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Early-onset breast cancer may be due to Li-Fraumeni Syndrome (LFS). Current national and international guidelines recommend that TP53 genetic testing should be considered for women with breast cancer diagnosed before the age of 31 years. However, large studies investigating TP53 mutation prevalence in this population are scarce. We collected nationwide laboratory records for all young breast cancer patients tested for TP53 mutations in the Netherlands. Between 2005 and 2016, 370 women diagnosed with breast cancer younger than 30 years of age were tested for TP53 germline mutations, and eight (2.2%) were found to carry a (likely) pathogenic TP53 sequence variant. Among BRCA1/BRCA2 mutation negative women without a family history suggestive of LFS or a personal history of multiple LFS-related tumours, the TP53 mutation frequency was < 1% (2/233). Taking into consideration that TP53 mutation prevalence was comparable or even higher in some studies selecting patients with breast cancer onset at older ages or HER2-positive breast cancers, raises the question of whether a very early age of onset is an appropriate single TP53 genetic testing criterion.

Importance: Limited information about the relationship between specific mutations in BRCA1 or BRCA2 (BRCA1/2) and cancer risk exists., Objective: To identify mutation-specific cancer risks for carriers of BRCA1/2., Design, Setting, and Participants: Observational study of women who were ascertained between 1937 and 2011 (median, 1999) and found to carry disease-associated BRCA1 or BRCA2 mutations. The international sample comprised 19,581 carriers of BRCA1 mutations and 11,900 carriers of BRCA2 mutations from 55 centers in 33 countries on 6 continents. We estimated hazard ratios for breast and ovarian cancer based on mutation type, function, and nucleotide position. We also estimated RHR, the ratio of breast vs ovarian cancer hazard ratios. A value of RHR greater than 1 indicated elevated breast cancer risk; a value of RHR less than 1 indicated elevated ovarian cancer risk., Exposures: Mutations of BRCA1 or BRCA2., Main Outcomes and Measures: Breast and ovarian cancer risks., Results: Among BRCA1 mutation carriers, 9052 women (46%) were diagnosed with breast cancer, 2317 (12%) with ovarian cancer, 1041 (5%) with breast and ovarian cancer, and 7171 (37%) without cancer. Among BRCA2 mutation carriers, 6180 women (52%) were diagnosed with breast cancer, 682 (6%) with ovarian cancer, 272 (2%) with breast and ovarian cancer, and 4766 (40%) without cancer. In BRCA1, we identified 3 breast cancer cluster regions (BCCRs) located at c.179 to c.505 (BCCR1; RHR = 1.46; 95% CI, 1.22-1.74; P = 2 × 10(-6)), c.4328 to c.4945 (BCCR2; RHR = 1.34; 95% CI, 1.01-1.78; P = .04), and c. 5261 to c.5563 (BCCR2', RHR = 1.38; 95% CI, 1.22-1.55; P = 6 × 10(-9)). We also identified an ovarian cancer cluster region (OCCR) from c.1380 to c.4062 (approximately exon 11) with RHR = 0.62 (95% CI, 0.56-0.70; P = 9 × 10(-17)). In BRCA2, we observed multiple BCCRs spanning c.1 to c.596 (BCCR1; RHR = 1.71; 95% CI, 1.06-2.78; P = .03), c.772 to c.1806 (BCCR1'; RHR = 1.63; 95% CI, 1.10-2.40; P = .01), and c.7394 to c.8904 (BCCR2; RHR = 2.31; 95% CI, 1.69-3.16; P = .00002). We also identified 3 OCCRs: the first (OCCR1) spanned c.3249 to c.5681 that was adjacent to c.5946delT (6174delT; RHR = 0.51; 95% CI, 0.44-0.60; P = 6 × 10(-17)). The second OCCR spanned c.6645 to c.7471 (OCCR2; RHR = 0.57; 95% CI, 0.41-0.80; P = .001). Mutations conferring nonsense-mediated decay were associated with differential breast or ovarian cancer risks and an earlier age of breast cancer diagnosis for both BRCA1 and BRCA2 mutation carriers., Conclusions and Relevance: Breast and ovarian cancer risks varied by type and location of BRCA1/2 mutations. With appropriate validation, these data may have implications for risk assessment and cancer prevention decision making for carriers of BRCA1 and BRCA2 mutations.

BRCA1-associated breast and ovarian cancer risks can be modified by common genetic variants. To identify further cancer risk-modifying loci, we performed a multi-stage GWAS of 11,705 BRCA1 carriers (of whom 5,920 were diagnosed with breast and 1,839 were diagnosed with ovarian cancer), with a further replication in an additional sample of 2,646 BRCA1 carriers. We identified a novel breast cancer risk modifier locus at 1q32 for BRCA1 carriers (rs2290854, P = 2.7 × 10(-8), HR = 1.14, 95% CI: 1.09-1.20). In addition, we identified two novel ovarian cancer risk modifier loci: 17q21.31 (rs17631303, P = 1.4 × 10(-8), HR = 1.27, 95% CI: 1.17-1.38) and 4q32.3 (rs4691139, P = 3.4 × 10(-8), HR = 1.20, 95% CI: 1.17-1.38). The 4q32.3 locus was not associated with ovarian cancer risk in the general population or BRCA2 carriers, suggesting a BRCA1-specific association. The 17q21.31 locus was also associated with ovarian cancer risk in 8,211 BRCA2 carriers (P = 2×10(-4)). These loci may lead to an improved understanding of the etiology of breast and ovarian tumors in BRCA1 carriers. Based on the joint distribution of the known BRCA1 breast cancer risk-modifying loci, we estimated that the breast cancer lifetime risks for the 5% of BRCA1 carriers at lowest risk are 28%-50% compared to 81%-100% for the 5% at highest risk. Similarly, based on the known ovarian cancer risk-modifying loci, the 5% of BRCA1 carriers at lowest risk have an estimated lifetime risk of developing ovarian cancer of 28% or lower, whereas the 5% at highest risk will have a risk of 63% or higher. Such differences in risk may have important implications for risk prediction and clinical management for BRCA1 carriers., Competing Interests: The authors have declared that no competing interests exist.

Background: Mutations in the CHEK2 gene confer a moderately increased breast cancer risk. The risk for female carriers of the CHEK2*1100delC mutation is twofold increased. Breast cancer risk for carrier women is higher in a familial breast cancer setting which is due to coinheritance of additional genetic risk factors. This study investigated the occurrence of homozygosity for the CHEK2*1100delC allele among familial breast cancer cases and the associated breast cancer risk., Methods and Results: Homozygosity for the CHEK2*1100delC allele was identified in 8/2554 Dutch independent familial non-BRCA1/2 breast cancer cases. The genotype relative risk for breast cancer of homozygous and heterozygous familial breast cancer cases was 101.34 (95% CI 4.47 to 121 000) and 4.04 (95% CI 0.88 to 21.0), respectively. Female homozygotes appeared to have a greater than twofold increased breast cancer risk compared to familial CHEK2*1100delC heterozygotes (p=0.044). These results and the occurrence of multiple primary tumours in 7/10 homozygotes indicate a high cancer risk in homozygous women from non-BRCA1/2 families., Conclusions: Intensive breast surveillance is therefore justified in these homozygous women. It is concluded that diagnostic testing for biallelic mutations in CHEK2 is indicated in non-BRCA1/2 breast cancer families, especially in populations with a relatively high prevalence of deleterious mutations in CHEK2.

Background: Assessment of the clinical significance of unclassified variants (UVs) identified in BRCA1 and BRCA2 is very important for genetic counselling. The analysis of co-segregation of the variant with the disease in families is a powerful tool for the classification of these variants. Statistical methods have been described in literature but these methods are not always easy to apply in a diagnostic setting., Methods: We have developed an easy to use method which calculates the likelihood ratio (LR) of an UV being deleterious, with penetrance as a function of age of onset, thereby avoiding the use of liability classes. The application of this algorithm is publicly available http://www.msbi.nl/cosegregation. It can easily be used in a diagnostic setting since it requires only information on gender, genotype, present age and/or age of onset for breast and/or ovarian cancer., Results: We have used the algorithm to calculate the likelihood ratio in favour of causality for 3 UVs in BRCA1 (p.M18T, p.S1655F and p.R1699Q) and 5 in BRCA2 (p.E462G p.Y2660D, p.R2784Q, p.R3052W and p.R3052Q). Likelihood ratios varied from 0.097 (BRCA2, p.E462G) to 230.69 (BRCA2, p.Y2660D). Typing distantly related individuals with extreme phenotypes (i.e. very early onset cancer or old healthy individuals) are most informative and give the strongest likelihood ratios for or against causality., Conclusion: Although co-segregation analysis on itself is in most cases insufficient to prove pathogenicity of an UV, this method simplifies the use of co-segregation as one of the key features in a multifactorial approach considerably.

Introduction: Unclassified variants (UVs) in the BRCA1/BRCA2 genes are a frequent problem in counseling breast cancer and/or ovarian cancer families. Information about cancer family history is usually available, but has rarely been used to evaluate UVs. The aim of the present study was to identify which is the best combination of clinical parameters that can predict whether a UV is deleterious, to be used for the classification of UVs., Methods: We developed logistic regression models with the best combination of clinical features that distinguished a positive control of BRCA pathogenic variants (115 families) from a negative control population of BRCA variants initially classified as UVs and later considered neutral (38 families)., Results: The models included a combination of BRCAPRO scores, Myriad scores, number of ovarian cancers in the family, the age at diagnosis, and the number of persons with ovarian tumors and/or breast tumors. The areas under the receiver operating characteristic curves were respectively 0.935 and 0.836 for the BRCA1 and BRCA2 models. For each model, the minimum receiver operating characteristic distance (respectively 90% and 78% specificity for BRCA1 and BRCA2) was chosen as the cutoff value to predict which UVs are deleterious from a study population of 12 UVs, present in 59 Dutch families. The p.S1655F, p.R1699W, and p.R1699Q variants in BRCA1 and the p.Y2660D, p.R2784Q, and p.R3052W variants in BRCA2 are classified as deleterious according to our models. The predictions of the p.L246V variant in BRCA1 and of the p.Y42C, p.E462G, p.R2888C, and p.R3052Q variants in BRCA2 are in agreement with published information of them being neutral. The p.R2784W variant in BRCA2 remains uncertain., Conclusions: The present study shows that these developed models are useful to classify UVs in clinical genetic practice.

Introduction: Breast cancer patients with early disease and a natural humoral response to MUC1 have a favourable prognosis, suggesting a possible role of MUC1 antibodies (ab) in controlling haematogenous tumour dissemination and outgrowth. The aim of the study was to evaluate humoral immune responses to MUC1 in women at hereditary high risk of breast cancer to investigate whether this immune response could play a role in the prevention of disease., Materials and Methods: CA15.3 (U/mL), and IgG and IgM ab to MUC1 (arbitrary units per mL, Arb-U/mL) were measured in serum samples obtained from 422 women at hereditary high risk of breast/ovarian cancer, of whom 127 BRCA1/2 carriers, attending the Familial Cancer Clinic of the VU University Medical Centre, and from 370 age-matched healthy controls. Serum samples obtained from women who developed breast cancer (N=12) or breast cancer recurrence (N=17), and from women who underwent prophylactic mastectomy (N=12) and had no breast lesions were also tested., Results: CA15.3 ranked significantly higher in mutation carriers than in controls (P=0.03). MUC1 IgG ab levels ranked significantly lower in BRCA1/2 mutation carriers than in controls (P=0.003). MUC1 IgG levels were not significantly different (P=0.53) between women who developed primary breast cancer (median 0.72Arb-U/ml, range 0.52-2.44Arb-U/ml) and women who underwent prophylactic mastectomy and had no breast lesions (median 1.04Arb-U/ml, range 0.43-2.88Arb-U/ml)., Conclusion: Serum levels of natural IgG ab to MUC1 are lower in BRCA1/2 mutation carriers than in healthy controls. Furthermore, in contrast to previous results in women with sporadic breast cancer, no elevated MUC1 IgG ab were seen in women at hereditary high risk who developed breast cancer. Prophylactic immunotherapy with MUC1 substrates may be a strategy to reduce the risk of breast cancer in BRCA1/2 mutation carriers, strengthening tumour immune surveillance.

To analyse the prevalence of (pre) malignant lesions occurring in breast and adnexal tissue at prophylactic surgery in women at hereditary high risk of breast and/or ovarian cancers. Tissue was obtained from 85 women who underwent prophylactic bilateral salpingo-oophorectomy (pBSO) and from 59 women who underwent prophylactic mastectomy (pM). Control tissue samples were obtained from women undergoing breast reduction surgery (N = 99) or adnexal surgery for benign reasons (N = 72). In women with a BRCA1/2 mutation, the prevalence of a (pre) malignant adnexal lesion was 50% (95% CI 26-74) if older than 40 years and 14% (95% CI 0-58) if younger. The prevalences of (pre) malignant breast lesions in women older than 40 years, with and without a BRCA1/2 mutation, were 0% (95% CI 0-16) and 47% (95% CI 21-73), respectively. No association was found between (pre) malignant lesions in breast and adnexal tissue occurring in 28 women who underwent surgery on both organs (R = 0.155, p = 0.432), but the prevalence of lesions was significantly higher in adnexal tissue than in the breast (p = 0.023). Compared to controls, women at hereditary high risk had a higher chance of (pre) malignant lesions in the breast and an even higher chance of such lesions in the adnexal tissue. There was no indication for concomitant presence of such lesions in both organs at the time of prophylactic surgery. The high frequency of (pre) malignant lesions in the adnexal tissue stresses further the importance of pBSO from the age of 40 onwards in women at hereditary high risk.

Background: About 5% of all breast cancer cases are attributable to germline mutations in BRCA1 or BRCA2 genes. BRCA mutations in suspected carriers, however, may be missed, which hampers genetic counselling., Materials and Methods: Different clinicopathological features were compared between 22 breast cancers from carriers of proved BRCA1 mutations and 604 cancers from sporadic controls. In addition, 5 BRCA2-related breast cancers and 66 breast cancers of untested patients at intermediate risk and 19 breast cancers of untested patients at high risk of hereditary disease on the basis of family history were evaluated., Results: A "probably sporadic" class (age >or=54 years and epidermal growth factor receptor (EGFR) negative; 68% of cases) with a 0% chance of BRCA1-related breast cancer containing 79% of the sporadic cases was yielded by using a decision tree with age, Ki67 and EGFR. A 75% chance of BRCA1-related breast cancer was shown by the "probably BRCA1-related" class (age <54 years and Ki67 >or=25%; 8% of cases) with 82% of the BRCA1-related cases but only 1.4% of the sporadic cases. Most cases at intermediate or high risk of hereditary disease on the basis of family history could be classified with high probability as either probably BRCA1 related or probably sporadic., Conclusion: Breast carcinomas can be classified with a high level of certainty as sporadic or related to BRCA1 germline mutations by using a decision tree with age, Ki67 and EGFR. This can be clinically useful in mutation analysis in families with a borderline risk of hereditary disease.

Background: Although an increased cancer risk in Peutz-Jeghers syndrome is established, data on the spectrum of tumors associated with the disease and the influence of germ-line STK11/LKB1 (serine/threonine kinase) mutation status are limited., Experimental Design: We analyzed the incidence of cancer in 419 individuals with Peutz-Jeghers syndrome, and 297 had documented STK11/LKB1 mutations., Results: Ninety-six cancers were found among individuals with Peutz-Jeghers syndrome. The risk for developing cancer at ages 20, 30, 40, 50, 60, and 70 years was 2%, 5%, 17%, 31%, 60%, and 85%, respectively. The most common cancers represented in this analysis were gastrointestinal in origin, gastroesophageal, small bowel, colorectal, and pancreatic, and the risk for these cancers at ages 30, 40, 50, and 60 years was 1%, 9%, 15%, and 33%, respectively. In women with Peutz-Jeghers syndrome, the risk of breast cancer was substantially increased, being 8% and 31% at ages 40 and 60 years, respectively. Kaplan-Meier analysis showed that cancer risks were similar in Peutz-Jeghers syndrome patients with identified STK11/LKB1 mutations and those with no detectable mutation (log-rank test of difference chi2 = 0.62; 1 df; P = 0.43). Furthermore, the type or site of STK11/LKB1 mutation did not significantly influence cancer risk., Conclusions: The results from our study provide quantitative information on the spectrum of cancers and risks of specific cancer types associated with Peutz-Jeghers syndrome.

Breast cancer is the most common malignancy which affects women. In 5-10% of all cases, breast cancer presents as a hereditary cancer syndrome. Since 1996, 68 families with suspicion of familial breast cancer have been referred to our department. In 5 of the 68 families (7.4%), the clinical diagnosis was hereditary breast ovarian cancer syndrome. In 17 families (25%), two or more breast cancer cases were present. Mutation screening of BRCA1 and BRCA2 in these families revealed a BRCA1 mutation (185delAG) in one family. Three families (4.4%) had a diagnosis of Li-Fraumeni syndrome and germline mutations in TP53 (Lys292Ile, Pro278Ser and Pro278Thr). Breast cancer occurred in a family with hereditary nonpolyposis colon carcinoma (HNPCC; Lynch syndrome) carrying an MLH1 mutation (IVS17-3G>C). Most of our families (41 families; 60.2%) had only one case with breast cancer or cystic adenoma (or both) and did not need counseling and DNA testing. In summary, in 10 of the 68 families in our series (14.7%), a germline mutation in a breast cancer predisposing gene was detected. Our data show the importance of detailed examination of clinical data, pedigree analyses, and molecular germline diagnostics for the counseling of breast cancer cases.

Background & Aims: There is limited data on the spectrum and risk for cancer associated with germline serine/threonine protein kinase 11 (STK11) mutations that cause Peutz-Jeghers syndrome (PJS)., Methods: We analyzed the incidence of cancer in 240 individuals with PJS possessing germline mutations in STK11., Results: Fifty-four cancers were found among carriers. Overall, the risk for developing cancer at ages 20, 30, 40, 50, 60, and 70 years was 1%, 3%, 19%, 32%, 63%, and 81%, respectively. Kaplan-Meier analysis showed similar cancer risks between missense and truncating mutation carriers (log-rank chi(2) = 2.48; P = 0.12). There was some evidence that mutations in exon 3 of STK11 were associated with a higher cancer risk than mutations within other regions of the gene. We found no difference in overall cancer risk between male and female carriers (log-rank chi(2) = 1.31; P = 0.25) or between familial and sporadic cases (log-rank chi(2) = 1.16, with 1 df; P = 0.28). The most common cancers represented were gastrointestinal in origin--gastroesophageal, small bowel, colorectal, and pancreatic--and the risk for these cancers at ages 30, 40, 50, and 60 years was 1%, 10%, 18%, and 42%, respectively. In women, the risk for breast cancer was substantially increased, being 32% by age 60 years., Conclusions: These results quantitatively show the spectrum of cancer risk associated with STK11 germline mutations in the context of PJS and provide a valuable reference for defining surveillance regimens.

We applied a novel method to detect single or multiple exon deletions and amplifications in the BRCA1 gene. The test, called multiplex ligation-dependent probe amplification (MLPA), uses probes designed to hybridize adjacently to the target sequence. After ligation, the joined probes are amplified and quantified. Our two diagnostic laboratories have tested in the recent years 805 families by conventional PCR-based techniques, and found 116 BRCA1 and 28 BRCA2 mutation-positive families. Using MLPA, we have tested the remaining 661 noninformative breast cancer families and identified five distinct BRCA1 germ-line mutations in five families: a deletion of exon 8, a deletion of exons 20-22, a duplication of exon 13 and exons 21-23, respectively, and a triplication, encompassing exons 17-19. Genomic deletions of BRCA1 constitute a substantial fraction of mutations in Dutch breast cancer families. If MLPA had been included in our initial BRCA1 testing, 33 families with a deletion or duplication would have been identified, representing 27% of the total 121 BRCA1 mutation-positive families. The MLPA test for BRCA1 ensures a sensitive and comprehensive high-throughput screening test for genomic rearrangement and can easily be implemented in the molecular analysis of BRCA1.

A 46 year old woman with a family history of breast and ovarian cancer presented with multiple fibroadenomas in both breasts. From three fibroadenomas removed from the left breast carcinoma in situ (CIS) had developed. One fibroadenoma gave rise to ductal CIS, whereas the other two harboured lobular CIS. This is the first report of three fibroadenomas simultaneously giving rise to CIS. In addition, synchronous fibroadenomas harbouring different types of CIS from one fibroadenoma to the other have never been described. Direct sequencing revealed a mutation (5075G-->A) in the BRCA1 gene, but retention of BRCA1 immunohistochemical staining and no loss of heterozygosity at the BRCA1 locus by polymerase chain reaction made a pathogenic mutation in BRCA1 unlikely. Furthermore, in this family no cosegregation of breast cancer with this BRCA1 mutation was seen. Indeed, this mutation is now regarded as a polymorphism. This case stresses the need for histological evaluation of all breast masses in women with a strong positive family history for breast and/or ovarian cancer.

We have identified 79 mutations in BRCA1 in a set of 643 Dutch and 23 Belgian hereditary breast and ovarian cancer families collected either for research or for clinical diagnostic purposes. Twenty-eight distinct mutations have been observed, 18 of them not previously reported and 12 of them occurring more than once. Most conspicuously, a 2804delAA mutation has been found 19 times and has never been reported outside the Netherlands. A common haplotype spanning > or = 375 kb could be identified for each of the nine examined recurrent mutations, indicating the presence of multiple BRCA1 founder mutations in the Dutch population. The 2804delAA mutation has been estimated to have originated approximately 32 generations ago. No specific breast or ovarian cancer phenotype could be assigned to any of the common mutations, and the ovarian cancer incidence among 18 families with the 2804delAA mutation was heterogeneous.