Reinhard Buettner

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

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Publications (504)1961.3 Total impact

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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 : an official journal of the American Association for Cancer Research. 12/2014;
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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.
    Virchows Archiv : an international journal of pathology. 12/2014;
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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; · 6.64 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. · 4.47 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.
    09/2014;
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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. · 27.14 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). · 0.33 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
  • Virchows Archiv : an international journal of pathology. 07/2014;
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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: Lung neuroendocrine tumors (LNETs) comprise small-cell lung cancer (SCLC), large-cell neuroendocrine tumors (LCNEC), and pulmonary carcinoids (PCA), and account for 25% of all lung cancer cases. The low 5-year survival rate of the highly aggressive LNETs (SCLC and LCNEC) combined with the lack of an effective treatment, suggest that understanding how these tumors arise and identifying therapeutic targets are unmet needs. We performed genome/exome, and transcriptome sequencing of 29 SCLC (Ref.1), 60 LCNEC, and 45 PCA to better understand their molecular origin and identify altered genes that may offer therapeutic opportunities. In contrast to SCLC and LCNEC, we found that RB1 and TP53 mutations were rare events in PCA suggesting that PCA are not early progenitor lesions of SCLC or LCNEC, but arise through independent mechanisms. Moreover, GSEA analysis showed that genes of the RB1 pathway were downregulated in SCLC but not in PCA. Our data also show that inactivation of chromatin-remodeling genes, specifically genes involved in histone methylation and subunits of the SWI/SNF complex, is sufficient to drive transformation in PCA. In a preliminary analysis of 15 LCNEC (Ref.2) we observed a predominance of mutations typical of SCLC, such as RB1, TP53, and CREBBP/EP300. In this larger series, we additionally found samples with mutations frequent in adenocarcinoma (AD) or squamous (SQ) lung cancer. We could then distinguish two well-defined groups of LCNEC: a SCLC-like group, carrying MYCL1 amplifications and mutations in both RB1 and TP53 genes; and an AD/SQ-like group, harbouring CDKN2A deletions, TTF1 amplifications, and frequent mutations in KEAP1 and STK11. Interestingly, RB1, STK11, and KEAP1 mutations happened in an almost mutually exclusive way. These data suggest that LCNEC might represent an evolutionary trunk that can branch to SCLC or AD/SQ, and also that LNETs and non-LNETs are not completely different entities. This is already suggested by the fact that one of the resistance mechanisms of EGFR-mutant adenocarcinomas to tyrosine-kinase inhibitors is through trans-differentiation to SCLC (Ref.3). Finally, we also identified new targetable driver genes in SCLC and LCNEC: FGFR1 amplifications were observed in 6% and 18% of the cases respectively; PTEN mutations were identified in 10% of the SCLC cases; and interestingly, one of the LCNEC samples (belonging to the SCLC-like group) harboured an activating RFWD2-NTRK1 fusion gene suggesting that fusions affecting NTRK1 may not only be a targetable opportunity for AD (Ref.4) but also for LCNEC and, based on the molecular similarities, also SCLC. (1) Peifer and Fernandez-Cuesta et al. Nat Genetics 2012 (2) The Clinical Lung Cancer Genome Project (CLCGP) and Network Genomic Medicine (NGM) Sci Transl Med 2013 (3) Sequist et al., Sci Transl Med 2011 (4) Vaishnavi et al. Nat Medicine 2013
    American Association for Cancer Research Annual Meeting 2014, San Diego, USA; 04/2014
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    ABSTRACT: In nearly 50% of all lung adenocarcinomas the oncogenic driver is still unknown. We performed transcriptome sequencing on 25 pan-negative lung adenocarcinomas of never smokers leading to the identification of the novel CD74-NRG1 gene fusion. In an additional cohort of 102 adenocarcinomas we could identify and validate four additional cases bearing the CD74-NRG1 fusion gene - all of the invasive mucinous subtype. We then transduced NIH-3T3 cells with the fusion and found it localized on the cell surface in FACS experiments. As we found the identical breakpoint in CD74 in our fusion as in CD74-ROS1, we hypothesized that the functional active part needs to be NRG1. (Soda et al. 2012) The NRG1 isoform III-β3 that we found is generally not expressed in lung tissue - therefore the CD74 part leads to the expresseion of NRG1 III-β3 in the lung. As Neuregulins are known interaction partners of HER receptor family members we transduced the human adenocarcinoma cell lines H322 and H1568, that are HER receptor and KRAS wildtype, with the fusion and found phosphorylation of HER2 and HER3 in the transduced cells under starvation conditions compared to empty vector controls. (Hobbs et al. 2002) This holds true as well in the patient situation in which phospho-HER3 was only found in tumors expressing the fusion (p<0.0001). We could further show that the CD74-NRG1 fusion is associated with activation of the phosphoinsitide-3-kinase pathway activation and the mitogen-activated protein (MAP) kinase pathway . As functional consequence we could observe in several human adenocarcinoma cell lines increased colony forming ability in soft-agar assays showing the CD74-NRG1 fusion being a new oncogenic driver in lung adenocarcinoma. This finding may lead to new treatment options for patients harboring this fusion gene who do not benefit from any effective treatment option up to now.
    American Association for Cancer Research Annual Meeting 2014, San Diego, USA; 04/2014
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    ABSTRACT: Recent work has identified dysfunctional Hippo signaling to be involved in maintenance and progression of various human cancers, although data on clear cell renal cell carcinoma (ccRCC) have been limited. Here, we provide evidence implicating aberrant Hippo signaling in ccRCC proliferation, invasiveness, and metastatic potential. Nuclear overexpression of the Hippo target Yes-associated protein (YAP) was found in a subset of patients with ccRCC. Immunostaining was particularly prominent at the tumor margins and highlighted neoplastic cells invading the tumor-adjacent stroma. Short hairpin RNA-mediated knockdown of YAP significantly inhibited proliferation, migration, and anchorage-independent growth of ccRCC cells in soft agar and led to significantly reduced murine xenograft growth. Microarray analysis of YAP knockdown versus mock-transduced ccRCC cells revealed down-regulation of endothelin 1, endothelin 2, cysteine-rich, angiogenic inducer, 61 (CYR61), and c-Myc in ccRCC cells as well as up-regulation of the cell adhesion molecule cadherin 6. Signaling pathway impact analysis revealed activation of the p53 signaling and cell cycle pathways as well as inhibition of mitogen-activated protein kinase signaling on YAP down-regulation. Our data suggest CYR61 and c-Myc as well as signaling through the endothelin axis as bona fide downstream effectors of YAP and establish aberrant Hippo signaling as a potential therapeutic target in ccRCC.
    Translational oncology 04/2014; 7(2):309-21. · 3.40 Impact Factor
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    ABSTRACT: Hamartin (TSC1) and tuberin (TSC2), encoded by the tuberous sclerosis complex (TSC) genes, form a tumor-suppressor heterodimer which is implicated in PI3K-Akt signaling and acts as a functional inhibitor of the mammalian target of rapamycin (mTOR). Dysregulation of mTOR has been assigned to carcinogenesis and thus may be involved in cancer development. We have addressed the role of hamartin, phospho-tuberin (p-TSC2) and phospho-mTOR (p-mTOR) in a series of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) samples. We collected 166 NSCLC and SCLC samples for immunohistochemical studies and performed western blot analyses in NSCLC and SCLC cell lines as well as comparative analyses with EGFR phosphorylation and downstream effectors. In cell lines we found an inverse correlation between hamartin and p-mTOR expression. In surgical specimens cytoplasmic hamartin expression was observed in more than 50% of adenocarcinoma (AC) and squamous cell carcinoma (SCC) compared to 14% of SCLC. P-mTOR and p-TSC2 staining was found in a minority of cases.There was a significant correlation between p-EGFR Tyr-1068, p-EGFR Tyr-992 and hamartin, and also between p-mTOR and p-EGFR Tyr-1173 in AC. In SCC an inverse correlation between hamartin and p-EGFR Tyr-992 was detected. Phosphorylation of TSC2 was associated with expression of MAP-Kinase. Hamartin, p-TSC2 and p-mTOR expression was not dependant of the EGFR mutation status. Hamartin expression is associated with poorer survival in SCC and SCLC. Our findings confirm the inhibitory role of the tuberous sclerosis complex for mTOR activation in lung cancer cell lines. These results reveal hamartin expression in a substantial subset of NSCLC and SCLC specimens, which may be due to EGFR signaling but is not dependant on EGFR mutations. Our data provide evidence for a functional role of the tuberous sclerosis complex in lung cancer.Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9274845161175223.
    Diagnostic Pathology 03/2014; 9(1):48. · 2.41 Impact Factor
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    ABSTRACT: The detection of a wide range of genomic alterations plays an important role in the diagnostics improving individual therapeutic approaches of cancer patients. Technologies that help to identify therapeutic relevant targets in tumor samples are a major factor on the way to a personalized medicine. The number of predictive and prognostic markers that influence the therapeutic outcome is continuously increasing. Therefore, parallel sequencing also named next generation sequencing (NGS), allowing the simultaneous analysis of numerous cancer related hotspots in many patients starting from a limited amount of DNA, is urgently needed to be established in cancer diagnostics. Different methods of target library preparation are commercially available and offer the opportunity to sequence tumor relevant hotspot mutations in a panel of genes. In the present study, a multiplex PCR approach, targeting a lung cancer and a leukemia gene panel, each consisting of 20 disease and therapy relevant genes, was investigated. Twelve formalin fixed and paraffin embedded lung tumors and twelve native Chronic Lymphocytic Leukemia (CLL) samples, respectively, were analyzed. Samples were sequenced on the MiSeq sequencer platform. The results showed a very high quality of each run. In spite of the low DNA input, each multiplex approach allowed a simultaneous analysis of 20 genes, in total covered by around 1,000 amplicons, in up to twelve cancer samples. Thus, the application of NGS on amplicon targets revealed an excellent performance in detecting a wide range of genetic alterations, combined with a high sensitivity.
    International Journal of Genomic Medicine. 03/2014; 2(1).
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    ABSTRACT: We discovered a novel somatic gene fusion, CD74-NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74-NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-β3, thereby providing the ligand for ERBB2-ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (p<0.0001). Ectopic expression of CD74-NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K-AKT pathway, and led to increased colony formation in soft agar. Thus, CD74-NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment.
    Cancer Discovery 01/2014; · 15.93 Impact Factor
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    ABSTRACT: The approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma. Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response. Therefore, the precise identification of the underlying somatic mutations is essential. Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations. Samples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples. DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction. BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC). All mutations were independently reassessed by Sanger sequencing. Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively). There was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%). All mutations down to 6.6% allele frequency could be detected with 100% specificity. In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity. The allele specific PCR failed to detect 17.9% of the mutations eligible for therapy with vemurafenib. NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity. IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM. Therefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients.
    BMC Cancer 01/2014; 14(1):13. · 3.33 Impact Factor

Publication Stats

10k Citations
1,961.30 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