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  • 1
    Keywords: CANCER ; EXPRESSION ; INHIBITOR ; proliferation ; SURVIVAL ; CELL ; Germany ; RISK ; GENE ; GENES ; HYBRIDIZATION ; microarray ; PROTEIN ; transcription ; TISSUE ; COMPLEX ; TRANSCRIPTION FACTOR ; IMPACT ; primary ; cell cycle ; CELL-CYCLE ; DOWN-REGULATION ; PHOSPHORYLATION ; COMPARATIVE GENOMIC HYBRIDIZATION ; PROMOTER ; UP-REGULATION ; ARRAYS ; PROMOTERS ; RATES ; MUTATIONS ; protein expression ; PROTEOMICS ; C-KIT ; RETINOBLASTOMA PROTEIN ; molecular ; ONCOLOGY ; ARRAY ; p16(INK4A) ; quantitative RT-PCR ; mRNA ; LEVEL ; TARGET GENES ; PHASE ; NUCLEAR ; LOSSES ; ENGLAND ; quantitative ; PLATFORM ; Rb ; GIST ; gastrointestinal ; DEPENDENT KINASES ; E2F1 ; E2FS ; PROGNOSTIC IMPLICATIONS ; RPPA
    Abstract: Loss of chromosome 9p is a reliable predictor of malignant behaviour in gastrointestinal stromal tumours (GISTS). p16(INK4A) located at 9p21 inhibits the CDK4/6/cyclin D complex from phosphorylating RB. Phosphorylation of RB through CDK4/6/cyclin D in early G(1) phase frees the transcription factor E2F1 from RB and enables mRNA transcription of genes essential for G(1)/S phase transition. This study aims to determine the impact of 9p loss on mRNA and protein expression of p16(INK4A) and further key cell cycle regulators in the different phases of the cell cycle. Sixty primary GISTS previously characterized for 9p loss by comparative genomic hybridization were analysed for mRNA expression of p16(INK4A), p15(INK4B), CDK4, CDK6, cyclin D, p 21(CIP1)p27(KIP1), CDK2, cyclin E, cyclin B, RB and E2F1, using quantitative RT-PCR. The protein expression of CDK6, CDK2, p2(CIP1), p27(KIP1) and phosphorylated RB (S807/S811) was evaluated using protein arrays as a novel and highly sensitive platform for profiling of protein abundance and protein phosphorylation. In parallel, the nuclear percentages of immunohistochemical staining for p16(INK4A), cyclin D, E2F1 and RB were quantified on a tissue microarray. GISTS with 9p loss had significantly higher proliferation rates, higher metastatic behaviour and shorter disease-free survival. On the molecular level, GISTS with 9p loss had a significantly reduced mRNA as well as nuclear protein expression of p16(INK4A). RB was significantly more phosphorylated in these tumours, together with increased mRNA expression and nuclear staining for E2F1 Furthermore, GISTS with 9p loss had up-regulation of the late G(1)/S phase promoters CDK2 and cyclin E. We conclude that loss of 9p accompanied by early G(1) phase inhibitor p16(INK4A) down-regulation in GISTS facilitates phosphorylation of RB, enabling F2F1-dependent transcription of genes essential for late G(1)/S phase transition. This study provides a possible basis for the accelerated proliferation and particularly malignant behaviour in GISTS with 9p loss. Copyright (C) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd
    Type of Publication: Journal article published
    PubMed ID: 18438954
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  • 2
    Keywords: CANCER ; EXPRESSION ; tumor ; Germany ; PATHWAY ; PATHWAYS ; QUANTIFICATION ; TOOL ; GENOME ; microarray ; PROTEIN ; PROTEINS ; SAMPLE ; DRUG ; BIOLOGY ; DISCOVERY ; FIELD ; TARGET ; microarrays ; ARRAYS ; SIGNALING PATHWAY ; MASS-SPECTROMETRY ; systems biology ; CONSUMPTION ; PROTEOMICS ; protein microarray ; GASTROINTESTINAL STROMAL TUMORS ; signaling ; review ; RE ; CAPACITY ; ARRAY ; DRUG DISCOVERY ; IMMUNOSORBENT-ASSAY ELISA ; PHASE ; TECHNOLOGY ; ENGLAND ; quantitative ; PLATFORM ; TUMOR BIOLOGY ; 1PAQ ; biomarker identification ; CANCER-CELL LINES ; CODED AFFINITY TAGS ; quantitative biology ; QUANTITATIVE PROTEOMICS ; reverse phase microarray ; reverse phase protein array ; REVERSE-PHASE
    Abstract: Background: Protein microarrays have the potential to join the field of quantitative proteomics as a standard method. This antibody-based experimental platform allows the highly sensitive and highly specific quantification of selected target proteins and excels with high sample capacity. As a key feature, numerous arrays can be produced in parallel, thus minimizing sample consumption. Objective: The recent progress made in the field of reverse phase protein arrays is summarized, with a focus on the introduction of normalization measures, as introduced in the authors' infrared-based protein arrays with quantitative readout approach. Conclusion: Tumor biology as well as drug discovery applications will soon benefit from the comprehensive description of signaling pathways and protein microarrays are an appropriate tool to achieve this goal
    Type of Publication: Journal article published
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  • 3
    Keywords: Application, BIOLOGY, BREAST-CANCER, CANCER, CANCER PROGRESSION, cancer research, CELL LUNG-CANCER,
    Abstract: A detailed and quantitative analysis of disease-relevant signaling will greatly contribute to our understanding of tumorigenesis and cancer progression, and thus open new strategies for drug discovery. However, throughput and sensitivity of currently established methods available for proteome profiling do not comply with the needs of clinical research such as high sample capacity and low sample consumption. Protein microarrays emerged as a promising alternative to analyze the abundance of proteins and their phosphorylation status on a high-throughput level. Here we summarize recent methodological advancements in the field of reverse-phase protein arrays and demonstrate their potential for clinical research as well as for in vitro applications
    Type of Publication: Journal article published
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  • 4
    Keywords: THERAPY ; LUNG-CANCER ; adenocarcinoma ; SOCIETY ; GEFITINIB ; erlotinib
    Abstract: Detection of activating EGFR mutations in NSCLC is the prerequisite for individualised therapy with receptor tyrosine kinase inhibitors (TKI). In contrast, mutant downstream effector KRAS is associated with TKI resistance. Accordingly, EGFR mutation status is routinely examined in NSCLC specimens, but the employed methods may have a dramatic impact on the interpretation of results and, consequently, therapeutic decisions. Specimens with low tumour cell content are at particular risk for false-negative EGFR mutation reporting by sequencing with Sanger chemistry. To improve reliability of detecting clinically relevant mutant variants of EGFR and KRAS, we took full advantage of 454 deep sequencing and developed a two-step amplification protocol for the analysis of EGFR exons 18-21 and KRAS exons 2 and 3. We systematically addressed the sensitivity, reproducibility and specificity of the developed assay. Mutations could be reliably identified down to an allele frequency of 0.2-1.5 %, as opposed to 10-20 % detection limit of Sanger sequencing. High reproducibility (0-2.1 % variant frequency) and very low background level (0.4-0.8 % frequency) further complement the reliability of this assay. Notably, re-evaluation of 16 NSCLC samples with low tumour cell content 〈/=40 % and EGFR wild type status according to Sanger sequencing revealed clinically relevant EGFR mutations at allele frequencies of 0.9-10 % in seven cases. In summary, this novel two-step amplification protocol with 454 deep sequencing is superior to Sanger sequencing with significantly increased sensitivity, enabling reliable analysis of EGFR and KRAS in NSCLC samples independent of the tumour cell content.
    Type of Publication: Journal article published
    PubMed ID: 23468066
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  • 5
    Keywords: EXPRESSION ; transcription ; TISSUE ; TUMORS ; IDENTIFICATION ; PATTERN ; SPECTRUM ; NAB2 ; STAT6 ; HEMANGIOPERICYTOMA
    Abstract: Recurrent somatic fusions of the two genes, NGFI-A-binding protein 2 (NAB2) and STAT6, located at chromosomal region 12q13, have been recently identified to be presumable tumor-initiating events in solitary fibrous tumors. Herein, we evaluated a cohort of 52 solitary fibrous tumors/hemangiopericytomas by whole-exome sequencing (one case) and multiplex RT-PCR (all 52 cases), and identified 12 different NAB2-STAT6 fusion variants in 48 cases (92%). All 52 cases showed strong and diffuse nuclear positivity for STAT6 by IHC. We categorized the fusion variants according to their potential functional effects within the predicted fusion protein and found strong correlations with relevant clinicopathological features. Tumors with the most common fusion variant, NAB2ex4-STAT6ex2/3, corresponded to classic pleuropulmonary solitary fibrous tumors with diffuse fibrosis and mostly benign behavior and occurred in older patients (median age, 69 years). In contrast, tumors with the second most common fusion variant, NAB2ex6-STAT6ex16/17, were found in much younger patients (median age, 47 years) and represented typical hemangiopericytomas from deep soft tissue with a more aggressive phenotype and clinical behavior. In summary, these molecular genetic findings support the concept that classic pleuropulmonary solitary fibrous tumor and deep-seated hemangiopericytoma are separate entities that share common features but correlate to different clinical outcome.
    Type of Publication: Journal article published
    PubMed ID: 24513261
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  • 6
    Keywords: EXPRESSION ; GENE ; FUSION ; PATTERN ; CAVITY
    Abstract: AIMS: Sinonasal haemangiopericytoma (SN-HPC) is a rare sinonasal mesenchymal neoplasm of perivascular myoid cell origin. Solitary fibrous tumour (SFT) occurs only very rarely in the sinonasal tract. SFT and soft tissue HPC have been considered a single entity. Recently, recurrent gene fusions involving NAB2-STAT6 resulting in differential expression of STAT6 were characterized as central molecular events in SFT. However, no data exist for NAB2-STAT6 status or STAT6 expression in SN-HPC. METHODS AND RESULTS: We examined six SN-HPCs and two sinonasal SFTs by immunohistochemistry and RT-PCR for NAB2-STAT6 fusions. SN-HPC affected three females and three males (mean age: 72 years). They expressed smooth muscle actin, lacked strong CD34 reactivity and were negative for nuclear STAT6 expression. RT-PCR analysis confirmed the absence of NAB2-STAT6 fusions in all cases. Conversely, both sinonasal SFTs (in males aged 39 and 52 years) displayed classical features of pleuropulmonary and soft-tissue SFTs (uniformly CD34-positive with strong nuclear expression of STAT6). RT-PCR revealed NAB2-STAT6 fusions in both cases. CONCLUSIONS: These findings confirm the molecular and phenotypical distinctness of these two entities. While SN-HPC is a site-specific sinonasal neoplasm of as yet unknown molecular pathogenesis, sinonasal SFTs show phenotypical and molecular identity to their pleural/extrapleural counterparts.
    Type of Publication: Journal article published
    PubMed ID: 24807787
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  • 7
    Keywords: GENES ; METHYLATION ; germline mutations ; GASTROINTESTINAL STROMAL TUMORS ; MOLECULAR-GENETICS ; pheochromocytomas ; FAMILIAL PARAGANGLIOMA ; STRATAKIS-SYNDROME
    Abstract: Carney triad (CT) is a rare condition with synchronous or metachronous occurrence of gastrointestinal stromal tumors (GISTs), paragangliomas (PGLs), and pulmonary chondromas in a patient. In contrast to Carney-Stratakis syndrome (CSS) and familial PGL syndromes, no germline or somatic mutations in the succinate dehydrogenase (SDH) complex subunits A, B, C, or D have been found in most tumors and/or patients with CT. Nonetheless, the tumors arising among patients with CT, CSS, or familial PGL share a similar morphology with loss of the SDHB subunit on the protein level. For the current study, we employed massive parallel bisulfite sequencing to evaluate DNA methylation patterns in CpG islands in proximity to the gene loci of all four SDH subunits. For the first time, we report on a recurrent aberrant dense DNA methylation at the gene locus of SDHC in tumors of patients with CT, which was not present in tumors of patients with CSS or PGL, or in sporadic GISTs with KIT mutations. This DNA methylation pattern was correlated to a reduced mRNA expression of SDHC, and concurrent loss of the SDHC subunit on the protein level. Collectively, these data suggest epigenetic inactivation of the SDHC gene locus with functional impairment of the SDH complex as a plausible alternate mechanism of tumorigenesis in CT.
    Type of Publication: Journal article published
    PubMed ID: 24859990
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  • 8
    Keywords: CANCER ; CELLS ; ACTIVATION ; PROGRESSION ; TERM-FOLLOW-UP ; OSTEOPONTIN ; KIT ; PDGFRA MUTATIONS ; EPIGENETICS ; RECEPTOR-ALPHA MUTATIONS
    Abstract: Gastrointestinal stromal tumors (GISTs) have distinct gene expression patterns according to localization, genotype and aggressiveness. DNA methylation at CpG dinucleotides is an important mechanism for regulation of gene expression. We performed targeted DNA methylation analysis of 1.505 CpG loci in 807 cancer-related genes in a cohort of 76 GISTs, combined with genome-wide mRNA expression analysis in 22 GISTs, to identify signatures associated with clinicopathological parameters and prognosis. Principal component analysis revealed distinct DNA methylation patterns associated with anatomical localization, genotype, mitotic counts and clinical follow-up. Methylation of a single CpG dinucleotide in the non-CpG island promoter of SPP1 was significantly correlated with shorter disease-free survival. Hypomethylation of this CpG was an independent prognostic parameter in a multivariate analysis compared to anatomical localization, genotype, tumor size and mitotic counts in a cohort of 141 GISTs with clinical follow-up. The epigenetic regulation of SPP1 was confirmed in vitro, and the functional impact of SPP1 protein on tumorigenesis-related signaling pathways was demonstrated. In summary, SPP1 promoter methylation is a novel and independent prognostic parameter in GISTs, and might be helpful in estimating the aggressiveness of GISTs from the intermediate-risk category.
    Type of Publication: Journal article published
    PubMed ID: 25046773
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  • 9
    Keywords: EXPRESSION ; GENE ; VARIANTS ; FUSION ; BENIGN ; ANNOTATION ; GENOMES ; SOLITARY FIBROUS TUMOR ; DESMOID TUMORS ; FIBROMATOSIS
    Abstract: Sinonasal hemangiopericytoma (SN-HPC) is an uncommon, site-specific, low-grade mesenchymal neoplasm of probable perivascular myoid cell origin. In contrast to solitary fibrous tumors of soft tissue and sinonasal tract origin, SN-HPCs were recently shown to lack recurrent NAB2-STAT6 fusion variants. Other molecular alterations known to occur in some of soft tissue perivascular myoid cell neoplasms were also absent in SN-HPC; thus, the molecular pathogenesis of SN-HPCs remained unknown. Guided by whole-genome sequencing combined with RNA sequencing of an index case, we analyzed a total of six SN-HPCs for mutations within the amino-terminal region of the gene catenin (cadherin-associated protein), beta 1, 88 kDa (CTNNB1), encoding for beta-catenin. All six cases showed missense mutations, with amino acid substitutions clustering at positions 33 to 45, corresponding to the recognition site of the beta-catenin destruction complex. Similar CTNNB1 mutations have been described in a variety of epithelial and mesenchymal neoplasms. These mutations prevent beta-catenin phosphorylation and proteasomal degradation but promote its nuclear accumulation and subsequent increased transcription of Wingless-related integration site target genes. Consistent with these molecular findings, beta-catenin IHC showed consistent diffuse and strong nuclear staining of the tumor cells in all six SN-HPCs. Our results highlight, for the first time, CTNNB1 mutations as the likely initiating molecular events driving SN-HPC tumorigenesis, which places SN-HPC among the growing family of beta-catenin-driven mesenchymal neoplasms.
    Type of Publication: Journal article published
    PubMed ID: 25482924
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  • 10
    Keywords: BREAST-CANCER ; PLASMA ; MARKERS ; METHYLATION
    Abstract: Identification of a single molecular trait that is determinant of common malignancies may serve as a powerful diagnostic supplement to cancer type-specific markers. Here, we report a DNA methylation mark that is characteristic of seven studied malignancies, namely cancers of lung, breast, prostate, pancreas, colorectum, glioblastoma and B cell chronic lymphocytic leukaemia (CLL) (n = 137). This mark was defined by substantial hypermethylation at the promoter and first exon of growth hormone secretagouge receptor (GHSR) through bisulfite pyrosequencing. The degree of aberrant methylation was capable of accurate discrimination between cancer and control samples. The highest sensitivity and specificity of cancer detection was achieved for cancers of pancreas, lung, breast and CLL yielding the area under the curve (AUC) values of 1.0000, 0.9952, 0.9800 and 0.9400, respectively. Narrowing to a single CpG site within the gene's promoter or four consecutive CpG units of the highest methylation levels within the first exon improved the detection power. GHSR hypermethylation was detected already at the early stage tumors. The accurate performance of this marker was further replicated in an independent set of pancreatic cancer and control samples (n = 78). These findings support the candidature of GHSR methylation as a highly accurate pan-cancer marker.
    Type of Publication: Journal article published
    PubMed ID: 25557172
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