Kerstin Becker

Three-Dimensional Cell Growth Confers Radioresistance by Chromatin Density Modification

Cell shape and architecture are determined by cell-extracellular matrix interactions and have profound effects on cellular behavior, chromatin condensation, and tumor cell resistance to radiotherapy and chemotherapy. To evaluate the role of chromatin condensation for radiation cell survival, tumor cells grown in three-dimensional (3D) cell cultures as xenografts and monolayer cell cultures were compared. Here, we show that increased levels of heterochromatin in 3D cell cultures characterized by histone H3 deacetylation and induced heterochromatin protein 1alpha expression result in increased radiation survival and reduced numbers of DNA double strand breaks (DSB) and lethal chromosome aberrations. Intriguingly, euchromatin to heterochromatin-associated DSBs were equally distributed in irradiated 3D cell cultures and xenograft tumors, whereas irradiated monolayer cultures showed a 2:1 euchromatin to heterochromatin DSB distribution. Depletion of histone deacetylase (HDAC) 1/2/4 or application of the class I/II pharmacologic HDAC inhibitor LBH589 induced moderate or strong chromatin decondensation, respectively, which was translated into cell line-dependent radiosensitization and, in case of LBH589, into an increased number of DSBs. Neither growth conditions nor HDAC modifications significantly affected the radiation-induced phosphorylation of the important DNA repair protein ataxia telangiectasia mutated. Our data show an interrelation between cell morphology and cellular radiosensitivity essentially based on chromatin organization. Understanding the molecular mechanisms by which chromatin structure influences the processing of radiation-induced DNA lesions is of high relevance for normal tissue protection and optimization of cancer therapy.

De novo microdeletions of chromosome 6q14.1-q14.3 and 6q12.1-q14.1 in two patients with intellectual disability - further delineation of the 6q14 microdeletion syndrome and review of the literature.

Interstitial 6q deletions can cause a variable phenotype depending on the size and location of the deletion. 6q14 deletions have been associated with intellectual disability and a distinct pattern of minor anomalies, including upslanted palpebral fissures with epicanthal folds, a short nose with broad nasal tip, anteverted nares, long philtrum, and thin upper lip. In this study we describe two patients with overlapping 6q14 deletions presenting with developmental delay and characteristic dysmorphism. Molecular karyotyping using array CGH analysis revealed a de novo 8.9 Mb deletion at 6q14.1-q14.3 and a de novo 11.3 Mb deletion at 6q12.1-6q14.1, respectively. We provide a review of the clinical features of twelve other patients with 6q14 deletions detected by array CGH analysis. By assessing all reported data we could not identify a single common region of deletion. Possible candidate genes in 6q14 for intellectual disability might be FILIP1, MYO6, HTR1B, and SNX14.

Comprehensive molecular characterization of multifocal glioblastoma proves its monoclonal origin and reveals novel insights into clonal evolution and heterogeneity of glioblastomas

Abstract Background. The evolution of primary glioblastoma (GBM) is poorly understood. Multifocal GBM (ie, multiple synchronous lesions in one patient) could elucidate GBM development. Methods. We present the first comprehensive study of 12 GBM foci from 6 patients using array-CGH, spectral karyotyping, gene expression arrays, and next-generation sequencing. Results. Multifocal GBMs genetically resemble primary GBMs. Comparing foci from the same patient proved their monoclonal origin. All tumors harbored alterations in the 3 GBM core pathways: RTK/PI3K, p53, and RB regulatory pathways with aberrations of EGFR and CDKN2A/B in all (100%) patients. This unexpected high frequency reflects a distinct genetic signature of multifocal GBMs and might account for their highly malignant and invasive phenotype. Surprisingly, the types of mutations in these genes/pathways were different in tumor foci from the same patients. For example, we found distinct mutations/aberrations in PTEN, TP53, EGFR, and CDKN2A/B, which therefore must have occurred independently and late during tumor development. We also identified chromothripsis as a late event and in tumors with wild-type TP53. Only 2 events were found to be early in all patients: single copy loss of PTEN and TERT promoter point mutations. Conclusions. Multifocal GBMs develop through parallel genetic evolution. The high frequency of alterations in 3 main pathways suggests that these are essential steps in GBM evolution; however, their late occurrence indicates that they are not founder events but rather subclonal drivers. This might account for the marked genetic heterogeneity seen in primary GBM and therefore has important implications for GBM therapy.

Molecular and clinical analyses of 16q24.1 duplications involving FOXF1 identify an evolutionarily unstable large minisatellite

BackgroundPoint mutations or genomic deletions of FOXF1 result in a lethal developmental lung disease Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins. However, the clinical consequences of the constitutively increased dosage of FOXF1 are unknown.MethodsCopy-number variations and their parental origin were identified using a combination of array CGH, long-range PCR, DNA sequencing, and microsatellite analyses. Minisatellite sequences across different species were compared using a gready clustering algorithm and genome-wide analysis of the distribution of minisatellite sequences was performed using R statistical software.ResultsWe report four unrelated families with 16q24.1 duplications encompassing entire FOXF1. In a 4-year-old boy with speech delay and a café-au-lait macule, we identified an ~15 kb 16q24.1 duplication inherited from the reportedly healthy father, in addition to a de novo ~1.09 Mb mosaic 17q11.2 NF1 deletion. In a 13-year-old patient with autism and mood disorder, we found an ~0.3 Mb duplication harboring FOXF1 and an ~0.5 Mb 16q23.3 duplication, both inherited from the father with bipolar disorder. In a 47-year old patient with pyloric stenosis, mesenterium commune, and aplasia of the appendix, we identified an ~0.4 Mb duplication in 16q24.1 encompassing 16 genes including FOXF1. The patient transmitted the duplication to her daughter, who presented with similar symptoms. In a fourth patient with speech and motor delay, and borderline intellectual disability, we identified an ~1.7 Mb FOXF1 duplication adjacent to a large minisatellite. This duplication has a complex structure and arose de novo on the maternal chromosome, likely as a result of a DNA replication error initiated by the adjacent large tandem repeat. Using bioinformatic and array CGH analyses of the minisatellite, we found a large variation of its size in several different species and individuals, demonstrating both its evolutionarily instability and population polymorphism.ConclusionsOur data indicate that constitutional duplication of FOXF1 in humans is not associated with any pediatric lung abnormalities. We propose that patients with gut malrotation, pyloric or duodenal stenosis, and gall bladder agenesis should be tested for FOXF1 alterations. We suggest that instability of minisatellites greater than 1 kb can lead to structural variation due to DNA replication errors.

Germline truncating-mutations in BRCA1 and MSH6 in a patient with early onset endometrial cancer

BackgroundHereditary Breast and Ovarian Cancer Syndrome (HBOCS) and Hereditary Non-Polyposis Colorectal Cancer Syndrome (HNPCC, Lynch Syndrome) are two tumor predisposition syndromes responsible for the majority of hereditary breast and colorectal cancers. Carriers of both germline mutations in breast cancer genes BRCA1 or BRCA2 and in mismatch repair (MMR) genes MLH1, MSH2, MSH6 or PMS2 are very rare.Case presentationWe identified germline mutations in BRCA1 and in MSH6 in a patient with increased risk for HBOC diagnosed with endometrial cancer at the age of 46 years.ConclusionsAlthough carriers of mutations in both MMR and BRCA genes are rare in Caucasian populations and anamnestical and histopathological findings may guide clinicians to identify these families, both syndromes can only be diagnosed through a complete gene analysis of the respective genes.

Spectrum of genetic variants of BRCA1 and BRCA2 in a German single center study

BackgroundDetermination of mutation status of BRCA1 and BRCA2 has become part of the clinical routine. However, the spectrum of genetic variants differs between populations. The aim of this study was to deliver a comprehensive description of all detected variants.MethodsIn families fulfilling one of the German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC) criteria for genetic testing, one affected was chosen for analysis. DNA of blood lymphocytes was amplified by PCR and prescreened by DHPLC. Aberrant fragments were sequenced. All coding exons and splice sites of BRCA1 and BRCA2 were analyzed. Screening for large rearrangements in both genes was performed by MLPA.ResultsOf 523 index patients, 121 (23.1%) were found to carry a pathogenic or likely pathogenic (class 4/5) mutation. A variant of unknown significance (VUS) was detected in 73/523 patients (13.9%). Two mutations p.Gln1756Profs*74 and p.Cys61Gly comprised 42.3% (n = 33/78) of all detected pathogenic mutations in BRCA1. Most of the other mutations were unique mutations. The most frequently detected mutation in BRCA2 was p.Val1283Lys (13.9%; n = 6/43). Altogether, 101 different neutral genetic variants were counted in BRCA1 (n = 35) and in BRCA2 (n = 66).ConclusionThe two most frequently detected mutations are founder mutations in Poland and Czech Republic. More similarities seem to be shared with our direct neighbor countries compared to other European countries. For comparison of the extended genotype, a shared database is needed.

BRCA1/2 missense mutations and the value of in-silico analyses

INTRODUCTION The clinical implications of genetic variants in BRCA1/2 in healthy and affected individuals are considerable. Variant interpretation, however, is especially challenging for missense variants. The majority of them are classified as variants of unknown clinical significance (VUS). Computational (in-silico) predictive programs are easy to access, but represent only one tool out of a wide range of complemental approaches to classify VUS. With this single-center study, we aimed to evaluate the impact of in-silico analyses in a spectrum of different BRCA1/2 missense variants. METHODS We conducted mutation analysis of BRCA1/2 in 523 index patients with suspected hereditary breast and ovarian cancer (HBOC). Classification of the genetic variants was performed according to the German Consortium (GC)-HBOC database. Additionally, all missense variants were classified by the following three in-silico prediction tools: SIFT, Mutation Taster (MT2) and PolyPhen2 (PPH2). RESULTS Overall 201 different variants, 68 of which constituted missense variants were ranked as pathogenic, neutral, or unknown. The classification of missense variants by in-silico tools resulted in a higher amount of pathogenic mutations (25% vs. 13.2%) compared to the GC-HBOC-classification. Altogether, more than fifty percent (38/68, 55.9%) of missense variants were ranked differently. Sensitivity of in-silico-tools for mutation prediction was 88.9% (PPH2), 100% (SIFT) and 100% (MT2). CONCLUSION We found a relevant discrepancy in variant classification by using in-silico prediction tools, resulting in potential overestimation and/or underestimation of cancer risk. More reliable, notably gene-specific, prediction tools and functional tests are needed to improve clinical counseling.

Thrombocytopenia Microcephaly Syndrome - a novel phenotype associated with ACTB mutations

Until recently missense germ-line mutations in ACTB, encoding the ubiquitously expressed β-cytoplasmic actin (CYA), were exclusively associated with Baraitser-Winter Cerebrofrontofacial syndrome (BWCFF), a complex developmental disorder1,2. Here, we report six patients with previously undescribed heterozygous variants clustered in the 3’-coding region of ACTB. These patients present with clinical features different from BWCFF, including thrombocytopenia, microcephaly, and mild developmental disability. Patient derived cells are morphologically and functionally distinct from controls. Assessment of cytoskeletal constituents identified a discrete filament population altered in these cells, which comprises force generating and transmitting actin binding proteins (ABP) known to be associated with thrombocytopenia3–8. In silico modelling and molecular dynamics (MD)-simulations support altered interactions between these ABP and mutant β-CYA. Our results describe a new clinical syndrome associated with ACTB mutations with a distinct genotype-phenotype correlation, identify a cytoskeletal protein interaction network crucial for thrombopoiesis, and provide support for the hypomorphic nature of these actinopathy mutations.

Abstract 3415: Genetic analysis of multifocal glioblastoma multiforme foci points to their monoclonal evolution and highlights early and late events in their development

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Glioblastoma (GBM) is the most common and malignant type of brain tumor in adults with an average survival time of less than a year. A hallmark of GBM is its infiltrating growth hampering successful surgical treatment and precluding curative therapy. Multifocal GBMs - multiple synchronous lesions in the same patient - represent approximately 8-10% of all gliomas and generally have a poor prognosis. There are two theories as to the nature of these multifocal GBMs: they could either have originated from one main GBM or they can be independent tumors. Genetic studies of multifocal GBMs are very rare and such studies could elucidate the nature of these tumors. We have analyzed eleven tumor foci of six patients with multifocal GBMs using array comparative genome hybridization (aCGH), spectral karyotyping (SKY), and Sanger sequencing of PTEN, TP53, and IDH1/2. Array CGH analysis of ten of the tumors revealed multiple genetic aberrations. The most frequent were gain of chromosome 7 (9/10), amplification of EGFR (10/10), loss of chromosome 10 (10/10), and partial loss of chromosome 9 (7/10). SKY analysis identified multiple translocations including complex rearrangements. None of the tumors carried a mutation in IDH1/2 and they were genetically similar to primary GBMs although they exhibited unique characteristics; e.g. all carried an EGFR amplification, indicating that it is important for the multifocal phenotype. Tumor foci derived from the same patient always shared identical aberrations, proving monoclonal origin of these tumors but also exhibited additional aberrations, which were specific for each focus and therefore must have occurred later in tumor development. Moreover, our results show that events important for gliomagenesis could occur at different time points in tumor evolution: for example, the region involved in the EGFR amplification was different in both foci from three patients and identical in two foci from one patient. The same can be said of point mutations in TP53 and PTEN; TP53 mutations were identical in the different foci from one patient and different in another patient while PTEN mutations were different in the foci from two patients and similar in those of one. Interestingly, in each patient one focus always contained more aberrations than the other, highlighting the possibility that one derived from the other; this would further support the hypothesis that multifocal GBMs are of monoclonal origin and then develop independently of each other by clonal evolution. Taken together, multifocal GBM provide an excellent model for investigating tumor progression and invasion and might help to distinguish between driver and passenger alterations in GBM. Acknowledgements: This work was partially supported by a grant (MeDDrive program) of the Medizinische Fakultat Carl Gustav Carus, Technische Universitat Dresden. Citation Format: Khalil Abou-El-Ardat, Dietmar Krex, Michael Seifert, Kerstin Becker, Morten Hillmer, Sophie Eisenreich, Arleta Kasner-Frensel, Petra Freitag, Eva-Maria Gerlach, Karl Hackmann, Andreas Rump, Gabriele Schackert, Evelin Schrock, Barbara Klink. Genetic analysis of multifocal glioblastoma multiforme foci points to their monoclonal evolution and highlights early and late events in their development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3415. doi:10.1158/1538-7445.AM2014-3415