Ellen Honisch

Role of DNA methylation in miR-200c/141 cluster silencing in invasive breast cancer cells

BackgroundThe 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.FindingsWe 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.ConclusionsThe 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.

Diagnostic leukapheresis enables reliable detection of circulating tumor cells of nonmetastatic cancer patients

Significance The infrequent detection of circulating tumor cells (CTCs) has hindered their clinical implication and their potential use in the sense of a “liquid biopsy” for cancer diagnosis and therapy. Hypothesizing that the limited blood volume commonly used for CTC analysis (1–10 mL) accounts for variable detection rates, we used leukapheresis to screen large blood volumes for CTCs. This enabled a more reliable detection of CTCs at high frequency even in nonmetastatic cancer patients. Thus, diagnostic leukapheresis may facilitate the routine clinical use of CTCs as biomarkers for personalized medicine. Combined with technologies for single-cell molecular genetics or cell biology, it may significantly improve prediction of therapy response and monitoring, especially in early systemic cancer. 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.

Genomic High-Resolution Profiling of Single CKpos/CD45neg Flow-Sorting Purified Circulating Tumor Cells from Patients with Metastatic Breast Cancer

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.

Germline mutations in the PALB2 gene are population specific and occur with low frequencies in familial breast cancer

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 analysing 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 identidfied 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, favourable treatment options in familial breast cancer.

A Robust Method to Analyze Copy Number Alterations of Less than 100 kb in Single Cells Using Oligonucleotide Array CGH

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.

Association Between Loss-of-Function Mutations Within the FANCM Gene and Early-Onset Familial Breast Cancer

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.

Gene panel testing of 5589 BRCA1/2-negative index patients with breast cancer in a routine diagnostic setting: results of the German Consortium for Hereditary Breast and Ovarian Cancer

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.

BRIP1 loss-of-function mutations confer high risk for familial ovarian cancer, but not familial breast cancer

BackgroundGermline 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.MethodsTo 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.ResultsBRIP1 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.ConclusionsTo 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.