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BackgroundPALB2monoallelic loss-of-functiongerm-line variants confer a breast cancer risk comparable to the average BRCA2pathogenic variant. Recommendations for risk reduction strategies in carriers are similar. Elaborating robust criteria to identify loss-of-functionvariants in PALB2—without incurring overprediction—is thus of paramount clinical relevance. Towards this aim, we have performed a comprehensive characterisation of alternative splicing in PALB2, analysing its relevance for the classification of truncating and splice site variants according to the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines.MethodsAlternative splicing was characterised in RNAs extracted from blood, breast and fimbriae/ovary-related human specimens (n=112). RNAseq, RT-PCR/CE and CloneSeq experiments were performed by five contributing laboratories. Centralised revision/curation was performed to assure high-quality annotations. Additional splicing analyses were performed in PALB2c.212–1G>A, c.1684+1G>A, c.2748+2T>G, c.3113+5G>A, c.3350+1G>A, c.3350+4A>C and c.3350+5G>A carriers. The impact of the findings on PVS1 status was evaluated for truncating and splice site variant.ResultsWe identified 88 naturally occurring alternative splicing events (81 newly described), including 4 in-frame events predicted relevant to evaluate PVS1 status of splice site variants. We did not identify tissue-specific alternate gene transcripts in breast or ovarian-related samples, supporting the clinical relevance of blood-based splicing studies.ConclusionsPVS1 is not necessarily warranted for splice site variants targeting four PALB2acceptor sites (exons 2, 5, 7 and 10). As a result, rare variants at these splice sites cannot be assumed pathogenic/likely pathogenicwithout further evidences. Our study puts a warning in up to five PALB2genetic variants that are currently reported as pathogenic/likely pathogenicin ClinVar.

Purpose:CHEK2*1100delC is a founder variant in European populations that confers a two- to threefold increased risk of breast cancer (BC). Epidemiologic and family studies have suggested that the risk associated with CHEK2*1100delC is modified by other genetic factors in a multiplicative fashion. We have investigated this empirically using data from the Breast Cancer Association Consortium (BCAC).Methods:Using genotype data from 39,139 (624 1100delC carriers) BC patients and 40,063 (224) healthy controls from 32 BCAC studies, we analyzed the combined risk effects of CHEK2*1100delC and 77 common variants in terms of a polygenic risk score (PRS) and pairwise interaction.Results:The PRS conferred odds ratios (OR) of 1.59 (95% CI: 1.21–2.09) per standard deviation for BC for CHEK2*1100delC carriers and 1.58 (1.55–1.62) for noncarriers. No evidence of deviation from the multiplicative model was found. The OR for the highest quintile of the PRS was 2.03 (0.86–4.78) for CHEK2*1100delC carriers, placing them in the high risk category according to UK NICE guidelines. The OR for the lowest quintile was 0.52 (0.16–1.74), indicating a lifetime risk close to the population average.Conclusion:Our results confirm the multiplicative nature of risk effects conferred by CHEK2*1100delC and the common susceptibility variants. Furthermore, the PRS could identify carriers at a high lifetime risk for clinical actions.Genet Med advance online publication 06 October 2016

This study examined the utility of sets of single-nucleotide polymorphisms (SNPs) in familial but non-BRCA-associated breast cancer (BC).

Male breast cancer is poorly understood with a large proportion arising in the familial context particularly with the BRCA2 germline mutation. As phenotypic and genotypic differences between sporadic and familial male breast cancers have been noted, we investigated the importance of a hypoxic drive in these cancers as this pathway has been shown to be of importance in familial female breast cancer. Expression of two major hypoxia-induced proteins, the hypoxia-inducible factor-1α (HIF1A) and the carbonic anhydrase IX (CA9), examined within a large cohort including 61 familial (3 BRCA1, 28 BRCA2, 30 BRCAX) and 225 sporadic male breast cancers showed that 31% of all male breast cancers expressed either HIF1A (25%) and/or CA9 (8%) in the combined cohort. Expression of HIF1A correlated with an increased incidence of a second-major malignancy (P=0.04), histological tumor type (P=0.005) and basal phenotype (P=0.02). Expression of CA9 correlated with age (P=0.004) in sporadic cases and an increased tumor size (P=0.003). Expression of HIF1A was prognostic for disease-specific survival in sporadic male breast cancers (HR: 3.8, 95% CI: 1.5–9.8, P=0.006) but not within familial male breast cancer, whereas CA9 was only prognostic in familial male breast cancers (HR: 358.0, 95% CI: 9.3–13781.7, P=0.002) and not in sporadic male breast cancer. This study found that hypoxic drive is less prevalent in male breast cancer compared with female breast cancer, possibly due to a different breast microenvironment. The prognostic impact of HIF1A is greatest in sporadic male breast cancers with an alternate dominant mechanism for the oncogenic drivers suggested in high risk familial male breast cancers.

Male breast cancer is poorly understood with a large proportion arising in the familial context particularly with the BRCA2 germline mutation. As phenotypic and genotypic differences between sporadic and familial male breast cancers have been noted, we investigated the importance of a hypoxic drive in these cancers as this pathway has been shown to be of importance in familial female breast cancer. Expression of two major hypoxia-induced proteins, the hypoxia-inducible factor-1α(HIF1A) and the carbonic anhydrase IX (CA9), examined within a large cohort including 61 familial (3 BRCA1, 28 BRCA2, 30 BRCAX) and 225 sporadic male breast cancers showed that 31% of all male breast cancers expressed either HIF1A (25%) and/or CA9 (8%) in the combined cohort. Expression of HIF1A correlated with an increased incidence of a second-major malignancy (P=0.04), histological tumor type (P=0.005) and basal phenotype (P=0.02). Expression of CA9 correlated with age (P=0.004) in sporadic cases and an increased tumor size (P=0.003). Expression of HIF1A was prognostic for disease-specific survival in sporadic male breast cancers (HR: 3.8, 95% CI: 1.5–9.8, P=0.006) but not within familial male breast cancer, whereas CA9 was only prognostic in familial male breast cancers (HR: 358.0, 95% CI: 9.3–13781.7, P=0.002) and not in sporadic male breast cancer. This study found that hypoxic drive is less prevalent in male breast cancer compared with female breast cancer, possibly due to a different breast microenvironment. The prognostic impact of HIF1A is greatest in sporadic male breast cancers with an alternate dominant mechanism for the oncogenic drivers suggested in high risk familial male breast cancers.

Many of the DNA sequence variants identified in the breast cancer susceptibility gene BRCA1remain unclassified in terms of their potential pathogenicity. Both multifactorial likelihood analysis and functional approaches have been proposed as a means to elucidate likely clinical significance of such variants, but analysis of the comparative value of these methods for classifying all sequence variants has been limited. We have compared the results from multifactorial likelihood analysis with those from several functional analyses for the four BRCA1 sequence variants A1708E, G1738R, R1699Q, and A1708V. Our results show that multifactorial likelihood analysis, which incorporates sequence conservation, co-inheritance, segregation, and tumour immunohistochemical analysis, may improve classification of variants. For A1708E, previously shown to be functionally compromised, analysis of oestrogen receptor, cytokeratin 5/6, and cytokeratin 14 tumour expression data significantly strengthened the prediction of pathogenicity, giving a posterior probability of pathogenicity of 99%. For G1738R, shown to be functionally defective in this study, immunohistochemistry analysis confirmed previous findings of inconsistent 'BRCA1-like' phenotypes for the two tumours studied, and the posterior probability for this variant was 96%. The posterior probabilities of R1699Q and A1708V were 54% and 69%, respectively, only moderately suggestive of increased risk. Interestingly, results from functional analyses suggest that both of these variants have only partial functional activity. R1699Q was defective in foci formation in response to DNA damage and displayed intermediate transcriptional transactivation activity but showed no evidence for centrosome amplification. In contrast, A1708V displayed an intermediate transcriptional transactivation activity and a normal foci formation response in response to DNA damage but induced centrosome amplification. These data highlight the need for a range of functional studies to be performed in order to identify variants with partially compromised function. The results also raise the possibility that A1708V and R1699Q may be associated with a low or moderate risk of cancer. While data pooling strategies may provide more information for multifactorial analysis to improve the interpretation of the clinical significance of these variants, it is likely that the development of current multifactorial likelihood approaches and the consideration of alternative statistical approaches will be needed to determine whether these individually rare variants do confer a low or moderate risk of breast cancer.

Abstract Introduction : Prospective collection of epidemiological, psychosocial and outcome data in large breast cancer family cohorts should provide less biased data than retrospective studies regarding penetrance of breast cancer and modifiers of genetic risk. Methods: The Kathleen Cuningham Foundation for Research into Breast Cancer (kConFab) recently commenced 3-yearly follow-up on over 750 families with multiple cases of breast cancer. Clinical follow-up was by mailed self-report questionnaire to all participants, while psychosocial follow-up was only of unaffected women and consisted of two components: a mailed questionnaire and an interview regarding stressful life events. Results: To date, 1928 of 2748 (70%) participants returned the clinical follow-up questionnaire (10% opted out, 16% were non-responders, and 4% were not contactable). Of the unaffected females who returned the clinical follow-up questionnaire, 91% participated in the psychosocial follow-up. In multivariate analyses, sex, personal cancer status, marital status, age and educational status were independent predictors of response to the clinical follow-up questionnaire, and number of female children, age, and family history of breast cancer were independent predictors of response to the psychosocial follow-up. Conclusions: A first round of 3-yearly clinical and psychosocial follow-up using a mailed questionnaire was feasible in this cohort. High response rates were achieved by employing intensive tracing and reminder strategies. The predictors of response for the clinical and psychosocial follow-up components of this study should be considered in designing similar follow-up strategies for other family cancer cohorts.