Lisa Devereux

Panel Testing for Familial Breast Cancer: Calibrating the Tension Between Research and Clinical Care

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.

Prevalence of PALB2 mutations in Australian familial breast cancer cases and controls

IntroductionPALB2 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.MethodsIn 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.ResultsWe 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).ConclusionsOur 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.

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 Fishers 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

A community-based model of rapid autopsy in end-stage cancer patients

To the Editor: Systematic genomic studies, including the Cancer Genome Atlas (TCGA)1 and the International Cancer Genome Consortium (ICGC)2, have provided an unprecedented catalog of driver mutations in human cancer. However, these studies use mainly primary, pre-treatment tumor material obtained at surgery with curative intent. There is an urgent need to identify and characterize resistance mechanisms to understand how cancers can evade even the best medical efforts and kill patients; therefore, access to end-stage disease is important. Solid cancers show considerable spatial3, temporal4,5 and genomic heterogeneity at diagnosis. Selective pressure and mutagenic impact of treatment6 drives intra-patient evolution of cancer cell populations4,7. Understanding acquired resistance requires access to paired preand post-treatment samples4,7; however, curative surgery is typically confined to patients with locoregional disease, and opportunities for tumor sampling in advanced disseminated disease are limited. Here, we describe Cancer Tissue Collection After Death (CASCADE), an autopsy program that overcomes logistical challenges to enable collection of samples at end stage for research in melanoma and breast, ovarian and prostate cancers. For the CASCADE study, we aimed to recruit cancer patients close to the end of life, including those outside the minority of patients who die in hospitals. To preserve tissue integrity, autopsies must commence within a few hours of death, requiring access to around-the-clock services. Intervention in the emotionally charged end-of-life environment must be managed in an ethical manner and to a high standard. Finally, we aimed for the study to be highly cost-effective. We believe our approach to meeting these challenges is applicable to researchers in other large urban centers. Here we summarize the main steps in CASCADE’s operating protocol and our experiences from the initial 3 years and 30 autopsies performed (Fig. 1). Information about institutional review board approvals (including a detailed patient informationand-consent form), the autopsy procedure and certain laboratory processes is given in Supplementary Methods and Supplementary Figure 1. Recruitment of participants was led by the clinicians. Such discussions require careful consideration, in timing and in language, and were initiated only if there was a perception that tissue donation would be acceptable to the patients and their families. Factors suggesting acceptability include the emotional stability of the participant and family members and their clarity about and acceptance of the terminal nature of the disease. On occasion, participants prompted discussion by asking about organ or body donation. Consent discussions typically involved oncologists and/or palliative care physicians employed at recruiting hospitals who had established a care relationship with the participant and their family during the patient’s cancer journey. Frequently, the study was introduced at one meeting and discussed over several subsequent clinic visits, allowing patients and their families time to consider participation. We view the involvement of family members in the consent process as essential to support the participant and facilitate decisionmaking. Involvement of family members also ensures that they are fully aware of the autopsy process and helps to clarify funeral arrangements for the study team. After obtaining consent, study investigators collated clinical information, including that related to past and current treatment and diagnostic procedures such as imaging, on an ongoing basis. Between September 2012 and August 2015, 40 patients were approached, and 37 (92.5%) expressed interest in participating. Of those 32 patients (80%) consented; the other 5 had rapid clinical deterioration precluding

Reevaluation of the BRCA2 truncating allele c.9976A > T (p.Lys3326Ter) in a familial breast cancer context.

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.

Loss of Heterozygosity Analysis in Ductal Lavage Samples from BRCA1 and BRCA2 Carriers: A Cautionary Tale

Background: Loss of heterozygosity (LOH) in breast ductal lavage (DL) fluid has been reported to be a potential biomarker of malignant change. Interpretation of LOH is reliant on sufficient quality and quantity of DNA. We investigated LOH of the BRCA1/2 loci in DL samples from BRCA1/2 mutation carriers, while also assessing the effect of DNA quantity. Methods: DNA yield was estimated using quantitative real-time PCR. Allelic status of DL DNA was determined using fluorescently tagged microsatellite markers with the subjects lymphocytic DNA serving as a control. Samples were scored as consistently heterozygous or as demonstrating LOH if the same result was observed in replicate experiments. Additionally, samples were scored as “discordant LOH” if they initially showed LOH, but in replicate experiments either showed heterozygosity or LOH of the opposite allele. Results: In 11 BRCA1 carriers, 46 ducts were assessable, and 39 ducts from 14 BRCA2 carriers were assessable. LOH was observed in 17% and 18% of ducts from BRCA1 and BRCA2, respectively. Discordant results were seen in 23 BRCA1 (50%) and 15 BRCA2 (38%) samples. DNA yield was significantly greater in samples that were consistently heterozygous than those that were either discordant or showed LOH in replicate experiments for both BRCA1 (P = 0.003) and BRCA2 (P = 0.003). Conclusions: DNA quantity is highly variable between DL samples, with low yields likely to detrimentally affect the interpretation of LOH. In conclusion, LOH may not be an adequate method to detect the early stages of malignant change in samples obtained via DL. (Cancer Epidemiol Biomarkers Prev 2006;15(7):1396–8)

Mutations in RECQL are not associated with breast cancer risk in an Australian population

To the Editor — The slow progress of intensive international efforts over the past two decades to identify the remaining genetic causes of familial breast cancer suggests that numerous predisposition genes exist, which are likely to be much rarer than BRCA1 and BRCA2, and to each account for a small proportion of families. Searching for very rare risk alleles in the context of population diversity further increases the challenges of identifying and validating new breast cancer–associated genes, as evidenced by the inability of subsequent studies to validate primary reports of new genes1–3. Here we argue that the previously reported breast cancer–susceptibility gene RECQL is not associated with breast cancer risk in an Australian population. Two loss-of-function (LoF) founder mutations in RECQL have been genotyped in Polish and French-Canadian subjects, and a greater-than-fivefold-increased risk has been reported for c.1667_1667+ 3delAGTA carriers in the Polish women (0.23% in 13,136 unselected breast cancer cases versus 0.04% in 4,702 controls)4,5. Likewise, a 16-fold-increased risk has been reported for c.634C> T carriers in FrenchCanadian subjects with early-onset breast cancer compared with population newborn controls (0.69% in 1,013 cases versus 0.014% in 7,136 controls). At that time, a smaller study of familial breast cancer cases and controls from Han Chinese individuals from Northern China (448 cases and 1,588 controls) supported a role for RECQL in breast cancer predisposition but was underpowered compared with the study reported in Polish and FrenchCanadian populations6. Another study has reported a 0.54% carrier frequency of RECQL LoF variants in 1,110 highrisk females from Hong Kong with breast cancer; however, very few controls were sequenced (88 controls)7. In contrast, a null result has been reported for the c.1667_1667+ 3delAGTA variant (reported to be enriched more than fivefold in Polish individuals with breast cancer) among 2,596 breast cancer cases and 2,132 controls (0.35% versus 0.28%, odds ratio 1.23, P = 0.69) from Central European individuals who were likely to have ancestry similar to that of the Polish population8. Because some groups are now suggesting that RECQL be included in germline breast cancer–predisposition testing panels, it is crucial to be certain that this gene has genuine relevance to breast cancer predisposition, because adverse outcomes could potentially arise if the conclusions of the original publications are incorrect or overestimated. To address this important clinical question, we sequenced all exons and at least 10 bp of the exon–intron flanking regions of RECQL in 9,112 subjects from Australia (Supplementary Note). The case subjects were female index patients diagnosed with breast cancer (> 95% of cases) or ovarian cancer from 4,536 families with breast cancer with a negative result after BRCA1 and BRCA2 mutation testing, and were ascertained from the Variants in Practice Study from the combined Victorian and Tasmanian Familial Cancer Centres, and Pathology North, NSW Health Pathology, Newcastle, Australia. The controls were 4,576 women in the LifePool cohort (see URLs) who were above 40 years of age and were cancer free as of May 2016. All exons and exon–intron boundaries of RECQL were sequenced with high and consistent coverage in cases and controls (average sequencing depths of 189.0 and 195.4, respectively). Overall, 96.7% of the bases among the cases and 98.4% of the bases among the controls were sequenced to a depth greater than tenfold. We identified 13 LoF mutations (defined as nonsense, frameshift or essential splice-site mutations) in the cases and 25 in the controls (0.29% versus 0.55%, odds ratio 0.52, 95% confidence interval 0.25–1.06, P = 0.072 by two-tailed Fisher’s exact test here and subsequently) (Table 1 and Supplementary Tables 1 and 2). One variant, c.1859C> G (p.Ser620Ter), located in the last exon and resulting in a stop codon 29 amino acids upstream of the normal stop codon, accounted for most of the LoF mutations (6 in the cases and 16 in the controls). No carriers of the Quebec founder mutation (c.634C> T) were identified in either the cases or controls, and only two cases and one control were carriers of the Polish founder mutation c.1667_1667+ 3delAGTA. The LoF mutation frequency in our controls was similar to that reported in non-Finnish Europeans in the Exome Aggregation Consortium (ExAC) browser (0.65% in ~27,000 non-Finnish European participants and 0.51% in ~53,000 all participants; ExAC version 0.3, excluding TCGA data, released 13 January 2015)9. The personal and family histories of patients with breast cancer and carrying RECQL LoF mutations are shown in Supplementary Table 3. A marginally higher frequency of rare (minor allele frequency ≤ 0.5%) missense variants was observed in the cases compared with the controls, but the difference was not statistically significant (54 cases, 1.19%, versus 37 controls, 0.81%, P = 0.073) (Supplementary Table 4). In addition, no significant difference was observed between the cases and controls when the variants were filtered for likely pathogenicity with the commonly used in silico tools Condel, PolyPhen-2, SIFT, CADD and REVEL (Supplementary Table 5). The variants c.644G>A (p.Arg215Gln), c.1363C>T (p.Arg455Cys), c.1373T>A (p.Met458Lys) and c.1685C>T (p.Thr562Ile), previously reported by Sun et al.6 to lack helicase activity and thus to potentially represent pathogenic alleles, were not detected in either our case or control cohorts, in agreement with the rarity of these variants reported in nonFinnish Europeans in ExAC. Given the rarity of the LoF alleles in RECQL, genetic heterogeneity among study subjects may distort the relativerisk estimation in case–control studies. Principal-component analysis with 74 ancestry-informative markers showed that the study subjects were predominantly of European ancestry (> 96%), and there was a significant overlap in ancestry distributions between cases and controls (Supplementary Fig. 1), thus strongly suggesting that our findings were not due to major differences in ancestry between our cohorts. It is generally accepted that a predisposition gene is considered actionable only if the 90% confidence limit of the estimated relative risk is greater than four10; thus, RECQL would be excluded, because our study had 80% power to detect an odds ratio of at least 1.89 (P < 0.05) for a variant with a population frequency of 0.65% (RECQL LoF variant frequency in ExAC

Population-based genetic testing of asymptomatic women for breast and ovarian cancer susceptibility

PurposeThe 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.MethodsGermline 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.ResultsThirty-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.ConclusionWithin 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.

Meeting abstracts from the Annual Conference on Hereditary Cancers 2015

A1 Panel Testing for Breast Cancer Risk Assessment: is it just because we can rather than should? Ella R. Thompson, Michelle Wong-Brown, Simone M. Rowley, Susan Dooley, Na Li1, Michael Hipwell, Simone McInerny, Cliff Meldrum, Lisa Devereux, David Mossman, Alison H. Trainer, Briar-Rose Millar, Gillian Mitchell, Cate Smith, Paul A. James, Ian G. Campbell, Rodney J. Scott Cancer Genetics Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia; The University of Newcastle and Hunter Medical Research Institute, Newcastle, Australia; Division of Genetics, Hunter Area Pathology Service, Newcastle, Australia; Hereditary Cancer Program, BC Cancer Agency, Vancouver, Canada Hereditary Cancer in Clinical Practice 2017, 15(Suppl 1):A1

Biobanking in Cancer Research

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.