, TP53, BRIP1, RAD51C, RAD51D) among 15,000 healthy women from the Australian population. Methods. All subjects are female participants in the LifePool cohort (www.lifepool.org) who had no personal history of breast or ovarian cancer at the time of DNA collection. Participants found to carry an actionable germline mutation were notified by letter with an invitation to contact the PeterMac telephone genetic counselling service for further information and/or also invited for counselling at an FCC. Only participants with an actionable mutation were notified of their genetic testing result. Results. Of the 5,557 women tested to date, 40 (0.72%) were carriers of mutations that are currently actionable in the Australian context (BRCA1 n=7, BRCA2 n=15, PALB2 n=15, ATM n=3). All 40 women accepted the invitation to attend a familial cancer centre for formal predictive testing. Less than 20% of the women would have met the minimum threshold for clinical genetic testing under current guidelines. A further 16 participants (0.29%) carried mutations in BRIP1, RAD51C and RAD51D but were not notified of the result as these genes are not currently actionable in Australia. No mutations were identified in CDH1, PTEN, STK11 or TP53. Conclusions. A relatively large proportion of cancer free-women from Australia carry high-risk mutations in BRCA genes and subsequent uptake of clinical genetic testing was very high. Population-based genetic testing is well accepted and can identify a much larger proportion of the at risk-population than contemporary family history based approaches. Citation Format: Campbell IG, Rowley S, Devereux L, McInerny S, Grewal N, Young M-A, Lee A, Trainer A, James P. Population genetic testing for breast cancer susceptibility [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD1-07.

Background: Identifying the missing hereditary factors underlying the familial risk of breast cancer could have a major and immediate impact on managing the breast cancer risk for these families. Methods: We identified candidate breast cancer predisposition genes through whole exome sequencing of BRCAx families and subsequently sequenced up to 1325 genes, along with 76 common low penetrance variants associated with breast cancer, in index cases from 6,000 BRCAx families and 6,000 cancer free women (ethnically matched on principal component analysis). Results: The role of recently described (PALB2) or suspected (MRE11A) moderately penetrant genes was confirmed. Conversely, the size of the cohort means that the absence of enrichment for loss of function (LoF mutations) provides strong evidence against other reported breast cancer genes (BRIP1, RINT1, RECQL). For further moderate risk variants (in CHEK2, ATM, BRCA2) we observed significant risk modification based on the polygenic risk score (PRS - calculated from the common variant data), with the risk restricted to the co-occurrence of the rare variant and high PRS. Novel candidate genes were identified based on LoF mutations, including NTHL1 (38 cases versus 15 controls, OR 2.5 p=0.002): a member of the base excision repair (BER) pathway. DNA sequencing of the breast carcinomas from 17 heterozygous NTHL1 mutation carriers revealed a strong bias towards a C:G>T:A (C>T) transitions, consistent with a BER defect, which confirmed the recent findings in colorectal carcinomas from bi-allelic NTHL1 mutation carriers. This data extends the cancer predisposition phenotype of NTHL1 to heterozygous carriers. In addition to NTHL1, there are a large number of candidate genes where the ratio of LoF mutations in cases versus controls indicates that they may convey an actionable level of risk; 46 genes (519 families) meet the basic criteria of multiple LoF variants and an OR >2 for cases versus controls – including previously proposed breast cancer genes MRE11A, BLM, MLH1, MYH, FANCD2 and functionally plausible candidates such as MLH3, PARP2 and ATR. Collectively the OR of breast cancer for LoF mutations in this group of genes is 3.3 (95% CI 2.7-3.9, P=3.5x10-41). Conclusion: Our data shows that the effect of rare variation in established and novel breast cancer genes, along with consideration of the background polygenic risk, together explains a substantial component of the heritable risk of breast cancer in our cohort. Citation Format: Campbell IG, Li N, Rowley S, Goode D, Devereux L, McInerny S, Grewal N, Lee A, Trainer A, Wong-Brown M, Scott R, Gorringe K, James P. The contribution of rare variants, polygenic risk, and novel candidate genes to the hereditary risk of breast cancer in a large cohort of breast cancer families [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD1-04.

Gene panel sequencing is revolutionizing germline risk assessment for hereditary breast cancer. Despite scant evidence supporting the role of many of these genes in breast cancer predisposition, results are often reported to families as the definitive explanation for their family history. We assessed the frequency of mutations in 18 genes commonly included in hereditary breast cancer panels among 2,000 index cases from breast cancer families and 1,997 population controls. Cases were predominantly breast cancer-affected women referred to specialized familial cancer centers (BRCA1 and BRCA2 wild-type). Controls were cancer-free women from the LifePool study (www.lifepool.org). Sequencing data were filtered for known pathogenic or novel loss of function mutations. The frequency of pathogenic mutations in BRCA1 and BRCA2 in the control group was 0.2% (4 mutations) and 0.4% (8 mutations), respectively, which is consistent with previous indirect estimates for Caucasian populations but to our knowledge this the largest direct assessment of their prevalence. Excluding 18 mutations identified in BRCA1 and BRCA2 among the cases and controls, a total of 69 cases (3.5%) and 26 controls (1.3%) were found to carry an "actionable mutation". PALB2 was most frequently mutated (22 cases, 3 controls), while no mutations were identified in PTEN or STK11. Among the remaining genes, loss of function mutations were rare with similar frequency between cases and controls. The frequency of mutations in most breast cancer panel genes among individuals selected for possible hereditary breast cancer is low and in many cases similar or even lower than that observed among cancer-free population controls. While multi-gene panels can significantly aid in cancer risk management, they equally have the potential to provide clinical misinformation and harm at the individual level if the data is not interpreted cautiously. Citation Format: Campbell IG, Thompson ER, Rowely SM, Li N, McInerny S, Devereux L, Wong-Brown MW, Trainer AH, Mitchell G, Scott RJ, James PA. Panel testing for familial breast cancer: Tension at the boundary of research and clinical care. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-09-02.

The traditional model of familial breast cancer practice involves ascertaining high-risk individuals based on family history. However, most individuals who carry a BRCA1 or BRCA2 mutation will not have a family history of breast or other cancer in a close relative. Moving germline testing from the familial cancer center to the population will result in many essentially unanticipated findings of great significance in regards to the risk of breast cancer in healthy relatives. To date there have been no large studies that have directly measured the frequency of BRCA1 and BRCA2 mutations in a cancer-free general population. As a first step toward population based BRCA1 or BRCA2 screening, we sequenced the entire coding region of these genes in 1,997 cancer-free Australian women recruited from the lifepool study (www.lifepool.org) which is a cohort of women attending the Australian population based mammographic screening program. Sequencing data were filtered for known pathogenic or novel loss of function mutations. Twelve individuals were identified with pathogenic mutations in either BRCA1 (4 mutations) or BRCA2 (8 mutations), which is consistent with previous indirect estimates for Caucasian populations but to our knowledge this is the largest direct assessment of their prevalence. Interestingly, the self-reported cancer family history of the majority of the 12 mutation positive women was unremarkable. All 12 women subsequently accepted an invitation to attend a Familial Cancer Centre for advice on whether to proceed with formal clinical genetic testing. A population carrier frequency of 0.6% for mutations in BRCA1 and BRCA2, coupled with the rapidly declining costs of gene panel sequencing, suggests that population-based screening for these genes will be a highly cost effective way of reducing the incidence of breast and ovarian cancer through preventative strategies. Citation Format: Campbell IG, Trainer AH, Devereux L, Young M-A, Mitchell G, Rowley S, Li N, James PA, Thompson E. Breast cancer prevention: Is it time for population-based mutation screening of high risk genes?. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-09-03.

The identification of carriers of hereditary breast and ovarian cancer (HBOC) gene variants through family cancer history alone is suboptimal, and most population-based genetic testing studies have been limited to founder mutations in high-risk populations. Here, we determine the clinical utility of identifying actionable variants in a healthy cohort of women. Germline DNA from a subset of healthy Australian women participating in the lifepool project was screened using an 11-gene custom sequencing panel. Women with clinically actionable results were invited to attend a familial cancer clinic (FCC) for post-test genetic counseling and confirmatory testing. Outcomes measured included the prevalence of pathogenic variants, and the uptake rate of genetic counseling, risk reduction surgery, and cascade testing. Thirty-eight of 5908 women (0.64%) carried a clinically actionable pathogenic variant. Forty-two percent of pathogenic variant carriers did not have a first-degree relative with breast or ovarian cancer and 89% pursued referral to an FCC. Forty-six percent (6/13) of eligible women pursued risk reduction surgery, and the uptake rate of cascade testing averaged 3.3 family members per index case. Within our cohort, HBOC genetic testing was well accepted, and the majority of high-risk gene carriers identified would not meet eligibility criteria for genetic testing based on their existing family history.

Background There is a growing interest in delivering more personalised, risk-based breast cancer screening protocols. This requires population-level validation of practical models that can stratify women into breast cancer risk groups. Few studies have evaluated the Gail model (NCI Breast Cancer Risk Assessment Tool) in a population screening setting; we validated this tool in a large, screened population. Methods We used data from 40,158 women aged 50–69 years (via the lifepool cohort) participating in Australia’s BreastScreen programme. We investigated the association between Gail scores and future invasive breast cancer, comparing observed and expected outcomes by Gail score ranked groups. We also used machine learning to rank Gail model input variables by importance and then assessed the incremental benefit in risk prediction obtained by adding variables in order of diminishing importance. Results Over a median of 4.3 years, the Gail model predicted 612 invasive breast cancers compared with 564 observed cancers (expected/observed (E/O) = 1.09, 95% confidence interval (CI) 1.00–1.18). There was good agreement across decile groups of Gail scores (χ2 = 7.1, p = 0.6) although there was some overestimation of cancer risk in the top decile of our study group (E/O = 1.65, 95% CI 1.33–2.07). Women in the highest quintile (Q5) of Gail scores had a 2.28-fold increased risk of breast cancer (95% CI 1.73–3.02, p

Background Genome-wide association studies (GWASs) have identified numerous single-nucleotide polymorphisms (SNPs) associated with small increases in breast cancer risk. Studies to date suggest that some SNPs alter the expression of the associated genes, which potentially mediates risk modification. On this basis, we hypothesised that some of these genes may be enriched for rare coding variants associated with a higher breast cancer risk. Methods The coding regions and exon-intron boundaries of 56 genes that have either been proposed by GWASs to be the regulatory targets of the SNPs and/or located

Introduction: Over 90% of breast cancers occur sporadically with no obvious hereditary factors at play, therefore highlighting the need for biomarkers that can identify women who are predisposed to developing breast cancer. DNA methylation of the BRCA1 promoter region is detectable at low levels in the peripheral blood of some women. This phenomenon is referred to as constitutional BRCA1 methylation. Peripheral blood methylation of the BRCA1 gene has been identified as a predisposition factor to the development of BRCA1 methylated tumors. We performed a case-control study to assess peripheral blood BRCA1 methylation from healthy women, and from women with breast cancer. This study aimed to establish the frequency of peripheral blood methylation of the BRCA1 gene in women with and without breast cancer, to further our understanding of the role of constitutional BRCA1 methylation in breast cancer predisposition. Methods: Constitutional BRCA1 methylation was assessed in peripheral blood DNA of breast cancer patients and healthy women. Whole blood DNA was obtained from healthy women (n=327 controls) and women with breast cancer (n=300 cases) as part of the LifePool Project. Blood was collected on the day of mammography with BreastScreen Victoria. We used a probe-based droplet digital PCR (ddPCR) assay designed to quantify methylated and unmethylated alleles at the BRCA1 promoter region. Results: Previous research using less-sensitive methodologies has reported a BRCA1 methylation frequency of 2-4% in peripheral blood of healthy women. In this study, there was no significant difference in the observed BRCA1 methylation frequency between cases and controls (6.6% and 6.4% respectively). The discrepancy between our observed methylation frequencies and previously reported data can be attributed to the higher sensitivity of the ddPCR methodology. Nevertheless, the level of BRCA1 methylation in cases was significantly higher than the level of methylation observed in controls, consistent with previous studies. In addition, the frequency of BRCA1 methylation was higher in women diagnosed with breast cancer under the age of 50 compared to women diagnosed over 50 years old (average methylation frequency = 12.5% and 5.3% respectively). Conclusions: ddPCR methodology enabled accurate quantification of methylated BRCA1 alleles in peripheral blood DNA down to less than 0.1%. In this selected population of women undergoing mammography, there was no difference in the frequency of detectable BRCA1 methylation between cases and controls. Once age was considered, detectable BRCA1 methylation showed an association with age of onset. Tumours for the women with breast cancer will be retrieved and tested for BRCA1 methylation, to further examine the link between the methylation observed in peripheral blood DNA and development of a BRCA1-methylated tumor. Citation Format: Basant Ebaid, Hongdo Do, Jia Min Pang, Lisa Devereux, Alexander Dobrovic. A case-control study of constitutional BRCA1 methylation in a mammographically screened cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 823.

Rad50 interactor 1 (RINT1) has recently been reported as an intermediate-penetrance (odds ratio 3.24) breast cancer susceptibility gene, as well as a risk factor for Lynch syndrome. The coding regions and exon-intron boundaries of RINT1 were sequenced in 2024 familial breast cancer cases previously tested negative for BRCA1, BRCA2, and PALB2 mutations and 1886 population-matched cancer-free controls using HaloPlex Targeted Enrichment Assays. Only one RINT1 protein-truncating variant was detected in a control. No excess was observed in the total number of rare variants (truncating and missense) (28, 1.38 %, vs. 27, 1.43 %. P > 0.999) or in the number of variants predicted to be pathogenic by various in silico tools (Condel, Polyphen2, SIFT, and CADD) in the cases compared to the controls. In addition, there was no difference in the incidence of classic Lynch syndrome cancers in RINT1 rare variant-carrying families compared to RINT1 wild-type families. This study had 90 % power to detect an odds ratio of at least 2.06, and the results do not provide any support for RINT1 being a moderate-penetrance breast cancer susceptibility gene, although larger studies will be required to exclude more modest effects. This study emphasizes the need for caution before designating a cancer predisposition role for any gene based on very rare truncating variants and in silico-predicted missense variants.

Background: High mammographic density (MD) is a known risk factor for breast cancer, but it is currently unknown whether this altered microenvironment leads to the development of genomically different tumours. Method: We used lifepool, an Australian prospective population-based cohort of over 53,000 women currently in a mammographic screening program, to identify >1000 cases of breast carcinoma (in situ and invasive breast cancer) for analysis. Pathology reports, detection modality (screen-detected/interval), MD data (AutoDensity, Nickson et al., 2013 DOI:10.1186/bcr3474), germline DNA and tumor tissue were available for a subset of cases. Tumours were classified into quintiles, depending on the relative MD ranking of the most recent normal mammogram. Tumour DNA was analysed using Affymetrix OncoScan MIP arrays or using a targeted Agilent SureSelect and Illumina sequencing panel of 105 genes relevant to breast cancer. Copy number and mutation data were compared between tumours from the highest (high-MD) and lowest (low-MD) quintiles of MD. Results: Preliminary analysis found no significant differences in clinico-pathological features between tumours from breasts with high-MD (n=38) and low-MD (n=35). Genomic analysis of 8 high-MD tumours and 8 low-MD tumours identified chromosome 15 loss as being significantly enriched in low-MD tumours. Only Low-MD tumours to date contained TP53 mutation (3/8). Data will be presented from at least an additional 9 low-MD and 15 high-MD tumours currently undergoing genomic assays. Conclusion: When complete, our data will be the first to evaluate the somatic genetic events in tumours arising in low-MD and high-MD breasts. Although our cohort to date is small, it may be possible that tumours from extremes of MD have different genomic characteristics. Citation Format: Gorringe KL, Hughes S, Nickson C, Devereux L, Pridmore V, Rowley SM, Byrne DJ, Cheasley D, Kavanagh AM, Mann GB, Fox SB, Campbell IG. Genomic analysis of cancers arising in breasts with different mammographic density [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-04-06.

Purpose Gene panel sequencing is revolutionizing germline risk assessment for hereditary breast cancer. Despite scant evidence supporting the role of many of these genes in breast cancer predisposition, results are often reported to families as the definitive explanation for their family history. We assessed the frequency of mutations in 18 genes included in hereditary breast cancer panels among index cases from families with breast cancer and matched population controls. Patients and Methods Cases (n = 2,000) were predominantly breast cancer-affected women referred to specialized Familial Cancer Centers on the basis of a strong family history of breast cancer and BRCA1 and BRCA2 wild type. Controls (n = 1,997) were cancer-free women from the LifePool study. Sequencing data were filtered for known pathogenic or novel loss-of-function mutations. Results Excluding 19 mutations identified in BRCA1 and BRCA2 among the cases and controls, a total of 78 cases (3.9%) and 33 controls (1.6%) were found to carry potentially actionable mutations. A significant excess of mutations was only observed for PALB2 (26 cases, four controls) and TP53 (five cases, zero controls), whereas no mutations were identified in STK11. Among the remaining genes, loss-of-function mutations were rare, with similar frequency between cases and controls. Conclusion The frequency of mutations in most breast cancer panel genes among individuals selected for possible hereditary breast cancer is low and, in many cases, similar or even lower than that observed among cancer-free population controls. Although multigene panels can significantly aid in cancer risk management and expedite clinical translation of new genes, they equally have the potential to provide clinical misinformation and harm at the individual level if the data are not interpreted cautiously.

The availability of a biological resource such as human tissue and its derivatives for research that is fit for purpose and linked to well-annotated clinical data under approved ethical protocols is an essential facility for biomedical research, especially in the present era of personalized, translational medicine. The importance of these facilities have been recognized in the popular media with Time Magazine (2009) identifying biobanks as one of the ten tools of significance in recent times that have contributed to health and well-being [1]. Recent investments to upgrade the health department’s databases held by government and institutional registries, with electronic data mining and linkage tools, now means it is possible to perform data linkage to a specific disease, such as a cancer diagnosis and the related treatments but in addition, to have access to the other non-cancer related conditions and treatments so the effect of co-morbidities can be researched and the overall influence of the treatments determined. This important data linkage can be routinely performed by a biobank with the participant’s informed consent whilst still protecting the privacy and security of all personal information [2]. Access to the national health department’s clinical databases also provides practical and great economies to a biobank whose routine task is to perform clinical follow-up on all recruited participants. The reason being, the national health records database provides the additional clinical history and treatment regimen information that a bio-bank cannot currently obtain, as it is impractical for the biobank team to know about, or even try to cover, all hospital and/or general practitioner interactions that a biobank participant may have.

The breast cancer predisposition gene, BRCA2, has a large number of genetic variants of unknown effect. The variant rs11571833, an A > T transversion in the final exon of the gene that leads to the creation of a stop codon 93 amino acids early (K3326*), is reported as a neutral polymorphism but there is some evidence to suggest an association with an increased risk of breast cancer. We assessed whether this variant was enriched in a cohort of breast cancer cases ascertained through familial cancer clinics compared to population-based non-cancer controls using a targeted sequencing approach. We identified the variant in 66/2634 (2.5%) cases and 33/1996 (1.65%) controls, indicating an enrichment in the breast cancer cases (p = 0.047, OR 1.53, 95% CI 1.00–2.34). This data is consistent with recent iCOGs data suggesting that this variant is not neutral with respect to breast cancer risk. rs11571833 may need to be included in SNP panels for evaluating breast cancer risk.

Introduction PALB2 is emerging as a high-penetrance breast cancer predisposition gene in the order of BRCA1 and BRCA2. However, large studies that have evaluated the full gene rather than just the most common variants in both cases and controls are required before all truncating variants can be included in familial breast cancer variant testing. Methods In this study we analyse almost 2000 breast cancer cases sourced from individuals referred to familial cancer clinics, thus representing typical cases presenting in clinical practice. These cases were compared to a similar number of population-based cancer-free controls. Results We identified a significant excess of truncating variants in cases (1.3 %) versus controls (0.2 %), including six novel variants (p = 0.0001; odds ratio (OR) 6.58, 95 % confidence interval (CI) 2.3–18.9). Three of the four control individuals carrying truncating variants had at least one relative with breast cancer. There was no excess of missense variants in cases overall, but the common c.1676A > G variant (rs152451) was significantly enriched in cases and may represent a low-penetrance polymorphism (p = 0.002; OR 1.24 (95 % CI 1.09–1.47). Conclusions Our findings support truncating variants in PALB2 as high-penetrance breast cancer susceptibility alleles, and suggest that a common missense variant may also lead to a low level of increased breast cancer risk.