Britta Hasemeier

Publications of Britta Hasemeier

• Quantitative cross-validation and content analysis of the 450k DNA methylation array from Illumina, Inc.

  Authors: Jessica Roessler, Ole Ammerpohl, Jana Gutwein, Britta Hasemeier, Sumadi Lukman Anwar, Hans H Kreipe, Ulrich Lehmann

  BMC research notes. 04/2012; 5(1):210.

  ABSTRACT: BACKGROUND: The newly released 450k DNA methylation array from Illumina, Inc. offers the possibility to analyze more than 480,000 individual CpG sites in a user friendly standardizedABSTRACT: BACKGROUND: The newly released 450k DNA methylation array from Illumina, Inc. offers the possibility to analyze more than 480,000 individual CpG sites in a user friendly standardized format. In this study the relationship between the beta-values provided by the Illumina, Inc. array for each individual CpG dinucleotide and the quantitative methylation levels obtained by pyrosequencing were analyzed. In addition, the representation of microRNA genes and imprinted loci on the Illumina, Inc. array was assessed in detail. Genomic DNA from 4 human breast cancer cell lines (IPH-926, HCC1937, MDA-MB-134, PMC42) and 18 human breast cancer specimens as well as 4 normal mammary epithelial fractions was analyzed on 450k DNA methylation arrays. The beta-values for 692 individual CpG sites from 62 different genes were cross-validated using conventional quantitative pyrosequencing. FINDINGS: The newly released 450k methylation array from Illumina, Inc. shows a high concordance with quantitative pyrosequencing if identical CpG sites are analyzed in cell lines (Spearman r = 0.88, p < 0.0001), which is somewhat reduced in primary tumor specimens (Spearman r = 0.86, p < 0.0001). 80.7% of the CpG sites show an absolute difference in methylation level of less than 15 percentage points. If different CpG sites in the same CpG islands are targeted the concordance is lower (r = 0.83 in cell lines and r = 0.7 in primary tumors). The number of CpG sites representing microRNA genes and imprinted loci is very heterogeneous (range: 1 - 70 CpG sites for microRNAs and 1 - 288 for imprinted loci). CONCLUSIONS: The newly released 450k methylation array from Illumina, Inc. provides a genome-wide quantitative representation of DNA methylation aberrations in a convenient format. Overall, the congruence with pyrosequencing data is very good. However, for individual loci one should be careful to translate the beta-values directly into percent methylation levels.

• Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution.

  Authors: Anna Potapova, Cord Albat, Britta Hasemeier, Katrin Haeussler, Stella Lamprecht, Sebastian Suerbaum, Hans Kreipe, Ulrich Lehmann

  BMC biotechnology. 01/2011; 11:6.

  New high-throughput sequencing technologies promise a very sensitive and high-resolution analysis of DNA methylation patterns in quantitative terms. However, a detailed and comprehensive comparisonNew high-throughput sequencing technologies promise a very sensitive and high-resolution analysis of DNA methylation patterns in quantitative terms. However, a detailed and comprehensive comparison with existing validated DNA methylation analysis methods is not yet available. Therefore, a systematic cross-validation of 454 sequencing and conventional pyrosequencing, both of which offer exact quantification of methylation levels with a single CpG dinucleotide resolution, was performed. To this end the methylation patterns of 12 loci (GSTπ1, p16INK4a, RASSF1A, SOCS1, MAL, hsa-mir-1-1, hsa-mir-9-3, hsa-mir-34a, hsa-mir-596, hsa-mir-663, MINT31, and LINE-1) were analyzed in ten primary hepatocellular carcinoma specimens. After applying stringent quality control criteria, 35749 sequences entered further analysis. The methylation level of individual CpG dinucleotides obtained by 454 sequencing was systematically compared with the corresponding values obtained by conventional pyrosequencing. Statistical analyses revealed an excellent concordance of methylation levels for all individual CpG dinucleotides under study (r2 = 0.927). Our results confirm that 454 sequencing of bisulfite treated genomic DNA provides reliable high quality quantitative methylation data and identify MAL, hsa-mir-9-3, hsa-mir-596, and hsa-mir-663 as new targets of aberrant DNA methylation in human hepatocellular carcinoma. In addition, the single molecule resolution of 454 sequencing provides unprecedented information about the details of DNA methylation pattern heterogeneity in clinical samples.

• Identification of differentially expressed microRNAs in human male breast cancer.

  Authors: Ulrich Lehmann, Thomas Streichert, Benjamin Otto, Cord Albat, Britta Hasemeier, Henriette Christgen, Elisa Schipper, Ursula Hille, Hans H Kreipe, Florian Länger

  BMC cancer. 03/2010; 10:109.

  The discovery of small non-coding RNAs and the subsequent analysis of microRNA expression patterns in human cancer specimens have provided completely new insights into cancer biology. Genetic andThe discovery of small non-coding RNAs and the subsequent analysis of microRNA expression patterns in human cancer specimens have provided completely new insights into cancer biology. Genetic and epigenetic data indicate oncogenic or tumor suppressor function of these pleiotropic regulators. Therefore, many studies analyzed the expression and function of microRNA in human breast cancer, the most frequent malignancy in females. However, nothing is known so far about microRNA expression in male breast cancer, accounting for approximately 1% of all breast cancer cases. The expression of 319 microRNAs was analyzed in 9 primary human male breast tumors and in epithelial cells from 15 male gynecomastia specimens using fluorescence-labeled bead technology. For identification of differentially expressed microRNAs data were analyzed by cluster analysis and selected statistical methods.Expression levels were validated for the most up- or down-regulated microRNAs in this training cohort using real-time PCR methodology as well as in an independent test cohort comprising 12 cases of human male breast cancer. Unsupervised cluster analysis separated very well male breast cancer samples and control specimens according to their microRNA expression pattern indicating cancer-specific alterations of microRNA expression in human male breast cancer. miR-21, miR519d, miR-183, miR-197, and miR-493-5p were identified as most prominently up-regulated, miR-145 and miR-497 as most prominently down-regulated in male breast cancer. Male breast cancer displays several differentially expressed microRNAs. Not all of them are shared with breast cancer biopsies from female patients indicating male breast cancer specific alterations of microRNA expression.

• Identification of differentially expressed microRNAs in human male breast cancer

  Authors: Ulrich Lehmann, Thomas Streichert, Benjamin Otto, Cord Albat, Britta Hasemeier, Henriette Christgen, Elisa Schipper, Ursula Hille, Hans Kreipe, Florian Länger

  BMC Cancer. 01/2010;

  Abstract Background The discovery of small non-coding RNAs and the subsequent analysis of microRNA expression patterns in human cancer specimens have provided completely new insights into cancerAbstract Background The discovery of small non-coding RNAs and the subsequent analysis of microRNA expression patterns in human cancer specimens have provided completely new insights into cancer biology. Genetic and epigenetic data indicate oncogenic or tumor suppressor function of these pleiotropic regulators. Therefore, many studies analyzed the expression and function of microRNA in human breast cancer, the most frequent malignancy in females. However, nothing is known so far about microRNA expression in male breast cancer, accounting for approximately 1% of all breast cancer cases. Methods The expression of 319 microRNAs was analyzed in 9 primary human male breast tumors and in epithelial cells from 15 male gynecomastia specimens using fluorescence-labeled bead technology. For identification of differentially expressed microRNAs data were analyzed by cluster analysis and selected statistical methods. Expression levels were validated for the most up- or down-regulated microRNAs in this training cohort using real-time PCR methodology as well as in an independent test cohort comprising 12 cases of human male breast cancer. Results Unsupervised cluster analysis separated very well male breast cancer samples and control specimens according to their microRNA expression pattern indicating cancer-specific alterations of microRNA expression in human male breast cancer. miR-21, miR519d, miR-183, miR-197, and miR-493-5p were identified as most prominently up-regulated, miR-145 and miR-497 as most prominently down-regulated in male breast cancer. Conclusions Male breast cancer displays several differentially expressed microRNAs. Not all of them are shared with breast cancer biopsies from female patients indicating male breast cancer specific alterations of microRNA expression.

• Epigenetic inactivation of tumour suppressor gene KLF11 in myelodysplastic syndromes*

  Authors: Anna Potapova, Britta Hasemeier, Daniel Römermann, Kathleen Metzig, Gudrun Göhring, Brigitte Schlegelberger, Florian Länger, Hans Kreipe, Ulrich Lehmann

  European journal of haematology. 11/2009;

  Abstract The identification of aberrantly hypermethylated genes may lead to the development of new diagnostic markers and the identification of novel targets of epigenetic therapy in myelodysplasticAbstract The identification of aberrantly hypermethylated genes may lead to the development of new diagnostic markers and the identification of novel targets of epigenetic therapy in myelodysplastic syndromes (MDS). We therefore investigated the methylation status of transcription factor genes KLF5, KLF11, and MAFB, shown to be aberrantly methylated in myelogeneous leukaemia cells, in a series of 115 myelodysplastic syndromes patient as well as in 25 control subjects. Using quantitative high-resolution pyrosequencing methodology KLF11, MAFB, and KLF5 were shown for the first time to be hypermethylated in 17 (15%), 8 (7%), and 2 (1.7%) cases, respectively, but not in any of the patients with an isolated 5q-deletion. Patient samples harboring KLF11 methylation displayed reduced KLF11 mRNA expression and KLF11 hypermethylation correlated with a high IPSS score (p < 0.05). In conclusion, epigenetic inactivation and subsequent transcriptional repression of the KLF11 gene is quite frequent in myelodysplastic syndromes. Patients with an isolated 5q-deletion seem to harbour a distinct epigenetic profile.

• Reliable microRNA profiling in routinely processed formalin-fixed paraffin-embedded breast cancer specimens using fluorescence labelled bead technology.

  Authors: Britta Hasemeier, Matthias Christgen, Hans Kreipe, Ulrich Lehmann

  BMC biotechnology. 12/2008; 8(1):90.

  ABSTRACT: BACKGROUND: During the last years the analysis of microRNA expression patterns has led to completely new insights into cancer biology. Furthermore, these patterns are a very promising toolABSTRACT: BACKGROUND: During the last years the analysis of microRNA expression patterns has led to completely new insights into cancer biology. Furthermore, these patterns are a very promising tool for the development of new diagnostic and prognostic markers. However, most human tumour samples for which long term clinical records are available exist only as formalin-fixed paraffin-embedded specimens. Therefore, the aim of this study was to examine the feasibility of microRNA profiling studies in routinely processed formalin-fixed paraffin-embedded human breast cancer specimens using fluorescence labelled bead technology. RESULTS: A statistically highly significant correlation (Spearman r: 0.78 - 0.90, p < 0.0001) was observed for the expression of 319 microRNAs in routinely processed FFPE breast cancer specimens and paired fresh frozen tissue samples (n = 5). Results were confirmed in a larger series analyzing a selection of 10 microRNAs reported to be deregulated in breast cancer (n = 12). The expression pattern of 3 microRNAs was independently validated in this cohort using real-time RT-PCR technology. CONCLUSIONS: Comprehensive microRNA expression patterns can be reliably derived from routinely processed FFPE breast cancer specimens using fluorescence labelled bead technology.

• Comprehensive genetic and functional characterization of IPH-926: a novel CDH1-null tumour cell line from human lobular breast cancer.

  Authors: Matthias Christgen, Henriette Bruchhardt, Catarina Hadamitzky, Cornelia Rudolph, Doris Steinemann, Dorothea Gadzicki, Britta Hasemeier, Daniel Römermann, Tim Focken, Till Krech, Matthias Ballmaier, Brigitte Schlegelberger, Hans Kreipe, Ulrich Lehmann

  The Journal of pathology. 11/2008;

  Infiltrating lobular breast cancer (ILBC) is a clinically and biologically distinct tumour entity defined by a characteristic linear cord invasion pattern and inactivation of the CDH1 tumourInfiltrating lobular breast cancer (ILBC) is a clinically and biologically distinct tumour entity defined by a characteristic linear cord invasion pattern and inactivation of the CDH1 tumour suppressor gene encoding for E-cadherin. ILBCs also lack beta-catenin expression and show aberrant cytoplasmic localization of the E-cadherin binding protein p120-catenin. The lack of a well-characterized ILBC cell line has hampered the functional characterization of ILBC cells in vitro. We report the establishment of a permanent ILBC cell line, named IPH-926, which was derived from a patient with metastatic ILBC. The DNA fingerprint of IPH-926 verified genetic identity with the patient and had no match among the human cell line collections of several international biological resource banks. IPH-926 expressed various epithelial cell markers but lacked expression of E-cadherin due to a previously unreported, homozygous CDH1 241ins4 frameshift mutation. Detection of the same CDH1 241ins4 mutation in archival tumour tissue of the corresponding primary ILBC proved the clonal origin of IPH-926 from this particular tumour. IPH-926 also lacked beta-catenin expression and showed aberrant cytoplasmic localization of p120-catenin. Array-CGH analysis of IPH-926 revealed a profile of genomic imbalances that included many distinct alterations previously observed in primary ILBCs. Spectral karyotyping of IPH-926 showed a hyperdiploid chromosome complement and numerous clonal, structural aberrations. IPH-926 cells were anti-cancer drug-resistant, clonogenic in soft agar, and tumourigenic in SCID mice. In xenograft tumours, IPH-926 cells recapitulated the linear cord invasion pattern that defines ILBCs. In summary, IPH-926 significantly extends the biological spectrum of the established breast cancer cell lines and will facilitate functional analyses of genuine human ILBC cells in vitro and in vivo. Copyright (c) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

• Complete cytogenetic remission after decitabine treatment in a patient with secondary AML harbouring high p15 ( INK4b ) gene methylation and high global DNA methylation.

  Authors: Ulrich Lehmann, Christiane Dobbelstein, Martin Fenner, Daniel Römermann, Britta Hasemeier, Kathleen Metzig, Doris Steinemann, Guntram Büsche, Jürgen Krauter, Arnold Ganser, Hans Kreipe

  Annals of hematology. 09/2008;

• Epigenetic defects of hepatocellular carcinoma are already found in non-neoplastic liver cells from patients with hereditary haemochromatosis.

  Authors: Ulrich Lehmann, Luzie U Wingen, Kai Brakensiek, Heiner Wedemeyer, Thomas Becker, Albert Heim, Kathleen Metzig, Britta Hasemeier, Hans Kreipe, Peer Flemming

  Human molecular genetics. 06/2007; 16(11):1335-42.

  Gene silencing through aberrant CpG island methylation is a frequent epigenetic defect in hepatocellular carcinoma (HCC). However, nothing is known as yet whether aberrant hypermethylation occursGene silencing through aberrant CpG island methylation is a frequent epigenetic defect in hepatocellular carcinoma (HCC). However, nothing is known as yet whether aberrant hypermethylation occurs already in non-neoplastic liver cells from patients with hereditary haemochromatosis who have a clearly elevated risk for developing HCC. Therefore, quantitative real-time PCR-based methylation analysis of six genes frequently hypermethylated in HCC (RASSF1A, cyclinD2, p16(INK4a), GSTpi1, SOCS-1, APC) was performed for liver biopsies from patients with hereditary haemochromatosis. For genotyping of the HFE gene restriction enzyme analysis and Pyrosequencing were used. Transcriptional repression of hypermethylated genes was assessed using real-time RT-PCR. Eighty-four percent of all samples with severe hepatic iron overload and a mutated HFE gene (but without HCC) had at least one gene hypermethylated. All six genes tested were affected by aberrant hypermethylation, albeit to a different extent: RASSF1A 55%, cyclinD2 45%, p16(INK4a) 32%, GSTpi1 10%, SOCS-1 6%, APC 8%. Concomitant transcriptional down-regulation was shown for RASSF1A, cyclinD2, GSTpi1 and SOCS-1. Biopsies from haemochromatosis patients showed significantly more aberrant hypermethylation than normal liver tissue or benign liver tumours (P < 0.001) and also to a higher degree. This effect is independent of patient age, cirrhosis or hepatitis infection. This is the first report demonstrating that longstanding severe iron overload is frequently associated with epigenetic defects characteristic of HCC, which reflects the increased risk of these lesions to progress to HCC. Thus, changes in DNA methylation patterns are an early event preceding morphological alterations of malignant transformation and represent promising targets for early detection.

• Down-regulation of the IGF-2/H19 locus during normal and malignant hematopoiesis is independent of the imprinting pattern.

  Authors: Mathewos Tessema, Florian Länger, Oliver Bock, Axel Seltsam, Kathleen Metzig, Britta Hasemeier, Hans Kreipe, Ulrich Lehmann

  International journal of oncology. 03/2005; 26(2):499-507.

  H19 and IGF-2 are two growth regulatory genes located on chromosome 11p15 implicated in tumorigenesis. Both genes are imprinted and regulated reciprocally under many circumstances. In order toH19 and IGF-2 are two growth regulatory genes located on chromosome 11p15 implicated in tumorigenesis. Both genes are imprinted and regulated reciprocally under many circumstances. In order to elucidate the contribution of H19 and IGF-2 to leukemogenesis, the mRNA expression level of both genes were quantitated in bone marrow biopsies and peripheral blood samples from normal (n=98), chronic myelomonocytic leukemia (CMML, n=43), chronic myelogenous leukemia (CML, n=40) and, acute myelogenous leukemia (AML, n=32) cases. A concomitant reduction of H19 and IGF-2 expression was observed in all leukemic samples compared to the healthy controls. This down-regulation was not accompanied by changes in methylation of the differentially methylated region (DMR). Whereas the H19 gene showed strict monoallelic expression in all informative normal (n=31) and leukemic (n=54) samples, the imprinting pattern of the IGF2 gene was found to be heterogeneous. No correlations between imprinting status (mono- versus biallelic expression), quantitative mRNA expression levels and course of disease were found for the IGF-2 gene. The data suggest a disturbed regulation of the IGF-2/H19 locus in myeloid leukemias which is not caused by loss of imprinting.

• Promoter hypermethylation of the death-associated protein kinase gene in breast cancer is associated with the invasive lobular subtype.

  Authors: Ulrich Lehmann, Gülhan Celikkaya, Britta Hasemeier, Florian Länger, Hans Kreipe

  Cancer research. 11/2002; 62(22):6634-8.

  Expression of death-associated protein (DAP) kinase, a proapoptotic serine/threonine protein kinase, is frequently lost in human tumors. In a study of 134 primary breast cancer specimensExpression of death-associated protein (DAP) kinase, a proapoptotic serine/threonine protein kinase, is frequently lost in human tumors. In a study of 134 primary breast cancer specimens hypermethylation of the DAP kinase gene was found in 13% of cases. A highly significant difference (P < 0.001) of DAP kinase inactivation was observed between invasive lobular cancer (n = 19) and invasive ductal cancer (n = 85; 53% versus 9%, respectively). Hypermethylation correlated with loss of RNA expression, estrogen receptor positivity (P < 0.01), and the absence of p53 overexpression (P < 0.01). In contrast to invasive lobular cancer, the in situ-growing precursor lesion lacked epigenetic modification of the DAP kinase promotor by aberrant methylation indicating a potential role in tumor progression. Unlike the DAP kinase gene, hypermethylation of the cyclin D2 and RASSF1A genes did not correlate with a particular histological subtype or to invasiveness [corrected]. We conclude that different histological subtypes of breast cancer may not only differ concerning specific chromosomal abnormalities and DNA mutations but also with regard to epigenetic inactivation patterns.

• Quantitative assessment of promoter hypermethylation during breast cancer development.

  Authors: Ulrich Lehmann, Florian Länger, Henning Feist, Sabine Glöckner, Britta Hasemeier, Hans Kreipe

  The American journal of pathology. 03/2002; 160(2):605-12.

  The aberrant methylation of cytosine residues in the promoter region of growth regulatory genes is now widely recognized as an additional mechanism for gene inactivation in cancer cells. In thisThe aberrant methylation of cytosine residues in the promoter region of growth regulatory genes is now widely recognized as an additional mechanism for gene inactivation in cancer cells. In this study we analyzed the methylation status of four growth regulatory genes (p16, RASSF1A, cyclinD2, 14-3-3zeta) during breast cancer progression. For this purpose invasive and noninvasive tumor cell populations as well as hyperplastic cell proliferations were isolated from a series of archival breast tissue specimens (n = 57) using laser-assisted microdissection. A new real-time polymerase chain reaction-based assay was used for the sensitive and quantitative determination of the cell-specific methylation status. We found that aberrant promoter methylation was already prevalent in pure intraductal carcinoma with different frequencies and different methylation levels for the four genes analyzed. For RASSF1A and 14-3-3zeta promoter methylation was also demonstrated in epithelial hyperplasia and intraductal papillomas. By contrast, aberrant methylation of cyclinD2 and p16 was restricted to cancerous epithelium. Increased methylation of the cyclinD2 gene was significantly associated with a higher van Nuys grade. Furthermore, when intraductal and invasive tumor cells were compared, significant quantitative changes in the methylation level were detected primarily within the cyclinD2 gene. These results demonstrate that promoter methylation is an early and frequent event in breast cancer development, but displays great quantitative and gene-specific differences, and changes in a gene-specific manner during tumor progression.