Reinhard Buettner

Institute for Pathology, Cologne, Köln, North Rhine-Westphalia, Germany

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Publications (506)2070.41 Total impact

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    ABSTRACT: This study examines the expression and the role of four-and-a-half LIM domains protein 2 (FHL2) and transforming growth factor β1 (TGF-β1) in human malignant melanoma. It is determined whether both proteins influence melanoma survival time. We analyzed the immunohistochemical staining intensities of FHL2 and TGF-β1 in normal skin and in 50 malignant melanomas with different mutation status (BRAF-V600E, NRAS codon 61 mutation, and wild type). Survival data were available for 45 cases. In melanocytes of nonneoplastic human skin, FHL2 expression was absent. In contrast, 38 (76%) of 50 melanomas showed strong cytoplasmic and partly nuclear FHL2 expression. At the invasion front, cytoplasmic TGF-β1 staining was observed in 32 (64%) of 50 melanomas, and a correlation of FHL2 and TGF-β1 staining intensities was detectable. In follow-up analyses, enhanced FHL2 and TGF-β1 staining intensities in the tumor invasion front were associated with poor survival. Enhanced FHL2 and TGF-β1 expression is correlated with poor survival in human malignant melanoma. Protumorigenic effects of autocrine TGF-β1 secretion might be exerted by induction of FHL2 expression in melanoma cells. Since melanomas treated with targeted therapies often do not show sufficient response rates, inhibition of FHL2 and/or TGF-β1 might be a promising therapeutic approach. Copyright© by the American Society for Clinical Pathology.
    American Journal of Clinical Pathology 02/2015; 143(2):248-56. · 2.88 Impact Factor
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    ABSTRACT: Small cell lung cancer and extrapulmonary small cell carcinomas are the most aggressive type of neuroendocrine carcinomas. Clinical treatment relies on conventional chemotherapy and radiotherapy; relapses are frequent. The PD-1/PD-L1/PD-L2 pathway is a major target of anti-tumour immunotherapy. Aberrant PD-L1 or PD-L2 expression may cause local immune-suppression. Here we investigated expression of PD-1 and its ligands by immunohistochemistry and RNA-seq in small cell carcinomas. PD-L1 and PD-1 protein expression were analysed in 94 clinical cases of small cell carcinomas (61 pulmonary, 33 extrapulmonary) by immunohistochemistry using two different monoclonal antibodies (5H1, E1L3N). RNA expression was profiled by RNA-seq in 43 clinical cases. None of the small cell carcinomas showed PD-L1 protein expression in tumour cells. PD-L1 and PD-1 expression was noticed in the stroma: Using immunohistochemistry, 18.5% of cases (17/92) showed PD-L1 expression in tumour-infiltrating macrophages and 48% showed PD-1 positive lymphocytes (45/94). RNA-seq showed moderate PD-L1 gene expression in 37.2% (16/43). PD-L1 was correlated with macrophage and T-cell markers. The second PD-1 ligand PD-L2 was expressed in 27.9% (12/43) and showed similar correlations. Thus, the PD-1/PD-L1 pathway seems activated in a fraction of small cell carcinomas. The carcinoma cells were negative in all cases, PD-L1 was expressed in tumour-infiltrating macrophages and was correlated with tumour-infiltrating lymphocytes. Patients with stromal PD-L1/PD-L2 expression may respond to anti-PD-1 treatment. Thus, evaluation of the composition of the tumour microenvironment should be included in clinical trials. Besides conventional immunohistochemistry, RNA-seq seems suitable for detection of PD-L1/PD-L2 expression and might prove to be more sensitive. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.
    European journal of cancer (Oxford, England: 1990) 01/2015; · 4.12 Impact Factor
  • Kirsten Kübler, Sally Heinenberg, Christian Rudlowski, Mignon-Denise Keyver-Paik, Alina Abramian, Sabine Merkelbach-Bruse, Reinhard Büttner, Walther Kuhn, Hans-Ulrich Schildhaus
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    ABSTRACT: Cervical carcinoma develops from preneoplasia by a multistep process. Although most low-grade dysplastic lesions will regress without intervention and even high-grade changes exhibit a substantial rate of regression, a small percentage of dysplasia will progress over time. Thus, indicators are needed to estimate the biological risk and to help avoid overtreatment in women who desire to preserve fertility. In addition to the classical biomarkers, PCR-ELISA-determined HPV genotype and immunohistochemically assessed p16INK4a and Ki-67 expression, cells with integrated HPV and copy number gain of TERC and c-myc were quantified in a panel of 104 benign, intraepithelial neoplastic (CIN I, II, III) and cancerous lesions using fluorescence in situ hybridization. Optimal cut-off values were calculated; Kaplan-Meier curves and a Cox proportional hazard regression model were used to evaluate prognostic signatures. The assay reliably identified HPV integration, TERC and c-myc copy number gain as determined by comparisons with established biomarkers. All biomarker levels increased with the progression of the disease. However, only c-myc copy number gain independently prognosticated a low probability of dysplastic regression. Our results suggest that c-myc plays a key role in the process of dysplastic transformation and might thus be exploited for treatment and follow-up decision-making of cervical dysplasia.
    Oncotarget 12/2014; · 6.63 Impact Factor
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    ABSTRACT: Four and a half LIM domain protein-2 (FHL2) is part of the focal adhesion structures modulating cell motility. FHL2 may translocate into the nucleus serving as a transcriptional cofactor binding several transcription factors. Overexpression of FHL2 has been linked to cancer progression in various neoplasias. The aim of the present study was to determine, whether FHL2's function as nuclear cofactor plays a prognostic role in invading tumor cells of sporadic and HNPCC-associated colorectal cancer (CRC). Immunohistochemical staining intensity of nuclear FHL2 was quantified by Remmele score analysing 47 sporadic and 42 HNPCC-associated colorectal cancers. Analysis was restricted to carcinoma cells of the tumoral invasion front. Confocal microscopy detected nuclear expression of FHL2 in colon cancer cells and absence of nuclear FHL2 signal in normal colon enterocytes. In colon cancer, nuclear FHL2 expression was predominantly observed in low-differentiated, often mucinous tumor areas. 42.55% of sporadic and 54.76% of HNPCC-associated CRC showed enhanced (Remmele score 6-12) nuclear FHL2 expression in the carcinoma cells of the tumoral advancing edge. Enhanced nuclear FHL2 expression was significantly linked to lymphatic metastasis in sporadic CRC (p=0.0197) and almost reached significance in HNPCC-associated CRC (p=0.0545). In contrast, nuclear FHL2 expression was neither associated with hematogenic metastasis in sporadic (p=0.7087) nor in HNPCC-associated colorectal cancer (p=0.3007). We recently demonstrated that enhanced nuclear FHL2 expression in tumor stroma of sporadic colon cancer is associated with lymphatic metastasis. The results of the present study indicate a synergistic effect of nuclear cofactor FHL2 in tumor cells as well as in peritumoral stroma cells promoting lymphatic metastasis in sporadic CRC. As HNPCC-associated tumors did not show a significant association between tumoral nuclear FHL2 expression and lymphatic metastasis we speculate, that the intensive lymphocytic immune response in HNPCC precludes a direct contact of tumor cells and stromal cells resulting in reduced lymphatic spread. Copyright © 2014 Elsevier GmbH. All rights reserved.
    Pathology - Research and Practice 12/2014; · 1.56 Impact Factor
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    ABSTRACT: Purpose: MET is a potential therapeutic target in lung cancer and both MET tyrosine kinase inhibitors and monoclonal antibodies have entered clinical trials. MET signaling can be activated by various mechanisms including gene amplification. In this study we aimed to investigate MET amplification status in adeno- and squamous cell carcinomas of the lung. We propose clearly defined amplification scores and provide epidemiologic data on MET amplification in lung cancer. Experimental design: We evaluated the prevalence of increased MET gene copy numbers in 693 treatment-naïve cancers by fluorescence in situ hybridization, defined clear cut-off criteria, and correlated FISH results to MET immunohistochemistry. Results: Two thirds (67%) of lung cancers do not have gains in MET gene copy numbers whereas 3% show a clear-cut high-level amplification (MET/centromer7 ratio ≥2.0 or average gene copy number per nucleus ≥6.0 or ≥10% of tumor cells containing ≥15 MET copies). The remaining cases can be subdivided into intermediate (6%) and low level gains (24%). Importantly, MET amplifications occur at equal frequencies in squamous and adenocarcinomas without or with EGFR or KRAS mutations. Conclusion: MET amplification is not a mutually exclusive genetic event in therapy-naïve NSCLC. Our data suggest that it might be useful to determine MET amplification a) before EGFR inhibitor treatment to identify possible primary resistance to anti-EGFR treatment, and b) to select cases that harbor KRAS mutations additionally to MET amplification and, thus, may not benefit from MET inhibition. Furthermore, our study provides comprehensive epidemiologic data for upcoming trials with various MET inhibitors. Copyright © 2014, American Association for Cancer Research.
    Clinical Cancer Research 12/2014; · 8.19 Impact Factor
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    ABSTRACT: Pilomatricoma is a tumour derived from hair matrix cells, which shows progressive keratin expression. Tumorigenesis is frequently associated with activating mutations in β-catenin gene inducing nuclear expression of β-catenin protein. The present study analysed the role of transforming growth factor-β1 (TGF-β1) and four-and-a-half LIM domain protein 2 (FHL2) in pilomatricoma in synopsis with their expression patterns in human anagen hair. Human anagen hair showed TGF-β1 and nuclear FHL2 expression in the outer root sheath layer separated from nuclear β-catenin staining, which was observed in cells of matrix and inner root sheath layers. Correspondingly, 41 out of 50 pilomatricomas showed co-labelling of TGF-β1 and nuclear FHL2 in tumour cells, which mostly lacked nuclear β-catenin expression. Tumoural proliferation (ki67) was associated with nuclear β-catenin staining but not with expression of nuclear FHL2. In early pilomatricomas, TGF-β1 expression was observed in few peripheral tumour cells showing absent or faint nuclear FHL2 co-staining. TGF-β1 expression extended in growing tumours going along with strong nuclear FHL2 co-labelling as well as progressive keratin 14 and keratin 1 expression. In vitro, cultured human keratinocytes showed weak to marked autocrine TGF-β1 expression; in case of enhanced TGF-β1 expression associated with keratin 10 staining. TGF-β1-treatment of cultured human keratinocytes induced nuclear and cytoplasmatic FHL2 staining as well as keratin 14 staining. Accordingly, siRNA-mediated FHL2 knockdown of TGF-β1-stimulated keratinocytes reduced keratin 14 staining. In conclusion, tumoural TGF-β1 secretion seems to induce nuclear translocation of co-factor FHL2 mediating progressive keratin expression in pilomatricoma.
    Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin 12/2014; · 2.56 Impact Factor
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    ABSTRACT: Background: Somatic mutations of the PIK3CA gene have been described in non-small cell lung cancer (NSCLC), but limited data is available on their biological relevance. This study was performed to characterize PIK3CA-mutated NSCLC clinically and genetically. Patients and methods: Tumor tissue collected consecutively from 1144 NSCLC patients within a molecular screening network between March 2010 and March 2012 was analyzed for PIK3CA mutations using dideoxy-sequencing and next-generation sequencing (NGS). Clinical, pathological, and genetic characteristics of PIK3CA-mutated patients are described and compared with a control group of PIK3CA-wildtype patients. Results: Among the total cohort of 1144 patients we identified 42 (3.7%) patients with PIK3CA mutations in exon 9 and exon 20. These mutations were found with a higher frequency in sqamous cell carcinoma (8.9%) compared to adenocarcinoma (2.9%, p<0.001). The most common PIK3CA mutation was exon 9 E545K. The majority of patients (57.1%) had additional oncogenic driver aberrations. With the exception of EGFR-mutated patients, non of the genetically defined subgroups in this cohort had a significantly better median overall survival. Further, PIK3CA-mutated patients had a significantly higher incidence of malignancy prior to lung cancer (p<0.001). Conclusion: PIK3CA-mutated NSCLC represents a clinically and genetically heterogeneous subgroup in adenocarcinomas as well as in squamous cell carcinomas with a higher prevalence of these mutations in sqamous cell carcinoma. PIK3CA mutations have no negative impact on survival after surgery or systemic therapy. However, PIK3CA mutated lung cancer frequently develops in patients with prior malignancies.
    Oncotarget 11/2014; · 6.63 Impact Factor
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    ABSTRACT: Lynch syndrome is the most frequent hereditary cancer syndrome, accounting for approximately 3-5 % of all colorectal cancers. In addition, it is the most frequent predisposing hereditary cause of endometrial cancer and is also associated with gastric cancer, ovarian cancer, cancer of the urinary tract as well as several other cancers. In clinical practise Lynch syndrome is frequently not detected and many clinicians admit uncertainties regarding diagnostic procedures. Also, counselling of patients is considered difficult regarding therapeutic - especially prophylactic surgical and chemopreventive options and recommendations. Based on a review of available literature we discuss optimized strategies for improved detection of suspected Lynch syndrome patients. The aim of this review is to establish a clinical algorithm of how to proceed on a diagnostic level and to discuss surgical options at the time of a colorectal cancer. In order to identify patients with Lynch syndrome, family history should be ascertained and evaluated in regards to fulfilment of the Amsterdam-II- and/or the revised Bethesda criteria. Subsequently immunohistochemical staining for the mismatch-repair-genes, BRAF testing for MLH1 loss of expression, as well as testing for microsatellite instability in some, followed by genetic counselling and mutation analysis when indicated, is recommended. Pathological identification of suspected Lynch syndrome is readily feasible and straightforward. However, the need of performing these analyses in the tumor biopsy at the time of (gastroenterological) diagnosis of CRC neoplasia is essential, in order to offer patients the option of a prophylactically extended surgery and - as recommended in the German S3 guidelines - to discuss the option of a merely prophylactical hysterectomy and oophorectomy (if postmenopausal) in women. Close cooperation between gastroenterologists, pathologists and surgeons is warranted, so that patients may benefit from options of extended or prophylactically extended surgery at the time of diagnosis of a colorectal primary. Patients nowadays must be involved in informed decision-making regarding prophylactic or extended prophylactic surgery at the time of a colorectal primary. To date, however, limitations in daily clinical practise, the failure to assess family history and the lack of awareness of this important hereditary syndrome is the major asset leading to severe underdiagnosis and putting to risk the indexpatients themselves and their families to (metachronous) CRC and the associated extracolonic cancers. If at all tumors of patients fulfilling Bethesda criteria will be analysed for MSI in the surgical specimen and therefore Lynch syndrome patients are not given the opportunity to opt for extended surgery. In clinical experience the postoperative MSI-analysis is inconsistently performed - even if the Bethesda criteria are fulfilled - and in case of suspected Lynch syndrome genetically counselling is not consistently recommended. Therefore affected cancer patients are left unaware of their increased genetic risk and in average 3 high-risk gene carriers per family miss the opportunity to actively engage in the recommended screening program.
    Zentralblatt fur Chirurgie, Supplement 11/2014;
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    ABSTRACT: Neoadjuvant multimodality treatment is frequently applied to improve the poor prognosis of locally advanced adenocarcinomas of the gastroesophageal junction. This study aimed to asses if serum microRNA profiles are useable as response indicators in this therapeutic setting.Fifty patients with locally advanced adenocarcinomas of the gastroesophageal junction were included in the study. All patients received neoadjuvant therapy and subsequently underwent surgical resection. Histomorphologic regression was defined as major histopathological response when resected specimens contained less than 10% vital residual tumor cells. Circulating RNA was isolated from pre-therapeutic/post-neoadjuvant blood serum samples. RNA from 9 patients was applied to PCR microarray analyses Based on these findings possible predictive miRNA markers were validated by quantitative RT-PCR analyses.Depending on the histomorphologic regression, a differential serum microRNA profile was identified by microarray analyses. Based on the divergent miRNA pattern, miR-21, miR-192, miR-222, miR-302c, miR-381 and miR-549 were selected for further validation. During neoadjuvant therapy, there was a significant increase of miR 222 and miR-549. Although on an expanded patient cohort, the six microRNAs could not be validated as markers for therapy response, there was a significant correlation between a high miR-192 and miR-222 expression with a high T-category as well as miR-302c and miR-222 expression significantly correlated with overall survival.Comprehensive miRNA profiling showed a differential microRNA expression pattern depending on the histomorphologic regression in the multimodality therapy of locally advanced adenocarcinomas of the gastroesophageal junction. Moreover, using single RT-PCR analyses a prognostic impact of miR-222 and miR-302c was detected. This article is protected by copyright. All rights reserved.
    International Journal of Cancer 11/2014; · 6.20 Impact Factor
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    ABSTRACT: Detection of anaplastic lymphoma kinase (ALK)-gene rearrangements in non-small-cell lung cancer (NSCLC) is mainly performed by fluorescence in-situ hybridization (FISH). The question was raised if FISH might be replaced by immunohistochemistry (IHC) in a reliable and reproducible manner across different laboratories. After calibration of the staining instruments and training of the observers to binary interpretation (positive versus negative), 15 NSCLC were independently tested for ALK protein expression by IHC only in a multicenter setting (16 institutes). Each laboratory utilized the VENTANA ALK-D5F3 IHC assay. As demonstrated by FISH the samples displayed unequivocal ALK break-positivity (6×) and negativity (7×), as well as ALK positive-"borderline" character (2×), which is challenging for FISH diagnosis and thus was RT-PCR-confirmed. All seven ALK FISH-negative cases were homogenously scored as ALK-IHC negative. All 16 participants scored the two ALK positive-"borderline" samples as unequivocally positive according to their protein expression. Concordant IHC interpretation was also noticed in four of six unequivocal ALK break positive cases. In two of six some observers described a weak/heterogeneous ALK-IHC staining. This would have resulted in a subsequent ALK-testing (FISH/PCR) in a routine diagnostic setting. This so-called "ALK-Harmonization-Study" shows for the first time that predictive semiquantitative IHC reveals reliable and reproducible results across several labs when methodology and interpretation are strictly defined and the pathologists are uniquely trained. The application of validated ALK IHC assays and its comparison to ALK-FISH is highly needed in future clinical trials. This might answer the question if ALK-IHC cannot only serve as a prescreening tool, but as a stand-alone test at least in cases displaying an unequivocally staining pattern as well as an alternative predictive test in samples with reduced FISH interpretability.
    Journal of Thoracic Oncology 11/2014; 9(11):1685-1692. · 5.80 Impact Factor
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    ABSTRACT: Context: Primary macronodular adrenal hyperplasia (PMAH) is a rare cause of Cushing's syndrome (CS), which may present in the context of different familial multitumor syndromes. Heterozygous inactivating germline mutations of armadillo repeat containing 5 (ARMC5) have very recently been described as cause for sporadic PMAH. Whether this genetic condition also causes familial PMAH in association with other neoplasias is unclear. Objective: The aim of the present study was to delineate the molecular cause in a large family with PMAH and other neoplasias. Patients and Methods: Whole genome sequencing and comprehensive clinical and biochemical phenotyping was performed in members of a PMAH affected family. Nodules derived from adrenal surgery and pancreatic and meningeal tumor tissue were analysed for accompanying somatic mutations in the identified target genes. Results: PMAH presenting either as overt or subclinical CS was accompanied by a heterozygous germline mutation in ARMC5 (p.A110fs*9) located on chromosome 16. Analysis of tumor tissue showed different somatic ARMC5 mutations in adrenal nodules supporting a "second hit" hypothesis with inactivation of a tumor suppressor gene. A damaging somatic ARMC5 mutation was also found in a concomitant meningioma (p.R502fs) but not in a pancreatic tumor suggesting biallelic inactivation of ARMC5 as causal also for the intracranial meningioma. Conclusions: Our analysis further confirms inherited inactivating ARMC5 mutations as a cause of familial PMAH and suggests an additional role for the development of concomitant intracranial meningiomas.
    The Journal of Clinical Endocrinology and Metabolism 10/2014; · 6.31 Impact Factor
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    ABSTRACT: In this study, immunohistochemical staining pattern of cytochrome c oxidase subunit 1 (CCO1) was investigated in the differentiation of renal oncocytoma (RO) from eosinophilic (EoC) and classic chromophobe renal cell carcinoma (ChRCC). A feature found in ChRCC/EoC but not in RO is the predominance of a perinuclear halo when stained for CCO1. In a cohort of 103 mixed cases including 44 RO, 37 classic ChRCC and 22 EoC, the diagnosis based on this immunohistochemical feature alone was consistent with the previous routine diagnosis in 95.7%. We reached 100% specificity and 81.4% sensitivity of this pattern in ChRCC. Specificity for RO was 93.2% and sensitivity correspondingly 95.5%. We propose a novel and easily interpretable immunohistochemical method for the discrimination of benign RO from certain subtypes of malignant ChRCC. Because of strong similarity in morphology of the 2 entities the diagnosis often cannot be made based on standard histopathology alone. The study describes for the first time the formation of a perinuclear halo in CCO1 immunohistochemistry as a highly specific marker for the diagnosis of ChRCC. We think this method can be a strong amendment for routine diagnostics in renal cell carcinoma.
    Applied immunohistochemistry & molecular morphology: AIMM / official publication of the Society for Applied Immunohistochemistry 09/2014; · 2.06 Impact Factor
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    ABSTRACT: Crizotinib, a selective tyrosine kinase inhibitor (TKI), shows marked activity in patients whose lung cancers harbor fusions in the gene encoding anaplastic lymphoma receptor tyrosine kinase (ALK), but its efficacy is limited by variable primary responses and acquired resistance. In work arising from the clinical observation of a patient with ALK fusion-positive lung cancer who had an exceptional response to an insulin-like growth factor 1 receptor (IGF-1R)-specific antibody, we define a therapeutic synergism between ALK and IGF-1R inhibitors. Similar to IGF-1R, ALK fusion proteins bind to the adaptor insulin receptor substrate 1 (IRS-1), and IRS-1 knockdown enhances the antitumor effects of ALK inhibitors. In models of ALK TKI resistance, the IGF-1R pathway is activated, and combined ALK and IGF-1R inhibition improves therapeutic efficacy. Consistent with this finding, the levels of IGF-1R and IRS-1 are increased in biopsy samples from patients progressing on crizotinib monotherapy. Collectively these data support a role for the IGF-1R-IRS-1 pathway in both ALK TKI-sensitive and ALK TKI-resistant states and provide a biological rationale for further clinical development of dual ALK and IGF-1R inhibitors.
    Nature medicine 09/2014; 20(9):1027-34. · 28.05 Impact Factor
  • Dr. A. Schultheis, J. Wolf, R. Büttner
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    ABSTRACT: Die Tumorimmuntherapie hat in den letzten Jahren rasante Fortschritte gemacht. So lieferten Studien zur Behandlung des Bronchialkarzinoms mit „cancer vaccines“ wie dem melanomassoziierten Antigen A3 (MAGE-A3) und liposomalem BLP25 vielversprechende Ergebnisse in den Stadien IB/II und III des nichtkleinzelligen Bronchialkarzinoms. Immunmodulatorische Agenzien wie Talactoferrin oder Ipilimumab scheinen v. a. in Verbindung mit einer platinbasierten Chemotherapie zu wirken, was andeutet, dass insbesondere die Kombination von Immuntherapeutika, konventioneller Chemotherapie und tumorspezifischen, zielgerichteten Agenzien das größte therapeutische Zukunftspotenzial besitzt. Das genaue Verständnis der Interaktion zwischen Tumor und Immunsystem bleibt essenziell für die Identifizierung potenzieller Biomarker. Im Idealfall ermöglicht es auch im Bereich der Immuntherapie die Entwicklung gezielter Ansätze.
    Der Internist 09/2014; 54(2). · 0.27 Impact Factor
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    ABSTRACT: The determination of the mutation status of many samples is of major interest for research and diagnostics of cancer and has become feasible through Next Generation Sequencing (NGS). The principle of high throughput mutaton analyses opens up the way to personalized therapeuticall strategies, addressing the patientsíndividual risk profiles or therapy potential can by determination of their cancer associated mutation status. The different NGS procedures include a wide variety of sample library preparation protocols that accelerate the sample processing by the opportunity of sample and target multiplexing. However, the sample processing steps consist of many single time-consuming, error-prone pipetting and incubation steps. Therefore, in the present study we aimed the automation of NGS sample library preparation for a MiSeq (Illumina) multiplex target sequencing assay of cancer diagnostics.
    23rd Biennial Congress of the European Association For Cancer Research (EACR), Munich, Germany; 07/2014
  • Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin 07/2014; · 2.56 Impact Factor
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    ABSTRACT: Recurrent mutations in tumor relevant genes have been identified and proposed as new indicators of prognostic subsets in patients with chronic lymphocytic leukemia (CLL). Next generation sequencing (NGS) technologies allow the simultaneous analysis of genomic mutations in numerous target regions in one single experiment and are able to reach a higher sensitivity than conventional sequencing technologies. We aimed to establish and validate a NGS panel of distinct genes, known to be potentially mutated and/or targeted in CLL, which can be run as a diagnostic screening assay in a high-throughput fashion for clinical research purposes and potential routine applications in the future. 2 CLL gene panels targeting the most disease related genes in CLL, i.e. ATM, MYD88, NOTCH1, SF3B1, and TP53, were designed and specific primers obtained for complete coding exon or hot spot regions of each gene. Using 4 primer pools resulting in 338 amplicons, a PCR-based multiplex technology was applied to prepare target-enriched DNA libraries from purified B-cell DNA from 136 CLL patients. Samples were sequenced using a MiSeq instrument (Illumina). Conventional Sanger sequencing served as a reference technology. Additionally, 2 dilution series of cell line DNA with known TP53 or ATM mutation were used as a positive control and for sensitivity determination. Alignment and variant calling was performed using an internal algorithm. Data was visually verified with integrative genomic viewer. We obtained >10 million reads per run covering 338 amplicons from 15 genes of interest. The median coverage per amplicon was approximately 2,000 reads (range: ≈15 to >25,000). 3 genes (ATM, NOTCH1, SF3B1) demonstrated regions of low amplicon representation, which had to be discarded from evaluation during quality assessment. Mutation hot spots were detected in ATM, NOTCH1, TP53, SF3B1, XPO1 and FBXW7, and are currently undergoing verification by Sanger sequencing. Series of mutated cell line DNA followed a linear relationship with increasing amounts of tumor DNA and sensitivity between 5-10%. We successfully established a CLL-dedicated sequencing panel based on one-step multiplexed amplification chemistry and applicable to primary CLL sample material and an Illumina MiSeq sequencing platform. Current sequencing results demonstrate overall acceptable gene coverage for diagnostic purposes in the majority of selected target regions, even though underrepresented amplicons/gene regions occur.
    98. Jahrestagung der Deutschen Gesellschaft für Pathologie, Berlin; 06/2014
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    ABSTRACT: Exposure to environmental cues such as cold or nutritional imbalance requires white adipose tissue (WAT) to adapt its metabolism to ensure survival. Metabolic plasticity is prominently exemplified by the enhancement of mitochondrial biogenesis in WAT in response to cold exposure or β3-adrenergic stimulation. Here we show that these stimuli increase the levels of lysine-specific demethylase 1 (LSD1) in WAT of mice and that elevated LSD1 levels induce mitochondrial activity. Genome-wide binding and transcriptome analyses demonstrate that LSD1 directly stimulates the expression of genes involved in oxidative phosphorylation (OXPHOS) in cooperation with nuclear respiratory factor 1 (Nrf1). In transgenic (Tg) mice, increased levels of LSD1 promote in a cell-autonomous manner the formation of islets of metabolically active brown-like adipocytes in WAT. Notably, Tg mice show limited weight gain when fed a high-fat diet. Taken together, our data establish LSD1 as a key regulator of OXPHOS and metabolic adaptation in WAT.
    Nature Communications 06/2014; 5:4093. · 10.74 Impact Factor
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    Michael Kloth, Reinhard Buettner
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    ABSTRACT: Traditionally, tumors are classified by histopathological criteria, i.e., based on their specific morphological appearances. Consequently, current therapeutic decisions in oncology are strongly influenced by histology rather than underlying molecular or genomic aberrations. The increase of information on molecular changes however, enabled by the Human Genome Project and the International Cancer Genome Consortium as well as the manifold advances in molecular biology and high-throughput sequencing techniques, inaugurated the integration of genomic information into disease classification. Furthermore, in some cases it became evident that former classifications needed major revision and adaption. Such adaptations are often required by understanding the pathogenesis of a disease from a specific molecular alteration, using this molecular driver for targeted and highly effective therapies. Altogether, reclassifications should lead to higher information content of the underlying diagnoses, reflecting their molecular pathogenesis and resulting in optimized and individual therapeutic decisions. The objective of this article is to summarize some particularly important examples of genome-based classification approaches and associated therapeutic concepts. In addition to reviewing disease specific markers, we focus on potentially therapeutic or predictive markers and the relevance of molecular diagnostics in disease monitoring.
    Genes. 06/2014; 5(2):444-459.
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    ABSTRACT: The LIM-only protein FHL2, also known as DRAL or SLIM3, has a function in fine-tuning multiple physiological processes. FHL2 is expressed in the vessel wall in smooth muscle cells (SMCs) and endothelial cells and conflicting data have been reported on the regulatory function of FHL2 in SMC phenotype transition. At present the function of FHL2 in SMCs in vascular injury is unknown. Therefore, we studied the role of FHL2 in SMC-rich lesion formation. In response to carotid artery ligation FHL2-deficient (FHL2-KO) mice showed accelerated lesion formation with enhanced Ki67 expression compared with wild-type (WT)-mice. Consistent with these findings, cultured SMCs from FHL2-KO mice showed increased proliferation through enhanced phosphorylation of extracellular-regulated kinase-1/2 (ERK1/2) and induction of CyclinD1 expression. Overexpression of FHL2 in SMCs inhibited CyclinD1 expression and CyclinD1-knockdown blocked the enhanced proliferation of FHL2-KO SMCs. We also observed increased CyclinD1 promoter activity in FHL2-KO SMCs, which was reduced upon ERK1/2 inhibition. Furthermore, FHL2-KO SMCs showed enhanced migration compared with WT SMCs. In conclusion, FHL2 deficiency in mice results in exacerbated SMC-rich lesion formation involving increased proliferation and migration of SMCs via enhanced activation of the ERK1/2-CyclinD1 signaling pathway.
    PLoS ONE 04/2014; 9(4):e94931. · 3.53 Impact Factor

Publication Stats

11k Citations
2,070.41 Total Impact Points

Institutions

  • 2011–2014
    • Institute for Pathology, Cologne
      Köln, North Rhine-Westphalia, Germany
    • Mount Sinai School of Medicine
      • Department of Pediatrics
      Manhattan, NY, United States
    • Bioscientia - Institut für Medizinische Diagnostik GmbH
      Frankfurt, Hesse, Germany
  • 2010–2014
    • University of Cologne
      • • Institute of Pathology
      • • Faculty of Medicine
      Köln, North Rhine-Westphalia, Germany
    • Medical University of Varna
      Odessos, Varna, Bulgaria
  • 2013
    • Centrum für Integrierte Onkologie
      Köln, North Rhine-Westphalia, Germany
  • 1998–2013
    • University of Bonn
      • • Institute of Human Genetics
      • • Institut für Pathologie
      • • Department of Neurobiology
      Bonn, North Rhine-Westphalia, Germany
  • 2003–2012
    • University of Bonn - Medical Center
      Bonn, North Rhine-Westphalia, Germany
  • 1999–2012
    • University of Freiburg
      • Institute of Pharmaceutical Sciences
      Freiburg, Lower Saxony, Germany
  • 2008–2011
    • University Hospital Essen
      • Institut für Pharmakogenetik
      Essen, North Rhine-Westphalia, Germany
    • University of Wuerzburg
      Würzburg, Bavaria, Germany
    • Targos Molecular Pathology GmbH
      Cassel, Hesse, Germany
    • German Cancer Research Center
      Heidelburg, Baden-Württemberg, Germany
    • Erasmus MC
      • Department of Pathology
      Rotterdam, South Holland, Netherlands
  • 2002–2009
    • Sigmund-Freud-Institut
      Frankfurt, Hesse, Germany
  • 2004–2008
    • Universitätsmedizin Göttingen
      • Department of General, Visceral and Child Surgery
      Göttingen, Lower Saxony, Germany
    • Klinikum Kassel
      Cassel, Hesse, Germany
  • 2000–2008
    • University Hospital RWTH Aachen
      • Division of Internal Medicine
      Aachen, North Rhine-Westphalia, Germany
    • University of California, San Francisco
      • Division of Hospital Medicine
      San Francisco, CA, United States
  • 2007
    • Universitätsklinikum Münster
      Muenster, North Rhine-Westphalia, Germany
  • 2006
    • University of Pretoria
      Πρετόρια/Πόλη του Ακρωτηρίου, Gauteng, South Africa
    • Heidelberg University
      Tiffin, Ohio, United States
  • 2000–2006
    • RWTH Aachen University
      • • Institute of Human Genetics
      • • Institute of Semiconductor Electronics
      • • Institute of Pathology
      Aachen, North Rhine-Westphalia, Germany
  • 1992–2006
    • Universität Regensburg
      • • Lehrstuhl für Innere Medizin I
      • • Institut für Psychologie
      Ratisbon, Bavaria, Germany
  • 2005
    • Università degli Studi di Messina
      • Dipartimento di Neuroscienze
      Messina, Sicily, Italy
  • 2000–2005
    • Max Planck Institute of Biochemistry
      • Department of Molecular Medicine
      München, Bavaria, Germany
  • 2003–2004
    • Universität Heidelberg
      • Gynecology and Obstetrics Polyclinic
      Heidelberg, Baden-Wuerttemberg, Germany
  • 1993–2001
    • University Hospital Regensburg
      • • Institut für Pathologie
      • • Klinik für Dermatologie
      Ratisbon, Bavaria, Germany
  • 1991–1994
    • University of Texas MD Anderson Cancer Center
      Houston, Texas, United States