Early-onset Breast Cancer Cases Research Articles

Abstract 6141: Genetic risk, health-associated lifestyle, and risk of early-onset total cancer and breast cancer: A prospective cohort study

Abstract Background Early-onset cancer (diagnosed under 50 years of age) is generally more aggressive. Its rising incidence is a global concern. The associations between genetic risk, lifestyle, and the risk of early-onset cancer may inform preventive strategies. Methods We studied the association between genetic risk, lifestyle and early-onset cancers among 66308 white British participants enrolled in the UK Biobank before age 50. Genetic risk was assessed using sex-specific composite total cancer polygenic risk scores (PRSs). We calculated composite PRSs as weighted sums of previously-developed cancer site-specific PRSs using weights obtained from lasso regression. We considered a breast cancer-specific PRS for analyses on breast cancer among women. A health-associated lifestyle score (HLS) was calculated based on baseline smoking, BMI (men only), physical activity, alcohol intake, and diet. Cox models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) of early-onset total cancer and breast cancer across genetic risk and HLS groups in women and men. Results A total of 1247 incident invasive early-onset cancer cases (820 in women, 427 in men) were documented, including 386 cases of early-onset breast cancer. In multivariable-adjusted analyses with 2-year latency, higher genetic risk (highest vs. lowest tertile of PRS) was associated with significantly increased risks of early-onset total cancer in women (composite PRS HR=1.85; 95% CI, 1.50-2.29), early onset total cancer in men (composite PRS HR=1.94; 95% CI, 1.45-2.59), and early-onset breast cancer in women (breast-cancer PRS HR=3.06; 95% CI, 2.20-4.25). The HRs (95% CI) of early-onset total cancer in women and men, and early-onset breast cancer in women associated with adopting an unfavorable lifestyle (highest vs. lowest tertile of HLS) were 1.49 (0.99, 2.25), 1.14 (0.67, 1.95), and 1.78 (0.97, 3.24), respectively, in the total study population; 1.85 (1.02, 3.36), 3.27 (0.78, 13.72), and 1.67 (0.71, 3.90), respectively, in those with high genetic risk; 1.25 (0.60, 2.57), 1.11 (0.42, 2.89), and 1.66 (0.54, 5.11), respectively, in those with intermediate genetic risk; and 1.15 (0.44, 2.98), 1.16 (0.39, 3.40), and 2.10 (0.57, 7.75), respectively, in those with low genetic risk. Compared to individuals with both low genetic risk and a favorable lifestyle, those with high genetic risk and an unfavorable lifestyle had substantially increased risks of early-onset total cancer in women (3.07; 1.64-5.78) and men (2.18; 0.78-6.11), and significantly higher early-onset breast cancer risk in women (4.11; 1.56-10.85). Conclusion Genetic and lifestyle factors were independently associated with risks of early-onset total cancer and breast cancer. Compared to those with low genetic risk, individuals with a high genetic risk may benefit more from adopting a healthy lifestyle in preventing early-onset cancer. Citation Format: Yin Zhang, Yuxi Liu, Kathryn L. Penney, Sara Lindström, Peter Kraft. Genetic risk, health-associated lifestyle, and risk of early-onset total cancer and breast cancer: A prospective cohort study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6141.

BRCA mutations: screening for germ-line founder mutations among early-onset Syrian breast cancer patients

BackgroundBreast cancer (BC) is the most common female cancers in many countries including Syria. Familial breast cancer or previous family cancer history are considered significant risk factors. Therefore, detecting the prevalence and founder mutations in the population facilitates genetic counselling, risk assessment and the development of a cost-effective screening strategy. In this study, we investigated the three germ-line founder mutations in the BRCA1/2 genes: [NM_007294.4 (BRCA1):c.68_69del (p.Glu23fs), NM_007294.4 (BRCA1):c.5266dup (p.Gln1756fs) and NM_000059.4 (BRCA2):c.5946del (p.Ser1982fs)], to examine their incidence and frequency in early-onset breast cancer cases and determine if they are connected to familial breast cancer. One hundred early diagnosed BC females (≤ 40 years old) with no other type of cancer were recruited. Genomic DNA was isolated from peripheral blood samples, and mutations were investigated using the Amplification-Created Restriction Site (ACRS) method.ResultsThe family history of cancer was observed in 61% of the cases, of which 35% were breast cancer; however, none of the screened mutations were detected among BC patients.ConclusionsThe investigated germ-line mutations were not common among Syrian female patients with early-onset BC and were not associated with familial BC. Other mutations in the BRCA1/2 genes or other genes may have a contributing role. Future studies and the need to launch nationwide mutation screening tests for BRCA 1/BRCA2 in the Syrian population are recommended.

Association of Genetic Markers with the Risk of Early-Onset Breast Cancer in Kazakh Women.

Breast cancer is a global health problem. It is an age-dependent disease, but cases of early-onset breast cancer (eBC) are gradually increasing. There are many unresolved questions regarding eBC risk factors, mechanisms of development and screening. Only 10% of eBC cases are due to mutations in the BRCA1/BRCA2 genes, and 90% have a more complex genetic background. This poses a significant challenge to timely cancer detection in young women and highlights the need for research and awareness. Therefore, identifying genetic risk factors for eBC is essential to solving these problems. This study represents an association analysis of 144 eBC cases and 163 control participants to identify genetic markers associated with eBC risks in Kazakh women. We performed a two-stage approach in association analysis to assess genetic predisposition to eBC. First-stage genome-wide association analysis revealed two risk intronic loci in the CHI3L2 gene (p = 5.2 × 10-6) and MGAT5 gene (p = 8.4 × 10-6). Second-stage exonic polymorphisms haplotype analysis showed significant risks for seven haplotypes (p < 9.4 × 10-4). These results point to the importance of studying medium- and low-penetrant genetic markers in their haplotype combinations for a detailed understanding of the role of detected genetic markers in eBC development and prediction.

Abstract 5772: Differences in clinical actionability by comparing mutational landscape of young and old breast cancer patients

Abstract Background: Early-onset breast cancer (BC) is associated with poor prognosis, which is also characterized by high recurrence risk, distant metastases and advanced stages at the time of diagnosis. While germline mutations in BRCA1/2 are known to underlie a subset of early-onset BC cases, the clinical and genetic characteristics in other young BC patients remain to be explored. Method: We profiled primary lesions from 440 Chinese female BC patients by using a 425-cancer-gene panel. The population was sub-divided into young (≤40 years old) and old (&amp;gt;40 years old) BC patients according to ages at diagnosis. Young and old BC patients were compared for their associations with genomic aberrations, with focus on existing BC targetable alterations, including those in the ERBB2 gene and PI3K and homologous recombination repair (HRR) pathways. Results: 137 (31.1%) patients presented with BC at a young age (median age=35 [18-40]) and 303 (68.9%) with BC diagnosed at an older age (median age=53 [41-82]) in our study cohort. TP53 (young, 61.3%; old, 67%), PIK3CA (33.6% and 41.3%), and GATA3 (9.5% and 8.3%) were among the top frequently altered genes in both subgroups. A total of 70 patients carried pathogenic germline mutations. Of these, 57.3% were alterations in the HRR pathway genes, including BRCA1/2, WRN, BLM, and PALB2, etc, with comparable frequencies in young and old patients. Germline BRCA1/2 mutations were detected in nine (6.6%) and 23 (7.6%) of the young and old patients, respectively. Somatic BRCA1/2 mutational frequencies were also comparable between young (8.8%) and old (9.9%) patients. Overall, somatic alterations in HRR pathway genes were detected in 32.1% young and 40.6% old BC patients. While similar frequencies of ERBB2 amplification (14.6% vs. 18.2%; P= 0.41) were detected in young and old BC patients, older patients carried more ERBB2 mutations (1.5% vs. 7.3%, P= 0.01). Interestingly, the highest rate of ERBB2 amplification was observed in the 20-30 years age group. Alterations in NF1 (10.6% vs. 3.6%, P=0.02) and PKHD1 (6.9% vs. 2.2%, P=0.04) were more frequent in older patients. Alterations in the PI3K pathway genes were more enriched in the older BC patients with overall mutational frequencies of 55.5% and 68.6% (P=0.01) in young and old subgroups, respectively. Finally, we observed an age-dependent increase in tumor mutational burden. Conclusion: BC in young and old patients exhibited different genomic characteristics that could be taken into consideration for treatment recommendations. Citation Format: Jiaqi Liu, Ziqi Jia, Gang Liu, Xiang Wang, Qi Meng, Sha Wang, Yang Shao. Differences in clinical actionability by comparing mutational landscape of young and old breast cancer patients [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 5772.

Identified potential pathogenic variants of BARD1 in 1449 Chinese high-risk breast cancer patients.

e22525 Background: BARD1 (BRCA1-associated ring domain 1), nuclear partner of BRCA1, has been recognized as a breast cancer predisposition gene. Recently, two large-scale population-based case–control studies confirmed that germline loss-of-function mutations in BARD1 were associated with a risk of breast cancer. Since the germline mutation frequency of BARD1 is much less than that of BRCA1/2, the distinct mutation spectrum of BARD1 is still obscure, especially in Chinese breast cancer patients. To verify the utility of BARD1 genetic testing in Chinese population, we assessed the mutational frequency and spectrum of BARD1 in a sequential series of Chinese high-risk breast cancer patients. Methods: A cohort of high-risk breast cancer patients (n = 1449) were collected in Zhejiang Cancer Hospital from 2008 through 2020, including 608 familial breast cancer (BC) cases, 173 bilateral BC cases, 222 triple negative BC cases, 436 early-onset BC cases (≤40 years) and 10 male BC cases. The complete coding sequence and intron–exon boundaries of BARD1 were screened by a 98-gene panel sequencing assay. The SIFT, Polyphen2 and MutationTaster prediction programs were used for analyzing the effect of the variants of unknown significance (VUS) on the BARD1 protein function. Results: A total of three BARD1 pathogenic mutations (c.1348_1349delinsCAT, c.70_71insGT and c.373G &gt; T) were identified, which had been all reported, and accounting for 0.2% (3/1449) in Chinese high-risk breast cancer patients. The frequencies were 0.33% (2/608) and 0.12% (1/841) in familial and non-familial breast cancer patients, respectively. In total, 19 VUSs including 17 missenses, one inframe deletion and one inframe insertion of BARD1 were identified in this study. Based on in silico analysis, nine BARD1 missense variants (c.76A &gt; G, c.1912G &gt; A, c.1693C &gt; T, c.2191C &gt; T, c.127C &gt; A, c.1601C &gt; T, c.443G &gt; A, c.233G &gt; A and c.1972C &gt; T) were classified as potentially damaging. However, the two newly variants c.420_422del and c.68_69insTCCGGGAACGAGCCTCGTTCC still remain VUS, and the other eight variants were classified as benign. Conclusions: Our data presented the germline mutations of BARD1 in a large-scale Chinese high-risk breast cancer population, which accounted for 0.2% in our cohort. Moreover nine potential pathogenic variants were found based on in silico analysis. Clinically, these data may be helpful in genetic counseling of breast cancer patients with BARD1 germline mutation. While the further research on the VUS are needed.

Prevalence of Recurrent Mutations Predisposing to Breast Cancer in Early-Onset Breast Cancer Patients from Poland.

There are twenty recurrent mutations in six breast-cancer-predisposing genes in Poland (BRCA1, BRCA2, CHEK2, PALB2, NBN, and RECQL). The frequencies of the twenty alleles have not been measured in a large series of early-onset breast cancer patients from Poland unselected for family history. We genotyped 2464 women with breast cancer diagnosed below age 41 years for twenty recurrent germline mutations in six genes, including BRCA1, BRCA2 CHEK2, PALB2, NBN, and RECQL. A mutation in one of the six genes was identified in 419 of the 2464 early-onset breast cancer cases (17%), including 22.4% of those cases diagnosed below age 31. The mutation frequency was 18.8% for familial breast cancer cases and 6% for non-familial cases. Among women with breast cancer below age 31, the mutation frequency was 23.6% for familial cases and 17.4% in non-familial cases. The majority of mutations (76.2%) were seen in BRCA1 and BRCA2. In Poland, a panel of twenty recurrent mutations in six genes can identify a genetic basis for a high percentage of early-onset cases and testing is recommended for all women with breast cancer at age 40 or below.

Spectrum and prevalence of BRCA1/2 germline mutations in Pakistani breast cancer patients: results from a large comprehensive study

BackgroundPathogenic germline mutations in BRCA1 and BRCA2 (BRCA1/2) account for the majority of hereditary breast and/or ovarian cancers worldwide. To refine the spectrum of BRCA1/2 mutations and to accurately estimate the prevalence of mutation in the Pakistani population, we studied 539 breast cancer patients selected for family history and age of diagnosis.MethodsComprehensive screening for BRCA1/2 germline mutations was performed using state-of-the-art technologies.ResultsA total of 133 deleterious mutations were identified in 539 families (24.7%), comprising 110 in BRCA1 and 23 in BRCA2. The prevalence of BRCA1/2 small-range mutations and large genomic rearrangements was 55.4% (36/65) for families with breast and ovarian cancer, 27.4% (67/244) for families with two or more cases of breast cancer, 18.5% (5/27) for families with male breast cancer, and 12.3% (25/203) for families with a single case of early-onset breast cancer. Nine mutations were specific to the Pakistani population. Eighteen mutations in BRCA1 and three in BRCA2 were recurrent and accounted for 68.2% (75/110) and 34.8% (8/23) of all identified mutations in BRCA1 and BRCA2, respectively. Most of these mutations were exclusive to a specific ethnic group and may result from founder effects.ConclusionsOur findings show that BRCA1/2 mutations account for one in four cases of hereditary breast/ovarian cancer, one in five cases of male breast cancer, and one in eight cases of early-onset breast cancer in Pakistan. Our study suggests genetic testing of an extended panel of 21 recurrent BRCA1/2 mutations for appropriately selected patients and their families in Pakistan.

Association study between polymorphism of breast cancer susceptibility gene 1 interacting protein C-terminal helicase 1 and early-onset breast cancer in Xinjiang women of Uygur

Objective To investigate the association between single Nucleotide polymorphisms (SNPs) of breast cancer susceptibility gene 1 interacting protein C-terminal helicase 1 (BACH1) gene and susceptibility of early-onset breast cancer in Uygur women in Xinjiang. Methods Detecting and analyzing BACH1 gene exon sites by DNA direct sequencing method and Snapshot genotyping technology, in 80 early-onset breast cancer cases and 240 cases of Uygur women (≤40 years). Results In breast cancer and control groups of Uygur women, the frequencies of genotypes of rs4986764、rs4986765 and c. 587A>G of BACHI gene showed differences between breast cancer and control groups (P=0.001, P=0.011, P=0.049), rs4986764 site of TC (OR=0.086), CC (OR=0.044)and TC+ CCdominant model (OR=0.058) significantly reduced the risk of breast cancer. rs4986765 site of GG (OR=5.617), AG+ GG dominant model (OR=4.254) and c. 587A>G site of GG (OR=7.590), AG+ GG dominant model (OR=7.590) significantly increased the risk of breast cancer. In addition, the SNP-SNP interactions of dominance-genotypes (TC+ CC, AG+ GG) reduced 0.4% risk of breast cancer. Conclusion SNP of rs4986764 of BACHl gene was associated with significantly reduces risk of early-onset breast cancer in Xinjiang women of Uygur, SNP of rs4986765 and c. 587A>G were associated with significantly increases risk of breast cancer in Uygur women. Key words: Breast cancer susceptibility gene 1 interacting protein C-terminal helicase 1 gene; Single nucleotide polymorphism; Uygur; Breast cancer

Is RNASEL:p.Glu265* a modifier of early-onset breast cancer risk for carriers of high-risk mutations?

BackgroundBreast cancer risk for BRCA1 and BRCA2 pathogenic mutation carriers is modified by risk factors that cluster in families, including genetic modifiers of risk. We considered genetic modifiers of risk for carriers of high-risk mutations in other breast cancer susceptibility genes.MethodsIn a family known to carry the high-risk mutation PALB2:c.3113G>A (p.Trp1038*), whole-exome sequencing was performed on germline DNA from four affected women, three of whom were mutation carriers.ResultsRNASEL:p.Glu265* was identified in one of the PALB2 carriers who had two primary invasive breast cancer diagnoses before 50 years. Gene-panel testing of BRCA1, BRCA2, PALB2 and RNASEL in the Australian Breast Cancer Family Registry identified five carriers of RNASEL:p.Glu265* in 591 early onset breast cancer cases. Three of the five women (60%) carrying RNASEL:p.Glu265* also carried a pathogenic mutation in a breast cancer susceptibility gene compared with 30 carriers of pathogenic mutations in the 586 non-carriers of RNASEL:p.Glu265* (5%) (p < 0.002). Taqman genotyping demonstrated that the allele frequency of RNASEL:p.Glu265* was similar in affected and unaffected Australian women, consistent with other populations.ConclusionOur study suggests that RNASEL:p.Glu265* may be a genetic modifier of risk for early-onset breast cancer predisposition in carriers of high-risk mutations. Much larger case-case and case-control studies are warranted to test the association observed in this report.

A low-frequency haplotype spanning SLX4/FANCP constitutes a new risk locus for early-onset breast cancer (

Only a fraction of breast cancer (BC) cases can be yet explained by mutations in genes or genomic variants discovered in linkage, genome-wide association and sequencing studies. The known genes entailing medium or high risk for BC are strongly enriched for a function in DNA double strand repair. Thus, aiming at identifying low frequency variants conferring an intermediate risk, we here investigated 17 variants (MAF: 0.01-0.1) in 10 candidate genes involved in DNA repair or cell cycle control. In an exploration cohort of 437 cases and 1189 controls, we show the variant rs3810813 in the SLX4/FANCP gene to be significantly associated with both BC (≤60 years; OR = 2.6(1.6-3.9), p = 1.6E-05) and decreased DNA repair capacity (≤60 years; beta = 37.8(17.9-57.8), p = 5.3E-4). BC association was confirmed in a verification cohort (N = 2441). Both associations were absent from cases diagnosed >60 years and stronger the earlier the diagnosis. By imputation we show that rs3810813 tags a haplotype with 5 additional variants with the same allele frequency (R2 > 0.9), and a pattern of association very similar for both phenotypes (cases <60 years, p < 0.001, the Bonferroni threshold derived from unlinked variants in the region). In young cases (≤60 years) carrying the risk haplotype, micronucleus test results are predictive for BC (AUC > 0.9). Our findings propose a risk variant with high penetrance on the haplotype spanning SLX4/FANCP to be functionally associated to BC predisposition via decreased repair capacity and suggest this variant is carried by a fraction of these haplotypes that is enriched in early onset BC cases.

Revertant mosaicism for family mutations is not observed in BRCA1/2 phenocopies.

In BRCA1/2 families, early-onset breast cancer (BrCa) cases may be also observed among non-carrier relatives. These women are considered phenocopies and raise difficult counselling issues concerning the selection of the index case and the residual risks estimate in negative family members. Few studies investigated the presence of potential genetic susceptibility factors in phenocopies, mainly focussing on BrCa-associated single-nucleotide polymorphisms. We hypothesized that, as for other Mendelian diseases, a revertant somatic mosaicism, resulting from spontaneous correction of a pathogenic mutation, might occur also in BRCA pedigrees. A putative low-level mosaicism in phenocopies, which has never been investigated, might be the causal factor undetected by standard diagnostic testing. We selected 16 non-carriers BrCa-affected from 15 BRCA1/2 families, and investigated the presence of mosaicism through MALDI-TOF mass spectrometry. The analyses were performed on available tumour samples (7 cases), blood leukocytes, buccal mucosa and urine samples (2 cases) or on blood only (7 cases). In one family (n.8), real-time PCR was also performed to analyse the phenocopy and her healthy parents. On the 16 phenocopies we did not detect the family mutations neither in the tumour, expected to display the highest mutation frequency, nor in the other analysed tissues. In family 8, all the genotyping assays did not detect mosaicism in the phenocopy or her healthy parents, supporting the hypothesis of a de novo occurrence of the BRCA2 mutation identified in the proband. These results suggest that somatic mosaicism is not likely to be a common phenomenon in BRCA1/2 families. As our families fulfilled high-risk selection criteria, other genetic factors might be responsible for most of these cases and have a significant impact on risk assessment in BRCA1/2 families. Finally, we found a de novo BRCA2 mutation, suggesting that, although rare, this event should be taken into account in the evaluation of high-risk families.

Abstract 796: ERCC3 R109X is a moderate risk breast cancer risk variant in Ashkenazi Jews

Abstract Background: Known gene mutations account for approximately 50% of the risk for breast cancer. However, a considerable fraction of heritable risk remains unexplained. Fifteen percent of the risk is accounted for by BRCA1/2 and another 3% by TP53, PTEN, LKB1 and CDH1. CHEK2, ATM, PALB2, BRIP1, RAD51C, RAD51D and BARD1 account for 4%, while SNPs discovered from large multicenter genome-wide association studies explain another 14% of the heritable risk. Founder mutations in the DNA repair pathway genes such as BRCA1 and BRCA2 account for the majority of AJ breast cancer mutations. Methods: We performed exome sequencing of 49 early onset (age &amp;lt;35) breast cancer cases, and 85 BRCA wild type familial breast cancer cases, all of Ashkenazi Jewish (AJ) from the New York City area. A recurrent truncating mutation was then analyzed in 3131 breast cancer cases and 2716 unaffected women of Ashkenazi ancestry from New York and Israel. Using CRISPR and overexpression systems on the human mammary epithelial cell line HMLE, transcript and protein levels were assayed for the mutant and compared to wild-type. Treatment with IlludinS and UVC were performed to assess DNA damage response. Finally, clonogenic survival assay was also performed. Results: Amongst the DNA repair pathway genes, exome sequencing revealed a heterozygous recurrent truncating mutation in ERCC3 (R109X) in 2 of 49 early onset breast cancer cases of AJ ancestry and 4 familial AJ probands. Taqman genotyping in a case control setting from New York and Israel revealed 54 mutation carriers in 3131 cases and 32 in the 2716 controls. In total, there were 60 heterozygotes detected in 3209 cases and 32 in 2716 controls [OR 1.59 (95% CI 1.01-2.50)]; p = 0.02 Fisher one- tailed). Functional studies using CRISPR and overexpression systems on human mammary epithelial cells, show that the mutation results in lower transcript levels and this reduction is effected by nonsense mediated decay of the mutant transcript. Western blotting showed that the mutation resulted in a smaller protein (∼12kDa). Clonogenic assays showed similar survival rate of mutant and wildtype under UVC exposure, however the mutant cell line showed significantly smaller colony size demonstrating a growth disadvantage that was further increased upon DNA damage. Treatment with fungal sesquiterpene IlludinS, a known sensitizer to mutant ERCC3 cell lines, showed drastically reduced survival when compared to the wild type human mammary epithelial cells. Conclusions: We demonstrate that ERCC3 is a moderate risk gene for breast cancer in individuals of Ashkenazi ancestry. ERCC3 is somatically mutated in multiple cancers including breast, ovarian and pancreatic cancers, however its role as a cancer susceptibility gene requires further elucidation. Additional functional and population genetic studies to further characterize this novel ERCC3 variant are underway. Citation Format: Joseph Vijai, Sabine Topka, Kara Maxwell, Vignesh Ravichandran, Tinu Thomas, Danylo Villano, Ann Maria, Pragna Gaddam, Anne Lincoln, Steven Hart, Susan Neuhausen, Mark Robson, Jeffrey Weitzel, Mark Daly, Katherine Nathanson, Fergus Couch, Gadi Rennert, Kenneth Offit. ERCC3 R109X is a moderate risk breast cancer risk variant in Ashkenazi Jews. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 796.

Multigene testing of moderate-risk genes: be mindful of the missense

BackgroundModerate-risk genes have not been extensively studied, and missense substitutions in them are generally returned to patients as variants of uncertain significance lacking clearly defined risk estimates. The fraction of...

Abstract 3973: Comparison of different highly sensitive miRNA array platforms for the investigation of circulating cell-free microRNAs in blood

Abstract Introduction: Circulating free (cf) microRNAs hold great promises in early detection and prognosis of human diseases such as cancer. However due to the very low amount of cf miRNA in plasma and serum, highly informative miRNAs are lost. Thus, there is an urgent need for highly efficient, sensitive, cost efficient technologies to measure low abundant miRNA in a quantitative manner. Previously, we have successfully measured and validated miRNA profiles of early onset breast cancer cases, healthy controls and breast cancer patients who had developed breast cancer metastasis during follow-up by applying TaqMan Low density arrays. We established and validated miRNA signatures for the early detection of early stage breast cancer, and prognosis and early detection of breast cancer metastasis. However more sensitive microarray platforms with highly dynamic ranges could even improve identified diagnostic signatures especially in respect of early detection of diseases and relapse. Here, we compared the sensitivity of the previously applied array platform TaqMan Low density array (TLDA) with Toray innovative 3D-Gene microarray technology by measuring plasma cf miRNA profiles of 40 individuals (10 healthy controls, 10 early stage breast cancer, 20 metastatic breast cancer patients). Experimental Procedures: Plasma samples were processed within 2 h after blood collection. EDTA blood was centrifuged for 15 min at 1200 g and the supernatant centrifuged again at 12.000 g for 10 min in order to remove any cell debris. Cf RNA was isolated from plasma. Cf miRNA profiles of plasma samples of 40 individuals (10 healthy controls, 10 early stage breast cancer, 20 metastatic breast cancer patients) were investigated A: via TaqMan low density arrays (TLDA) and B: via 3D-Gene microarray technology. Expression profiles between both array types and between cases, early stage breast cancer and metastatic breast cancer patients were compared. In addition selected miRNAs were measured with single TaqMan assays. These results were compared with the array based results. Conclusion: The result will be of importance for all scientists who intend to develop and improve diagnostic miRNA panels. Based on our results recommendations for a microarray platform for the investigation of cf miRNA will be given. Citation Format: Melanie Maierthaler, Eric Mennesson, Katarina Cuk, Dharanija Madhavan, Manuela Zucknick, Isabelle Fixe, Alexandra Foucher, Markus Wallwiener, Andreas Schneeweiss, Nadia Normand, Barbara Burwinkel. Comparison of different highly sensitive miRNA array platforms for the investigation of circulating cell-free microRNAs in blood. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3973. doi:10.1158/1538-7445.AM2015-3973

Recurrent CYP2C19 deletion allele is associated with triple-negative breast cancer.

BackgroundUsing a genome-wide approach, we have previously observed an increase in the frequency of rare copy number variants (CNVs) in familial and early-onset breast cancer cases when compared to controls. Moreover, the biological networks of the CNV disrupted genes differed between the two groups. Here, six of the previously observed CNVs were selected for further investigation. Four of these were singletons and disturbed the following genes: DCLRE1C, CASP3, DAB2IP and ITGA9, encoding proteins that are part of the TP53 and β-estradiol centered network. The two others were recurrent alleles and disrupted CDH19 and CYP2C19 genes. Of these, CDH19 encodes a cadherin functioning as a cell-cell adhesion receptor and CYP2C19 a CYP450 enzyme with a major function in estrogen catabolism.MethodsThe exact breakpoints of the six previously observed CNV deletion alleles were defined by using qPCR, nested PCR and sequencing. The prevalence of these CNVs was investigated in 842 Northern Finnish breast cancer cases, unselected for family history of cancer and age at disease onset, as well as in 497 healthy female controls by using multiplex PCR. Also the association of the relatively common CDH19 and CYP2C19 deletion alleles with different clinical parameters was studied.ResultsNo significant differences in the carrier frequencies between cases and controls were found for any of the studied CNVs. However, the deletion in CYP2C19 showed a significant association with triple-negative breast cancer (p = 0.021).ConclusionOur results indicate that inherited changes in CYP2C19 gene participating in estrogen catabolism have an influence on the molecular subtype of breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-902) contains supplementary material, which is available to authorized users.

Abstract 43: Distribution and frequency of BRCA gene mutations in Turkish population including Balkan Turks

Abstract Background: The distribution of hereditary breast and ovarian cancer in Turkish population has been reported in earlier publications. However, the regional distributions of breast cancer and/or BRCA1/2 gene mutations frequency are not known for Balkan Turks who came to Turkey for the forced migration in 1989 and for general Turkish populations in Turkey. Materials and Method: We recruited 401 patients with a family history of breast and/or ovarian cancers, and 126 early-onset breast cancer cases below age 40 with/without a family history of cancer between 1994-2013. These patients were referred to Cancer Genetics Department of Oncology Institute in University of Istanbul. Families were selected from consecutive referrals to the above-listed hospital meeting specific family-history criteria (either multiple cases of breast and or ovarian cancer in the family or the presence of an individual with a young age at diagnosis). Mean age of patients in the study was 41, 18±10, 24. Results: The distributions of patients participating in the study were evaluated in total 8 groups such as seven geographic regions of Turkey and the Balkan Turks. Overall frequency of BRCA1/2 gene mutations in Turkish population was 10%. The distribution of patients according to 8 groups were 26.6% in the Black Sea Region, 21.5% in the Marmara Region, 17.6% in the Eastern Anatolia Region, 8.6% in Central Anatolia, 3.1% in Aegean Region, 3.9% in the Mediterranean region, 2.7% in South Eastern Anatolia and 16% in Balkan Turks. BRCA1 and BRCA2 positivity was evaluated in the study groups: BRCA1/2 mutations ratio were 20% in the Black Sea Region, 16% in the Marmara Region, 20% in the Eastern Anatolia Region, 12% in Central Anatolia, % 4 in Aegean Region and 28% in the Balkan Turks. None of mutations in both genes were found in the patients participating in the study from South Eastern Anatolia and the Mediterranean regions. The observed BRCA1/2 mutations rate was found almost two-fold according to the expected mutation rate in the group of Balkan Turks. The highest breast cancer rates were seen in the city of Giresun in the Black Sea Region and in the city of Malatya in the Eastern Anatolia Region, respectively. Conclusions: In our study, breast cancer was more frequent in the regions which were the Black Sea, the Marmara, Eastern Anatolia and the Balkan Turks group. Although the Marmara region was industrialized, Bulgaria where Balkan Turks lived before forced-migration and the Black Sea Region are not industrialized areas. However, in 1986, these regions were the most affected by the Chernobyl disaster and these interactions was described with scientific data. Therefore, it might be concluded that the high rates of breast cancer are associated with the Chernobyl disaster in these regions. In the study, BRCA1/2 gene mutation rates were examined in Balkan Turks who came to Turkey by forced-migration after Chernobyl disaster. In the group of Balkan Turks, almost two fold expected incidence for BRCA1/2 gene mutations was observed. The investigated society was a small community living as a minority in Bulgaria. Therefore, it was suggested that the society was isolated as a nature and has been had high BRCA1/2 mutations rate. On the other hand, in the study, BRCA1/2 gene mutations and clinical/pathological data were also compared and survival analysis was performed in the general Turkish population. The diagnosis age for breast cancer was found 10 years earlier when compared to the other populations (p&amp;lt;0,005). BRCA1/2 mutations were not shown in the patients below age of 30. High frequency of the BRCA1 positivity was observed in triple negative patients (P &amp;lt;0.05). Citation Format: Demet Akdeniz, Bugra S. Tuncer, Seda Kilic, Ozge Sukruoglu, Leyla Kilic, Kevser Altundag, Rumeysa Ciftci, Sevda Ozel, Senem Karabulut, Meltem Ekenel, Pinar Saip, Hasan Karanlik, Neslihan Cabioglu, Hulya Yazici. Distribution and frequency of BRCA gene mutations in Turkish population including Balkan Turks. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 43. doi:10.1158/1538-7445.CANSUSC14-43

The changing landscape of genetic testing for hereditary breast and ovarian cancer

It took Angelina Jolie’s announcement in 2013 to bring broad awareness of hereditary breast and ovarian cancer (HBOC) to the general public. However, it has been 25 years since MaryClaire King mapped BRCA1 to chromosome 17 through linkage analysis involving painstaking collection of a large number of families with multiple cases of early-onset breast cancer. Though familial “clusters” of breast cancer had been described since the early 1800s, the concept that cancer can result from inheritance of a single faulty gene first made its way into the scientific literature when Knudson published his famous “2-hit hypothesis” in 1971. Lynch et al were one of the first to describe the association of breast with ovarian cancer in a 1978 article entitled “Familial Association of Breast and Ovarian.” However, it was the pioneering work of Gardner and Stephens initiated in the mid-1940s identifying and following large Mormon kindreds in Utah that ultimately drove the search for the “breast cancer gene.” Despite the work of Knudson, Lynch, Gardner, and others, the research and clinical communities remained skeptical well into the early 1980s that breast cancer could be hereditary, as opposed to primarily the result of environmental and lifestyle factors. The race to isolate the BRCA1 gene was ultimately won by Mark Skolnick, founder of Myriad Genetic Laboratories, Inc, in 1994, followed closely by the cloning of BRCA2 in 1995. At that time, despite the evidence of increased incidence of ovarian cancer in BRCA1 and BRCA2 families, many still believed there was an ovarian cancer–only gene that was responsible for pedigrees segregating multiple ovarian cancer cases, but no breast cancer. However, further study confirmed that there was not an “OVCA1” gene, and that BRCA1 and BRCA2 accounted for most familial site-specific ovarian cancer. In addition, the failure to find a BRCA1 and BRCA2 mutation in many apparently classic HBOC families led to the search for “BRCA3.” Ultimately it was determined that there likely did not exist another single major hereditary cancer gene responsible for most BRCA1and BRCA2-negative families.

Rare mutations in RINT1 predispose carriers to breast and Lynch syndrome-spectrum cancers.

Approximately half of the familial aggregation of breast cancer remains unexplained. A multiple-case breast cancer family exome-sequencing study identified three likely pathogenic mutations in RINT1 (NM_021930.4) not present in public sequencing databases: RINT1 c.343C>T (p.Q115X), c.1132_1134del (p.M378del), and c.1207G>T (p.D403Y). On the basis of this finding, a population-based case-control mutation-screening study was conducted that identified 29 carriers of rare (minor allele frequency < 0.5%), likely pathogenic variants: 23 in 1,313 early-onset breast cancer cases and six in 1,123 frequency-matched controls [OR, 3.24; 95% confidence interval (CI), 1.29-8.17; P = 0.013]. RINT1 mutation screening of probands from 798 multiple-case breast cancer families identified four additional carriers of rare genetic variants. Analysis of the incidence of first primary cancers in families of women carrying RINT1 mutations estimated that carriers were at increased risk of Lynch syndrome-spectrum cancers [standardized incidence ratio (SIR), 3.35; 95% CI, 1.7-6.0; P = 0.005], particularly for relatives diagnosed with cancer under the age of 60 years (SIR, 10.9; 95% CI, 4.7-21; P = 0.0003). The work described in this study adds RINT1 to the growing list of genes in which rare sequence variants are associated with intermediate levels of breast cancer risk. Given that RINT1 is also associated with a spectrum of cancers with mismatch repair defects, these findings have clinical applications and raise interesting biological questions.

BRCA1 gene exon 11 mutations in Uighur and Han women with early-onset sporadic breast cancer in the northwest region of China.

The prevalence of BRCA1 gene mutations in breast cancer differs between diverse ethnic groups. Relatively little information is known about patterns of BRCA1 mutations in early-onset breast cancer in women of Uighur or Han descent, the major ethnic populations of the Xinjiang region in China. The aim of this study was to identify BRCA1 mutations in Uighur and Han patients with early-onset (age <35 years), and sporadic breast cancer for genetic predisposition to breast cancer. For detection of BRCA1 mutations, we used a polymerase chain reaction single-stranded conformation polymorphism approach, followed by direct DNA sequencing in 22 Uighur and 13 Han women with early-onset sporadic breast cancer, and 32 women with benign breast diseases. The prevalence of BRCA1 mutations in this population was 22.9% (8/35) among early-onset sporadic breast cancer cases. Of these, 31.8% (7/22) of Uighur patients and 7.69% (1/13) of Han patients were found to have BRCA1 mutations. In 7 Uighur patients with BRCA1 mutations, there were 11 unique sequence alterations in the BRCA1 gene, including 4 clearly disease-associated mutations on exon 11 and 3 variants of uncertain clinical significance on exon 11, meanwhile 4 neutral variants on intron 20 or 2. None of the 11 BRCA1 mutations identified have been previously reported in the Breast Cancer Information Core database. These findings reflect the prevalence of BRCA1 mutations in Uighur women with early-onset and sporadic breast cancer, which will allow for provision of appropriate genetic counseling and treatment for Uighur patients in the Xinjiang region.

Rare key functional domain missense substitutions in MRE11A, RAD50, and NBN contribute to breast cancer susceptibility: results from a Breast Cancer Family Registry case-control mutation-screening study.

IntroductionThe MRE11A-RAD50-Nibrin (MRN) complex plays several critical roles related to repair of DNA double-strand breaks. Inherited mutations in the three components predispose to genetic instability disorders and the MRN genes have been implicated in breast cancer susceptibility, but the underlying data are not entirely convincing. Here, we address two related questions: (1) are some rare MRN variants intermediate-risk breast cancer susceptibility alleles, and if so (2) do the MRN genes follow a BRCA1/BRCA2 pattern wherein most susceptibility alleles are protein-truncating variants, or do they follow an ATM/CHEK2 pattern wherein half or more of the susceptibility alleles are missense substitutions?MethodsUsing high-resolution melt curve analysis followed by Sanger sequencing, we mutation screened the coding exons and proximal splice junction regions of the MRN genes in 1,313 early-onset breast cancer cases and 1,123 population controls. Rare variants in the three genes were pooled using bioinformatics methods similar to those previously applied to ATM, BRCA1, BRCA2, and CHEK2, and then assessed by logistic regression.ResultsRe-analysis of our ATM, BRCA1, and BRCA2 mutation screening data revealed that these genes do not harbor pathogenic alleles (other than modest-risk SNPs) with minor allele frequencies >0.1% in Caucasian Americans, African Americans, or East Asians. Limiting our MRN analyses to variants with allele frequencies of <0.1% and combining protein-truncating variants, likely spliceogenic variants, and key functional domain rare missense substitutions, we found significant evidence that the MRN genes are indeed intermediate-risk breast cancer susceptibility genes (odds ratio (OR) = 2.88, P = 0.0090). Key domain missense substitutions were more frequent than the truncating variants (24 versus 12 observations) and conferred a slightly higher OR (3.07 versus 2.61) with a lower P value (0.029 versus 0.14).ConclusionsThese data establish that MRE11A, RAD50, and NBN are intermediate-risk breast cancer susceptibility genes. Like ATM and CHEK2, their spectrum of pathogenic variants includes a relatively high proportion of missense substitutions. However, the data neither establish whether variants in each of the three genes are best evaluated under the same analysis model nor achieve clinically actionable classification of individual variants observed in this study.