Michelle W. Wong-Brown

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.

The relative mRNA expression of p53 isoforms in breast cancer is associated with clinical features and outcome.

Mutation of p53 is a common feature of cancer. Breast cancer is the most common malignancy that develops in women; however, somatic mutation of p53 is rare, suggesting that p53 becomes inactivated by other mechanisms. p53 is expressed as smaller isoforms, some of which inhibit wild-type p53. There are no studies that have examined the relative expression of all isoforms in this disease. We have analysed the relative messenger RNA expression of the p53 isoforms, Δ40, Δ133, β and γ in a panel of 6 breast cancer cell lines, 148 breast cancers specimens and 31 matched normal adjacent tissues by semi-quantitative real-time reverse transcription-PCR and analysed their relationship to clinical features and outcome. We have identified several important clinical associations, particularly with Δ40p53, which was expressed at levels that were ~50-fold higher than the least expressed isoform p53γ. Δ40p53 was significantly upregulated in tumour tissue when compared with the normal breast and was significantly associated with an aggressive breast cancer subtype-triple negative. Additionally, p53β expression was significantly negatively associated with tumour size and positively associated with disease-free survival, where high levels of p53β were protective, particularly in patients with a mutation in p53, suggesting p53β may counteract the damage inflicted by mutant p53. In conclusion, the relative expression of p53 isoforms is related to clinical features of breast cancer and outcome. These results have implications for the stratification of breast cancer based on p53 function and may provide an alternate explanation for deregulated p53 signalling in breast cancer.

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.

Novel genes associated with lymph node metastasis in triple negative breast cancer

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis and no targeted treatments. TNBC patients are more likely to develop metastases and relapse than patients with other breast cancer subtypes. We aimed to identify TNBC-specific genes and genes associated with lymph node metastasis, one of the first signs of metastatic spread. A total of 33 TNBCs were used; 17 of which had matched normal adjacent tissues available, and 15 with matched lymph node metastases. Gene expression microarray analysis was used to reveal genes that were differentially expressed between these groups. We identified and validated 66 genes that are significantly altered when comparing tumours to normal adjacent samples. Further, we identified 83 genes that are associated with lymph node metastasis and correlated these with miRNA-expression. Pathway analysis revealed their involvement in DNA repair, recombination and cell death, chromosomal instability and other known cancer-related pathways. Finally, four genes were identified that were specific for TNBC, of which one was associated with overall survival. This study has identified novel genes involved in LN metastases in TNBC and genes that are TNBC specific that may be used as treatment targets or prognostic indicators in the future.

Low prevalence of germline PALB2 mutations in Australian triple‐negative breast cancer

Triple‐negative breast cancer (TNBC) is a tumour classification that is defined by oestrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 receptor negativity. TNBCs share a similar gene expression profile to BRCA‐mutated tumours, have been shown to carry a high proportion of BRCA mutations and have a more adverse prognosis compared to other types of breast tumours. PALB2 has been shown to be a moderate‐penetrance breast cancer susceptibility gene and is involved in the same DNA damage repair pathway as BRCA1 and BRCA2; this raises the possibility that germline PALB2 mutations may be involved in the pathogenesis of TNBCs. In our study, we sequenced the coding regions of PALB2 (including intron/exon boundaries) in genomic DNA from 347 patients diagnosed with TNBC to determine the prevalence of deleterious mutations in this population. Two novel truncating mutations (c.758dup and c.2390del) and one previously detected truncating mutation (c.3113+5G>C) were found. In addition, five variants predicted to be protein‐affecting were also identified. Our study shows that the prevalence of PALB2 germline mutations in individuals with TNBC is ∼1%, similar to the prevalence of PALB2 germline mutation of 1% in familial non‐BRCA1/2 breast cancer cohorts.

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.

DNA methylation profile of triple negative breast cancer-specific genes comparing lymph node positive patients to lymph node negative patients

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with no targeted treatment available. Our previous study identified 38 TNBC-specific genes with altered expression comparing tumour to normal samples. This study aimed to establish whether DNA methylation contributed to these expression changes in the same cohort as well as disease progression from primary breast tumour to lymph node metastasis associated with changes in the epigenome. We obtained DNA from 23 primary TNBC samples, 12 matched lymph node metastases, and 11 matched normal adjacent tissues and assayed for differential methylation profiles using Illumina HumanMethylation450 BeadChips. The results were validated in an independent cohort of 70 primary TNBC samples. The expression of 16/38 TNBC-specific genes was associated with alteration in DNA methylation. Novel methylation changes between primary tumours and lymph node metastases, as well as those associated with survival were identified. Altered methylation of 18 genes associated with lymph node metastasis were identified and validated. This study reveals the important role DNA methylation plays in altered gene expression of TNBC-specific genes and lymph node metastases. The novel insights into progression of TNBC to secondary disease may provide potential prognostic indicators for this hard-to-treat breast cancer subtype.

The presence of the intron 3 16 bp duplication polymorphism of p53 (rs17878362) in breast cancer is associated with a low Δ40p53:p53 ratio and better outcome

Breast cancer is the most common female cancer, but it has relatively low rates of p53 mutations, suggesting other mechanisms are responsible for p53 inactivation. We have shown that the p53 isoform, Δ40p53, is highly expressed in breast cancer, where it may contribute to p53 inactivation. Δ40p53 can be produced by alternative splicing of p53 in intron 2 and this is regulated by the formation of G-quadruplex structures in p53 intron 3, from which the nucleotides forming these structures overlap with a common polymorphism, rs17878362. rs17878362 alters p53 splicing to decrease fully spliced p53 messenger RNA (mRNA) in vitro following ionizing radiation and this in turn alters Δ40p53:p53. Hence, the presence of rs17878362 may be important in regulating Δ40p53:p53 in breast cancer. This study aimed to determine if rs17878362 was associated with altered Δ40p53 and p53 expression and outcome in breast cancer. We sequenced p53 in breast tumours from 139 patients and compared this with Δ40p53 and p53 mRNA expression. We found that the ratio of Δ40p53:p53 was significantly lower in tumours homozygous for the polymorphic A2 allele compared with those who were wild-type (A1/A1). Furthermore, there was a lower proportion of breast cancers carrying the A2 allele from patients who subsequently developed metastasis compared with those that did not. Finally, we show that patients whose tumours carried the polymorphic A2 allele had significantly better disease-free survival. These results show that rs17878362 is associated with a low Δ40p53:p53 ratio in breast cancer and that this is associated with better outcome.

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

When is a mutation not a mutation: the case of the c.594-2A>C splice variant in a woman harbouring another BRCA1 mutation in trans.

Since the identification of BRCA1 there has only ever been described two bi-allelic mutation carriers, one of whom was subsequently shown to be a mono-allelic carrier. The second patient diagnosed with two BRCA1 mutations appears to be accurate but there remain some questions about the missense variant identified in that patient.In this report we have identified a woman who is a bi-allelic mutation carrier of BRCA1 and provide an explanation as to why this patient has a phenotype very similar to that of any mono-allelic mutation carrier. The splice variant identified in this patient appears to be associated with the up-regulation of a BRCA1 splice variant that rescues the lethality of being a double mutant. The consequences of the findings of this report may have implications for mutation interpretation and that could serve as a model for not only BRCA1 but also for other autosomal dominant disorders that are considered as being embryonically lethal.