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Considering polygenic risk scores (PRSs) in individual risk prediction is increasingly implemented in genetic testing for hereditary breast cancer (BC) based on next-generation sequencing (NGS). To calculate individual BC risks, the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) with the inclusion of the BCAC 313 or the BRIDGES 306 BC PRS is commonly used. The PRS calculation depends on accurately reproducing the variant allele frequencies (AFs) and, consequently, the distribution of PRS values anticipated by the algorithm. Here, the 324 loci of the BCAC 313 and the BRIDGES 306 BC PRS were examined in population-specific database gnomAD and in real-world data sets of five centers of the German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC), to determine whether these expected AFs can be reproduced by NGS-based genotyping. Four PRS loci were non-existent in gnomAD v3.1.2 non-Finnish Europeans, further 24 loci showed noticeably deviating AFs. In real-world data, between 11 and 23 loci were reported with noticeably deviating AFs, and were shown to have effects on final risk prediction. Deviations depended on the sequencing approach, variant caller and calling mode (forced versus unforced) employed. Therefore, this study demonstrates the necessity to apply quality assurance not only in terms of sequencing coverage but also observed AFs in a sufficiently large cohort, when implementing PRSs in a routine diagnostic setting. Furthermore, future PRS design should be guided by the technical reproducibility of expected AFs across commonly used genotyping methods, especially NGS, in addition to the observed effect sizes., (© 2024. The Author(s).)

Circulating tumor cells (CTCs) are constantly shed by tumor tissue and can serve as a valuable analyte for a gene expression analysis from a liquid biopsy. However, a high proportion of CTCs can be apoptotic leading to rapid mRNA decay and challenging the analysis of their transcriptome. We established a workflow to enrich, to identify, and to isolate single CTCs including the discrimination of apoptotic and non-apoptotic CTCs for further single CTC transcriptome analysis. Viable tumor cells-we first used cells from breast cancer cell lines followed by CTCs from metastatic breast cancer patients-were enriched with the CellSearch system from diagnostic leukapheresis products, identified by immunofluorescence analysis for neoplastic markers, and isolated by micromanipulation. Then, their cDNA was generated, amplified, and sequenced. In order to exclude early apoptotic tumor cells, staining with Annexin V coupled to a fluorescent dye was used. Annexin V staining intensity was associated with decreased RNA integrity as well as lower numbers of total reads, exon reads, and detected genes in cell line cells and CTCs. A comparative RNA analysis of single cells from MDA-MB-231 and MCF7 cell lines revealed the expected differential transcriptome profiles. Enrichment and staining procedures of cell line cells that were spiked into blood had only little effect on the obtained RNA sequencing data compared to processing of naïve cells. Further, the detection of transcripts of housekeeping genes such as GAPDH was associated with a significantly higher quality of expression data from CTCs. This workflow enables the enrichment, detection, and isolation of single CTCs for individual transcriptome analyses. The discrimination of apoptotic and non-apoptotic cells allows to focus on CTCs with a high RNA integrity to ensure a successful transcriptome analysis., (© 2023 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

Introduction: The purpose of this feasibility study was to select targeted therapies according to "ESMO Scale for Clinical Actionability of molecular Targets (ESCAT)". Data interpretation was further supported by a browser-based Treatment Decision Support platform (MH Guide, Molecular Health, Heidelberg, Germany)., Patients: We applied next generation sequencing based whole exome sequencing of tumor tissue and peripheral blood of patients with metastatic breast cancer (n = 44) to detect somatic as well as germline mutations., Results: In 32 metastatic breast cancer patients, data interpretation was feasible. We identified 25 genomic alterations with ESCAT Level of Evidence I or II in 18/32 metastatic breast cancer patients, which were available for evaluation: three copy number gains in HER2 , two g BRCA1 , two g BRCA2 , six PIK3CA, one ESR1 , three PTEN , one AKT1 and two HER2 mutations. In addition, five samples displayed Microsatellite instability high-H., Conclusions: Resulting treatment options were discussed in a tumor board and could be recommended in a small but relevant proportion of patients with metastatic breast cancer (7/18). Thus, this study is a valuable preliminary work for the establishment of a molecular tumor board within the German initiative "Center for Personalized Medicine" which aims to shorten time for analyses and optimize selection of targeted therapies., Competing Interests: Conflict of Interest The authors declare that they have no conflict of interest., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Biallelic germline mutations in BRCA2 occur in the Fanconi anemia (FA)-D1 subtype of the rare pediatric disorder, FA, characterized clinically by severe congenital abnormalities and a very high propensity to develop malignancies early in life. Clinical and genetic data from 96 FA-D1 patients with biallelic BRCA2 mutations were collected and used to develop a new cancer risk prediction score system based on the specific mutations in BRCA2. This score takes into account the location of frameshift/stop and missense mutations relative to exon 11 of BRCA2, which encodes the major sites for interaction with the RAD51 recombinase, and uses the MaxEnt and HBond splicing scores to analyze potential splice site perturbations. Among 75 FA-D1 patients with ascertained BRCA2 mutations, 66 patients developed 102 malignancies, ranging from one to three independent tumors per individual. The median age at the clinical presentation of peripheral embryonal tumors was 1.0, at the onset of hematologic malignancies 1.8 and at the manifestation of CNS tumors 2.7 years, respectively. Patients who received treatment lived longer than those without. Using our novel scoring system, we could distinguish three distinct cancer risk groups among FA-D1 patients: in the first, patients developed their initial malignancy at a median age of 1.3 years (n = 36, 95% CI = 0.9-1.8), in the second group at 2.3 years (n = 17, 95% CI = 1.4-4.4) and in the third group at 23.0 years (n = 22, 95% CI = 4.3-n/a). Therefore, this scoring system allows, for the first time, to predict the cancer manifestation of FA-D1 patients simply based on the type and position of the mutations in BRCA2., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The contribution of germline copy number variants (CNVs) to risk of developing cancer in individuals with pathogenic BRCA1 or BRCA2 variants remains relatively unknown. We conducted the largest genome-wide analysis of CNVs in 15,342 BRCA1 and 10,740 BRCA2 pathogenic variant carriers. We used these results to prioritise a candidate breast cancer risk-modifier gene for laboratory analysis and biological validation. Notably, the HR for deletions in BRCA1 suggested an elevated breast cancer risk estimate (hazard ratio (HR) = 1.21), 95% confidence interval (95% CI = 1.09-1.35) compared with non-CNV pathogenic variants. In contrast, deletions overlapping SULT1A1 suggested a decreased breast cancer risk (HR = 0.73, 95% CI 0.59-0.91) in BRCA1 pathogenic variant carriers. Functional analyses of SULT1A1 showed that reduced mRNA expression in pathogenic BRCA1 variant cells was associated with reduced cellular proliferation and reduced DNA damage after treatment with DNA damaging agents. These data provide evidence that deleterious variants in BRCA1 plus SULT1A1 deletions contribute to variable breast cancer risk in BRCA1 carriers., (© 2022. The Author(s).)

Background: The analysis of liquid biopsies, e.g., circulating tumor cells (CTCs) is an appealing diagnostic concept for targeted therapy selection. In this proof-of-concept study, we aimed to perform multiparametric analyses of CTCs to select targeted therapies for metastatic breast cancer patients., Methods: First, CTCs of five metastatic breast cancer patients were analyzed by whole exome sequencing (WES). Based on the results, one patient was selected and monitored by longitudinal and multiparametric liquid biopsy analyses over more than three years, including WES, RNA profiling, and in vitro drug testing of CTCs., Results: Mutations addressable by targeted therapies were detected in all patients, including mutations that were not detected in biopsies of the primary tumor. For the index patient, the clonal evolution of the tumor cells was retraced and resistance mechanisms were identified. The AKT1 E17K mutation was uncovered as the driver of the metastatic process. Drug testing on the patient's CTCs confirmed the efficacy of drugs targeting the AKT1 pathway. During a targeted therapy chosen based on the CTC characterization and including the mTOR inhibitor everolimus, CTC numbers dropped by 97.3% and the disease remained stable as determined by computer tomography/magnetic resonance imaging., Conclusion: These results illustrate the strength of a multiparametric CTC analysis to choose and validate targeted therapies to optimize cancer treatment in the future. Furthermore, from a scientific point of view, such studies promote the understanding of the biology of CTCs during different treatment regimens.

Pathogenic sequence variants (PSV) in BRCA1 or BRCA2 ( BRCA1/2 ) are associated with increased risk and severity of prostate cancer. We evaluated whether PSVs in BRCA1/2 were associated with risk of overall prostate cancer or high grade (Gleason 8+) prostate cancer using an international sample of 65 BRCA1 and 171 BRCA2 male PSV carriers with prostate cancer, and 3,388 BRCA1 and 2,880 BRCA2 male PSV carriers without prostate cancer. PSVs in the 3' region of BRCA2 (c.7914+) were significantly associated with elevated risk of prostate cancer compared with reference bin c.1001-c.7913 [HR = 1.78; 95% confidence interval (CI), 1.25-2.52; P = 0.001], as well as elevated risk of Gleason 8+ prostate cancer (HR = 3.11; 95% CI, 1.63-5.95; P = 0.001). c.756-c.1000 was also associated with elevated prostate cancer risk (HR = 2.83; 95% CI, 1.71-4.68; P = 0.00004) and elevated risk of Gleason 8+ prostate cancer (HR = 4.95; 95% CI, 2.12-11.54; P = 0.0002). No genotype-phenotype associations were detected for PSVs in BRCA1 . These results demonstrate that specific BRCA2 PSVs may be associated with elevated risk of developing aggressive prostate cancer. SIGNIFICANCE: Aggressive prostate cancer risk in BRCA2 mutation carriers may vary according to the specific BRCA2 mutation inherited by the at-risk individual., (©2019 American Association for Cancer Research.)

Mechanisms of intrinsic resistance of serous ovarian cancers to standard treatment with carboplatin and paclitaxel are poorly understood. Seventeen primary serous ovarian cancers classified as responders or nonresponders to standard treatment were screened with DigiWest protein array analysis for 279 analytes. Histone methyl transferase EZH2, an interaction partner of ataxia telangiectasia mutated (ATM), was found as one of the most significantly represented proteins in responsive tumors. Survival analysis of 616 patients confirmed a better outcome in patients with high EZH2 expression, but a worse outcome in patients with low EZH2 and high-ATM-expressing tumors compared with patients with low EZH2 and low-ATM-expressing tumors. A proximity ligation assay further confirmed an association between ATM and EZH2 in tumors of patients with an increased disease-free survival. Knockdown of EZH2 resulted in treatment-resistant cells, but suppression of both EZH2 and ATM, or ATM alone, had no effect. DigiWest protein analysis of EZH2-knockdown cells revealed a decrease in proteins involved in mitotic processes and checkpoint regulation, suggesting that deregulated ATM may induce treatment resistance. IMPLICATIONS: Ovarian cancer is a malignancy with high mortality rates, with to date, no successful molecular characterization strategies. Our study uncovers in a comprehensive approach the involvement of checkpoint regulation via ATM and EZH2, potentially providing a new therapeutic perspective for further investigations., (©2019 American Association for Cancer Research.)

Mutations in the ligand-binding domain (LBD) of the ESR1 gene result in resistance to estrogen deprivation therapy (EDT) in breast cancer. Their detection might enable optimization of therapy strategies. However, the predictive utility of the primary tumor (PT) is limited, and obtaining serial biopsies of metastatic lesions is challenging. To underline their application as a liquid biopsy, single circulating tumor cells (CTCs) were analyzed with a next-generation sequencing approach for the ESR1 coding region. CTCs from 46 metastatic luminal breast cancer patients were enriched using CellSearch system and isolated by micromanipulation. Their genomic DNA was amplified and the ESR1 gene was sequenced. Furthermore, tissue samples from corresponding PTs and/or metastatic lesions were investigated. ESR1 mutations were detected in 12 patients-exclusively in patients treated with EDT (P = 0.048). In seven cases mutations were located in the hotspot regions in the LBD. Six novel mutations were identified. ESR1 mutations were absent in PT tissue samples and were detected only in metastases obtained after CTC characterization. Single-cell CTC analysis for ESR1 mutations could be of clinical value to identify patients who progress under EDT and therefore benefit from an early switch to an alternative endocrine therapy or other treatment regimens. Furthermore, our data indicate that mutations outside the LBD's hotspot regions might also contribute to resistance to EDT., (Copyright © 2020 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification., (© 2019 Wiley Periodicals, Inc.)

The GILUPI CellCollector (CC) is a novel in vivo circulating tumor cell (CTC) detection device reported to overcome the limitations of small blood sample volumes. The aim of this prospective, blinded study was to evaluate the clinical application of the CC and to compare its performance to the CellSearch (CS) system in M0 and M1 colorectal cancer (CRC) patients. A total of 80 patients (31 M0, 49 M1) with CRC were enrolled. CTCs were simultaneously measured in the peripheral blood using CS and the CC, and the results of both assays were correlated to clinicopathological variables and overall survival. The total number of detected CTCs and CTC-positive patients did not significantly differ between both assays. In the M0 patients, the CC detected CTCs more frequently than CS. There was no significant difference in total CTC numbers detected with the CC between M0 and M1 patients. In addition, no significant correlation with clinicopathological parameters or overall survival was observed with CC CTCs. In contrast, detection of CTCs with CS was significantly correlated with Union for International Cancer Control stage and reduced overall survival. There was no correlation between CTCs detected by the CC and the CS system. Using in silico analysis, we estimate that CC screens a volume of 0.33-18 mL during in vivo application, in contrast to much higher volumes reported elsewhere. In conclusion, while being safe and easy to use, the CC did not outperform CS in terms of CTC yield or sensitivity. While CTC detection in M0 CRC patients was significantly increased with the CC, the clinical relevance of these CTCs appears inferior to the cells identified by the CS system., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Background: The role of the BARD1 gene in breast cancer (BC) and ovarian cancer (OC) predisposition remains elusive, as published case-control investigations have revealed controversial results. We aimed to assess the role of deleterious BARD1 germline variants in BC/OC predisposition in a sample of 4920 BRCA1/2-negative female BC/OC index patients of the German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC)., Methods: A total of 4469 female index patients with BC, 451 index patients with OC, and 2767 geographically matched female control individuals were screened for loss-of-function (LoF) mutations and potentially damaging rare missense variants in BARD1. All patients met the inclusion criteria of the GC-HBOC for germline testing and reported at least one relative with BC or OC. Additional control datasets (Exome Aggregation Consortium, ExAC; Fabulous Ladies Over Seventy, FLOSSIES) were included for the calculation of odds ratios (ORs)., Results: We identified LoF variants in 23 of 4469 BC index patients (0.51%) and in 36 of 37,265 control individuals (0.10%), resulting in an OR of 5.35 (95% confidence interval [CI] = 3.17-9.04; P < 0.00001). BARD1-mutated BC index patients showed a significantly younger mean age at first diagnosis (AAD; 42.3 years, range 24-60 years) compared with the overall study sample (48.6 years, range 17-92 years; P = 0.00347). In the subgroup of BC index patients with an AAD < 40 years, an OR of 12.04 (95% CI = 5.78-25.08; P < 0.00001) was observed. An OR of 7.43 (95% CI = 4.26-12.98; P < 0.00001) was observed when stratified for an AAD < 50 years. LoF variants in BARD1 were not significantly associated with BC in the subgroup of index patients with an AAD ≥ 50 years (OR = 2.29; 95% CI = 0.82-6.45; P = 0.11217). Overall, rare and predicted damaging BARD1 missense variants were significantly more prevalent in BC index patients compared with control individuals (OR = 2.15; 95% CI = 1.26-3.67; P = 0.00723). Neither LoF variants nor predicted damaging rare missense variants in BARD1 were identified in 451 familial index patients with OC., Conclusions: Due to the significant association of germline LoF variants in BARD1 with early-onset BC, we suggest that intensified BC surveillance programs should be offered to women carrying pathogenic BARD1 gene variants.

The prevalence and spectrum of germline mutations in BRCA1 and BRCA2 have been reported in single populations, with the majority of reports focused on White in Europe and North America. The Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) has assembled data on 18,435 families with BRCA1 mutations and 11,351 families with BRCA2 mutations ascertained from 69 centers in 49 countries on six continents. This study comprehensively describes the characteristics of the 1,650 unique BRCA1 and 1,731 unique BRCA2 deleterious (disease-associated) mutations identified in the CIMBA database. We observed substantial variation in mutation type and frequency by geographical region and race/ethnicity. In addition to known founder mutations, mutations of relatively high frequency were identified in specific racial/ethnic or geographic groups that may reflect founder mutations and which could be used in targeted (panel) first pass genotyping for specific populations. Knowledge of the population-specific mutational spectrum in BRCA1 and BRCA2 could inform efficient strategies for genetic testing and may justify a more broad-based oncogenetic testing in some populations., (© 2018 Wiley Periodicals, Inc.)

The prevalence of germ line mutations in non-BRCA1/2 genes associated with hereditary breast cancer (BC) is low, and the role of some of these genes in BC predisposition and pathogenesis is conflicting. In this study, 5589 consecutive BC index patients negative for pathogenic BRCA1/2 mutations and 2189 female controls were screened for germ line mutations in eight cancer predisposition genes (ATM, CDH1, CHEK2, NBN, PALB2, RAD51C, RAD51D, and TP53). All patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germ line testing. The highest mutation prevalence was observed in the CHEK2 gene (2.5%), followed by ATM (1.5%) and PALB2 (1.2%). The mutation prevalence in each of the remaining genes was 0.3% or lower. Using Exome Aggregation Consortium control data, we confirm significant associations of heterozygous germ line mutations with BC for ATM (OR: 3.63, 95%CI: 2.67-4.94), CDH1 (OR: 17.04, 95%CI: 3.54-82), CHEK2 (OR: 2.93, 95%CI: 2.29-3.75), PALB2 (OR: 9.53, 95%CI: 6.25-14.51), and TP53 (OR: 7.30, 95%CI: 1.22-43.68). NBN germ line mutations were not significantly associated with BC risk (OR:1.39, 95%CI: 0.73-2.64). Due to their low mutation prevalence, the RAD51C and RAD51D genes require further investigation. Compared with control datasets, predicted damaging rare missense variants were significantly more prevalent in CHEK2 and TP53 in BC index patients. Compared with the overall sample, only TP53 mutation carriers show a significantly younger age at first BC diagnosis. We demonstrate a significant association of deleterious variants in the CHEK2, PALB2, and TP53 genes with bilateral BC. Both, ATM and CHEK2, were negatively associated with triple-negative breast cancer (TNBC) and estrogen receptor (ER)-negative tumor phenotypes. A particularly high CHEK2 mutation prevalence (5.2%) was observed in patients with human epidermal growth factor receptor 2 (HER2)-positive tumors., (© 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Germline mutations in the BRIP1 gene have been described as conferring a moderate risk for ovarian cancer (OC), while the role of BRIP1 in breast cancer (BC) pathogenesis remains controversial., Methods: To assess the role of deleterious BRIP1 germline mutations in BC/OC predisposition, 6341 well-characterized index patients with BC, 706 index patients with OC, and 2189 geographically matched female controls were screened for loss-of-function (LoF) mutations and potentially damaging missense variants. All index patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germline testing and tested negative for pathogenic BRCA1/2 variants., Results: BRIP1 LoF mutations confer a high OC risk in familial index patients (odds ratio (OR) = 20.97, 95% confidence interval (CI) = 12.02-36.57, P < 0.0001) and in the subgroup of index patients with late-onset OC (OR = 29.91, 95% CI = 14.99-59.66, P < 0.0001). No significant association of BRIP1 LoF mutations with familial BC was observed (OR = 1.81 95% CI = 1.00-3.30, P = 0.0623). In the subgroup of familial BC index patients without a family history of OC there was also no apparent association (OR = 1.42, 95% CI = 0.70-2.90, P = 0.3030). In 1027 familial BC index patients with a family history of OC, the BRIP1 mutation prevalence was significantly higher than that observed in controls (OR = 3.59, 95% CI = 1.43-9.01; P = 0.0168). Based on the negative association between BRIP1 LoF mutations and familial BC in the absence of an OC family history, we conclude that the elevated mutation prevalence in the latter cohort was driven by the occurrence of OC in these families. Compared with controls, predicted damaging rare missense variants were significantly more prevalent in OC (P = 0.0014) but not in BC (P = 0.0693) patients., Conclusions: To avoid ambiguous results, studies aimed at assessing the impact of candidate predisposition gene mutations on BC risk might differentiate between BC index patients with an OC family history and those without. In familial cases, we suggest that BRIP1 is a high-risk gene for late-onset OC but not a BC predisposition gene, though minor effects cannot be excluded.

Importance: Germline mutations in established moderately or highly penetrant risk genes for breast cancer (BC) and/or ovarian cancer (OC), including BRCA1 and BRCA2, explain fewer than half of all familial BC and/or OC cases. Based on the genotyping of 2 loss-of-function (LoF) variants c.5101C>T (p.GIn1701Ter [rs147021911]) and c.5791C>T (p.Arg1931Ter [rs144567652]), the FANCM gene has been suggested as a novel BC predisposition gene, while the analysis of the entire coding region of the FANCM gene in familial index cases and geographically matched controls is pending., Objectives: To assess the mutational spectrum within the FANCM gene, and to determine a potential association of LoF germline mutations within the FANCM gene with BC and/or OC risk., Design, Setting, and Participants: For the purpose of identification and characterization of novel BC and/or OC predisposition genes, a total of 2047 well-characterized familial BC index cases, 628 OC cases, and 2187 geographically matched controls were screened for LoF mutations within the FANCM gene by next-generation sequencing. All patients previously tested negative for pathogenic BRCA1 and BRCA2 mutations. All data collection occurred between June 1, 2013, and April 30, 2016. Data analysis was performed from May 1, 2016, to July 1, 2016., Main Outcomes and Measures: FANCM LoF mutation frequencies in patients with BC and/or OC were compared with the FANCM LoF mutation frequencies in geographically matched controls by univariate logistic regression. Positive associations were stratified by age at onset and cancer family history., Results: In this case-control study, 2047 well-characterized familial female BC index cases, 628 OC cases, and 2187 geographically matched controls were screened for truncating FANCM alterations. Heterozygous LoF mutations within the FANCM gene were significantly associated with familial BC risk, with an overall odds ratio (OR) of 2.05 (95% CI, 0.94-4.54; P = .049) and a mutation frequency of 1.03% in index cases. In familial patients whose BC onset was before age 51 years, an elevated OR of 2.44 (95% CI, 1.08-5.59; P = .02) was observed. A more pronounced association was identified for patients with a triple-negative BC tumor phenotype (OR, 3.75; 95% CI, 1.00-12.85; P = .02). No significant association was detected for unselected OC cases (OR, 1.74; 95% CI, 0.57-5.08; P = .27)., Conclusions and Relevance: Based on the significant associations of heterozygous LoF mutations with early-onset or triple-negative BC, FANCM should be included in diagnostic gene panel testing for individual risk assessment. Larger studies are required to determine age-dependent disease risks for BC and to assess a potential role of FANCM mutations in OC pathogenesis.

Direct analysis of circulating tumor cells (CTCs) can inform on molecular mechanisms underlying systemic spread. Here we investigated promoter methylation of three genes regulating epithelial-to-mesenchymal transition (EMT), a key mechanism enabling epithelial tumor cells to disseminate and metastasize. For this, we developed a single-cell protocol based on agarose-embedded bisulfite treatment, which allows investigating DNA methylation of multiple loci via a multiplex PCR (multiplexed-scAEBS). We established our assay for the simultaneous analysis of three EMT-associated genes miR-200c/141, miR-200b/a/429 and CDH1 in single cells. The assay was validated in solitary cells of GM14667, MDA-MB-231 and MCF-7 cell lines, achieving a DNA amplification efficiency of 70% with methylation patterns identical to the respective bulk DNA. Then we applied multiplexed-scAEBS to 159 single CTCs from 11 patients with metastatic breast and six with metastatic castration-resistant prostate cancer, isolated via CellSearch (EpCAM pos /CK pos /CD45 neg /DAPI pos ) and subsequent FACS sorting. In contrast to CD45 pos white blood cells isolated and processed by the identical approach, we observed in the isolated CTCs methylation patterns resembling more those of epithelial-like cells. Methylation at the promoter of microRNA-200 family was significantly higher in prostate CTCs. Data from our single-cell analysis revealed an epigenetic heterogeneity among CTCs and indicates tumor-specific active epigenetic regulation of EMT-associated genes during blood-borne dissemination.

Fanconi anemia (FA) is a rare inherited disorder clinically characterized by congenital malformations, progressive bone marrow failure and cancer susceptibility. At the cellular level, FA is associated with hypersensitivity to DNA-crosslinking genotoxins. Eight of 17 known FA genes assemble the FA E3 ligase complex, which catalyzes monoubiquitination of FANCD2 and is essential for replicative DNA crosslink repair. Here, we identify the first FA patient with biallelic germline mutations in the ubiquitin E2 conjugase UBE2T. Both mutations were aluY-mediated: a paternal deletion and maternal duplication of exons 2-6. These loss-of-function mutations in UBE2T induced a cellular phenotype similar to biallelic defects in early FA genes with the absence of FANCD2 monoubiquitination. The maternal duplication produced a mutant mRNA that could encode a functional protein but was degraded by nonsense-mediated mRNA decay. In the patient's hematopoietic stem cells, the maternal allele with the duplication of exons 2-6 spontaneously reverted to a wild-type allele by monoallelic recombination at the duplicated aluY repeat, thereby preventing bone marrow failure. Analysis of germline DNA of 814 normal individuals and 850 breast cancer patients for deletion or duplication of UBE2T exons 2-6 identified the deletion in only two controls, suggesting aluY-mediated recombinations within the UBE2T locus are rare and not associated with an increased breast cancer risk. Finally, a loss-of-function germline mutation in UBE2T was detected in a high-risk breast cancer patient with wild-type BRCA1/2. Cumulatively, we identified UBE2T as a bona fide FA gene (FANCT) that also may be a rare cancer susceptibility gene., (© The Author 2015. Published by Oxford University Press.)

Background: Circulating tumor cells (CTCs) are promising surrogate markers for systemic disease, and their molecular characterization might be relevant to guide more individualized cancer therapies. To enable fast and efficient purification of individual CTCs, we developed a work flow from CellSearch(TM) cartridges enabling high-resolution genomic profiling on the single-cell level., Methods: Single CTCs were sorted from 40 CellSearch samples from patients with metastatic breast cancer using a MoFlo XDP cell sorter. Genomes of sorted single cells were amplified using an adapter-linker PCR. Amplification products were analyzed by array-based comparative genomic hybridization, a gene-specific quantitative PCR (qPCR) assay for cyclin D1 (CCND1) locus amplification, and genomic sequencing to screen for mutations in exons 1, 9, and 20 of the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene and exons 5, 7, and 8 of the tumor protein p53 (TP53) gene., Results: One common flow-sorting protocol was appropriate for 90% of the analyzed CellSearch cartridges, and the detected CTC numbers correlated positively with those originally detected with the CellSearch system (R(2) = 0.9257). Whole genome amplification was successful in 72.9% of the sorted single CTCs. Over 95% of the cells displayed chromosomal aberrations typical for metastatic breast cancers, and amplifications at the CCND1 locus were validated by qPCR. Aberrant CTCs from 2 patients harbored mutations in exon 20 of the PIK3CA gene., Conclusions: This work flow enabled effective CTC isolation and provided insights into genomic alterations of CTCs in metastatic breast cancer. This approach might facilitate further molecular characterization of rare CTCs to increase understanding of their biology and as a basis for their molecular screening in the clinical setting., (© 2014 American Association for Clinical Chemistry.)

Circulating tumor cells (CTCs) are promising biomarkers for diagnosis and therapy in systemic cancer. However, their infrequent and unreliable detection, especially in nonmetastatic cancer, currently impedes the clinical use of CTCs. Because leukapheresis (LA) targets peripheral blood mononuclear cells, which have a similar density to CTCs, and usually involves processing the whole circulating blood, we tested whether LA could substantially increase CTC detection in operable cancer patients. Therefore, we screened LA products generated from up to 25 L of blood per patient in two independent studies, and found that CTCs can be detected in more than 90% of nonmetastatic breast cancer patients. Interestingly, complete white blood cell sampling enabled determining an upper level for total CTC numbers of about 100,000 cells (median, 7,500 CTCs) per patient and identified a correlation of CTC numbers with anatomic disease spread. We further show that diagnostic leukapheresis can be easily combined with the US Food and Drug Administration-approved CellSearch system for standardized enumeration of CTCs. Direct comparison with 7.5 mL of blood revealed a significantly higher CTC frequency in matched LA samples. Finally, genomic single-cell profiling disclosed highly aberrant CTCs as therapy-escaping variants in breast cancer. In conclusion, LA is a clinically safe method that enabled a reliable detection of CTCs at high frequency even in nonmetastatic cancer patients, and might facilitate the routine clinical use of CTCs as in the sense of a liquid biopsy. Combined with technologies for single-cell molecular genetics or cell biology, it may significantly improve prediction of therapy response and monitoring of early systemic cancer.

Comprehensive genome wide analyses of single cells became increasingly important in cancer research, but remain to be a technically challenging task. Here, we provide a protocol for array comparative genomic hybridization (aCGH) of single cells. The protocol is based on an established adapter-linker PCR (WGAM) and allowed us to detect copy number alterations as small as 56 kb in single cells. In addition we report on factors influencing the success of single cell aCGH downstream of the amplification method, including the characteristics of the reference DNA, the labeling technique, the amount of input DNA, reamplification, the aCGH resolution, and data analysis. In comparison with two other commercially available non-linear single cell amplification methods, WGAM showed a very good performance in aCGH experiments. Finally, we demonstrate that cancer cells that were processed and identified by the CellSearch® System and that were subsequently isolated from the CellSearch® cartridge as single cells by fluorescence activated cell sorting (FACS) could be successfully analyzed using our WGAM-aCGH protocol. We believe that even in the era of next-generation sequencing, our single cell aCGH protocol will be a useful and (cost-) effective approach to study copy number alterations in single cells at resolution comparable to those reported currently for single cell digital karyotyping based on next generation sequencing data.

The Partner and Localizer of BRCA2 (PALB2) protein has been linked to Fanconi anemia and breast cancer predisposition. Here we present data of a comprehensive mutation screening of the PALB2 gene in 818 familial cases of breast cancer from Germany. By analyzing the entire coding region of PALB2, we found seven truncating mutations (six of them novel) in families tested negative for BRCA1/2-mutations. In addition, two novel potentially disease causing missense mutations were found. Remarkably, only one mutation reported previously in other populations, was also identified in the German population. No PALB2 mutation carriers were identified in 450 unaffected controls. Thus, our observations indicate a low prevalence of deleterious PALB2 mutations and a specific mutation profile within the German population. As PALB2-deficient tumors were shown to be sensitive to Poly(ADP-ribose) Polymerase (PARP) inhibitors, our study has implications for newly developed, favorable treatment options in familial breast cancer., (© 2011 Wiley-Liss, Inc.)

Background: The miR-200c/141 cluster has recently been implicated in the epithelial to mesenchymal transition (EMT) process. The expression of these two miRNAs is inversely correlated with tumorigenicity and invasiveness in several human cancers. The role of these miRNAs in cancer progression is based in part on their capacity to target the EMT activators ZEB1 and ZEB2, two transcription factors, which in turn repress expression of E-cadherin. Little is known about the regulation of the mir200c/141 cluster, whose targeting has been proposed as a promising new therapy for the most aggressive tumors., Findings: We show that the miR-200c/141 cluster is repressed by DNA methylation of a CpG island located in the promoter region of these miRNAs. Whereas in vitro methylation of the miR-200c/141 promoter led to shutdown of promoter activity, treatment with a demethylating agent caused transcriptional reactivation in breast cancer cells formerly lacking expression of miR-200c and miR-141. More importantly, we observed that DNA methylation of the identified miR-200c/141 promoter was tightly correlated with phenotype and the invasive capacity in a panel of 8 human breast cancer cell lines. In line with this, in vitro induction of EMT by ectopic expression of the EMT transcription factor Twist in human immortalized mammary epithelial cells (HMLE) was accompanied by increased DNA methylation and concomitant repression of the miR-200c/141 locus., Conclusions: The present study demonstrates that expression of the miR-200c/141 cluster is regulated by DNA methylation, suggesting epigenetic regulation of this miRNA locus in aggressive breast cancer cell lines as well as untransformed mammary epithelial cells. This epigenetic silencing mechanism might represent a novel component of the regulatory circuit for the maintenance of EMT programs in cancer and normal cells.

Germline mutations in a number of genes involved in the recombinational repair of DNA double-strand breaks are associated with predisposition to breast and ovarian cancer. RAD51C is essential for homologous recombination repair, and a biallelic missense mutation can cause a Fanconi anemia-like phenotype. In index cases from 1,100 German families with gynecological malignancies, we identified six monoallelic pathogenic mutations in RAD51C that confer an increased risk for breast and ovarian cancer. These include two frameshift-causing insertions, two splice-site mutations and two nonfunctional missense mutations. The mutations were found exclusively within 480 pedigrees with the occurrence of both breast and ovarian tumors (BC/OC; 1.3%) and not in 620 pedigrees with breast cancer only or in 2,912 healthy German controls. These results provide the first unambiguous evidence of highly penetrant mutations associated with human cancer in a RAD51 paralog and support the 'common disease, rare allele' hypothesis.