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1.  KRAS, EGFR, PDGFR-α, KIT and COX-2 status in carcinoma showing thymus-like elements (CASTLE) 
Diagnostic Pathology  2014;9:116.
Background
CASTLE (Carcinoma showing thymus-like elements) is a rare malignant neoplasm of the thyroid resembling lymphoepithelioma-like and squamous cell carcinoma of the thymus with different biological behaviour and a better prognosis than anaplastic carcinoma of the thyroid.
Methods
We retrospectively investigated 6 cases of this very rare neoplasm in order to investigate the mutational status of KRAS, EGFR, PDGFR-α and KIT, as well as the immunohistochemical expression pattern of CD117, EGFR and COX-2, and possibly find new therapeutic targets.
Results
Diagnosis was confirmed by a moderate to strong expression of CD5, CD117 and CK5/6, whereas thyroglobulin, calcitonin and TTF-1 were negative in all cases. Tumors were also positive for COX-2 and in nearly all cases for EGFR. In four cases single nucleotide polymorphisms (SNPs) could be detected in exon 12 of the PDGFR-α gene (rs1873778), in three cases SNPs were found in exon 20 of the EGFR gene (rs1050171). No mutations were found in the KIT and KRAS gene.
Conclusions
All tumors showed a COX-2 expression as well as an EGFR expression except for one case and a wild-type KRAS status. No activating mutations in the EGFR, KIT and PDGFR-α gene could be detected. Our data may indicate a potential for targeted therapies, but if these therapeutic strategies are of benefit in CASTLE remains to be determined.
Virtual Slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1658499296115016
doi:10.1186/1746-1596-9-116
PMCID: PMC4078982  PMID: 24934485
CASTLE; Thymic carcinoma; Mutational analysis; Immunohistochemistry; Thyroid gland
2.  Yes-Associated Protein Upregulates Jagged-1 and Activates the NOTCH Pathway in Human Hepatocellular Carcinoma 
Gastroenterology  2013;144(7):1530-1542.e12.
Background & Aims
Cancer cells often lose contact inhibition to undergo anchorage-independent proliferation and become resistant to apoptosis by inactivating the Hippo signaling pathway, resulting in activation of the transcriptional co-activator yes-associated protein (YAP). However, the oncogenic mechanisms of YAP are unclear.
Methods
Using cross-species analysis of expression data, the Notch ligand Jagged-1 (Jag-1) was identified as downstream target of YAP in hepatocytes and hepatocellular carcinoma (HCC) cells. We analyzed the functions of YAP in HCC cells via overexpression and RNA silencing experiments. We used transgenic mice that overexpressed a constitutively activated form of YAP (YAPS127A), and measured protein levels in HCC and colorectal and pancreatic tumor samples from patients.
Results
Human HCC cell lines and mouse hepatocytes that overexpress YAPS127A upregulated Jag-1, leading to activation of the Notch pathway and increased proliferation. Induction of Jag-1, activation of Notch, and cell proliferation required binding of YAP to its transcriptional partner TEAD4; TEAD4 binding required Mst1/2, but not WNT-β-catenin signaling. Levels of YAP correlated with Jag-1 expression and Notch signaling in human tumor samples and shorter survival times of patients with HCC or colorectal cancer.
Conclusion
The transcriptional regulator YAP upregulates Jag-1 to activate Notch signaling in HCC cells and mouse hepatocytes. YAP-dependent activity of Jag-1 and Notch correlate in human HCC and colorectal tumor samples with patient survival times, suggesting the use of YAP and Notch inhibitors as therapeutics for gastrointestinal cancer.
doi:10.1053/j.gastro.2013.02.009
PMCID: PMC3665638  PMID: 23419361
transcriptional regulators; liver cancer; colorectal cancer; pancreatic cancer
3.  Molecular analysis of pancreatic acinar cell cystadenomas: Evidence of a non-neoplastic nature 
Oncology Letters  2014;8(2):852-858.
The biology of pancreatic acinar cell cystadenomas has not been clearly defined. However, a non-neoplastic process, caused by a cell differentiation failure leading to a cystic transformation, has been discussed, as well as a benign neoplastic lesion. Pancreatic acinar cell cystadenomas usually consist of thin-walled unilocular or multilocular cysts, and mural nodules have been described in two cases of a recent series. In one of these nodules, chromosomal imbalances were detected, which provided preliminary evidence for a neoplastic process. The aim of the current study was to further characterize the lesions by molecular analyses. In four cases without mural nodules, the clonality was assessed by performing mutational analyses within the highly variable displacement-loop region of the mitochondrial DNA. As a result, no closer correlation was identified between different foci within the tumors than between the tumors and adjacent normal pancreatic acinar tissue, indicating polyclonality of these lesions. Further molecular analyses revealed no mutations of the β-catenin and K-ras genes. In addition, no immunohistochemical evidence was identified for mutations of Smad4 or p53. In conclusion, the results of the current study demonstrated that pancreatic acinar cell cystadenomas are non-neoplastic lesions, with the potential exception of those rare cases with mural nodules.
doi:10.3892/ol.2014.2163
PMCID: PMC4081433  PMID: 25009661
pancreatic tumor; cystic lesion; clonality; molecular
4.  Early aberrant DNA methylation events in a mouse model of acute myeloid leukemia 
Genome Medicine  2014;6(4):34.
Background
Aberrant DNA methylation is frequently found in human malignancies including acute myeloid leukemia (AML). While most studies focus on later disease stages, the onset of aberrant DNA methylation events and their dynamics during leukemic progression are largely unknown.
Methods
We screened genome-wide for aberrant CpG island methylation in three disease stages of a murine AML model that is driven by hypomorphic expression of the hematopoietic transcription factor PU.1. DNA methylation levels of selected genes were correlated with methylation levels of CD34+ cells and lineage negative, CD127-, c-Kit+, Sca-1+ cells; common myeloid progenitors; granulocyte-macrophage progenitors; and megakaryocyte-erythroid progenitors.
Results
We identified 1,184 hypermethylated array probes covering 762 associated genes in the preleukemic stage. During disease progression, the number of hypermethylated genes increased to 5,465 in the late leukemic disease stage. Using publicly available data, we found a significant enrichment of PU.1 binding sites in the preleukemic hypermethylated genes, suggesting that shortage of PU.1 makes PU.1 binding sites in the DNA accessible for aberrant methylation. Many known AML associated genes such as RUNX1 and HIC1 were found among the preleukemic hypermethylated genes. Nine novel hypermethylated genes, FZD5, FZD8, PRDM16, ROBO3, CXCL14, BCOR, ITPKA, HES6 and TAL1, the latter four being potential PU.1 targets, were confirmed to be hypermethylated in human normal karyotype AML patients, underscoring the relevance of the mouse model for human AML.
Conclusions
Our study identified early aberrantly methylated genes as potential contributors to onset and progression of AML.
doi:10.1186/gm551
PMCID: PMC4062060  PMID: 24944583
5.  TERT promoter hotspot mutations are recurrent in myxoid liposarcomas but rare in other soft tissue sarcoma entities 
Background
Recently, recurrent point mutations in the telomerase reverse transcriptase (TERT) promoter region have been found in many human cancers, leading to a new transcription factor binding site, increased induction of TERT and subsequently to telomere maintenance. We determined the prevalence of TERT promoter mutations in soft tissue sarcomas of 341 patients comprising 16 entities and in 16 sarcoma cell lines covering 7 different soft tissue sarcoma types.
Methods
The sarcoma tissue samples were collected from the archives of the Institute of Pathology, University of Heidelberg and were composed of 39 myxoid liposarcomas (MLS), 61 dedifferentiated liposarcomas, 15 pleomorphic liposarcomas, 27 leiomyosarcomas, 25 synovial sarcomas (SS), 35 malignant peripheral nerve sheath tumors (MPNST), 40 undifferentiated pleomorphic sarcomas, 17 myxofibrosarcomas, 9 low grade fibromyxoid sarcomas, 10 cases of dermatofibrosarcoma protuberans, 31 solitary fibrous tumors (SFT), 8 extraskeletal myxoid chondrosarcomas, 9 angiosarcomas, 6 alveolar soft part sarcomas, 5 clear cell sarcomas and 4 epithelioid sarcomas. Sarcoma cell lines were obtained from the raising laboratories. A 193 bp fragment of the TERT promoter region covering the hot-spot mutations C228T and C250T was amplified, and direct sequencing of the PCR products was performed.
Results
TERT promoter mutations were detected in 36/341 sarcomas. They were highly recurrent in MLS (29/39; 74%) and were in the present MLS series not associated with the phenotype (myxoid vs. round cell variant), tumor grade, tumor site and patients’ median age or gender. In the remaining cases, TERT promoter mutations were found only in 7/302 sarcoma samples and confined to SFTs (4/31; 13%), MPNSTs (2/35; 6%), and SSs (1/25; 4%). Within the collection of sarcoma cell lines examined, TERT promoter mutations were detected in two MLS and in one of three MPNST cell lines.
Conclusions
TERT promoter mutations are frequent in MLSs including their round cell variants, representing the most prevalent mutation identified in this sarcoma entity to date, and in a minor fraction of SFTs, MPNSTs and SSs. The majority of sarcomas are devoid of TERT promoter hotspot mutations. These data suggest that telomere maintenance through increased expression of telomerase plays an important role in the pathogenesis especially of MLS.
doi:10.1186/1756-9966-33-33
PMCID: PMC4022359  PMID: 24726063
TERT; Promoter; Mutation; Soft tissue; Sarcoma; Myxoid liposarcoma; Solitary fibrous tumor
6.  Integrative DNA methylation and gene expression analysis in high-grade soft tissue sarcomas 
Genome Biology  2013;14(12):r137.
Background
High-grade soft tissue sarcomas are a heterogeneous, complex group of aggressive malignant tumors showing mesenchymal differentiation. Recently, soft tissue sarcomas have increasingly been classified on the basis of underlying genetic alterations; however, the role of aberrant DNA methylation in these tumors is not well understood and, consequently, the usefulness of methylation-based classification is unclear.
Results
We used the Infinium HumanMethylation27 platform to profile DNA methylation in 80 primary, untreated high-grade soft tissue sarcomas, representing eight relevant subtypes, two non-neoplastic fat samples and 14 representative sarcoma cell lines. The primary samples were partitioned into seven stable clusters. A classification algorithm identified 216 CpG sites, mapping to 246 genes, showing different degrees of DNA methylation between these seven groups. The differences between the clusters were best represented by a set of eight CpG sites located in the genes SPEG, NNAT, FBLN2, PYROXD2, ZNF217, COL14A1, DMRT2 and CDKN2A. By integrating DNA methylation and mRNA expression data, we identified 27 genes showing negative and three genes showing positive correlation. Compared with non-neoplastic fat, NNAT showed DNA hypomethylation and inverse gene expression in myxoid liposarcomas, and DNA hypermethylation and inverse gene expression in dedifferentiated and pleomorphic liposarcomas. Recovery of NNAT in a hypermethylated myxoid liposarcoma cell line decreased cell migration and viability.
Conclusions
Our analysis represents the first comprehensive integration of DNA methylation and transcriptional data in primary high-grade soft tissue sarcomas. We propose novel biomarkers and genes relevant for pathogenesis, including NNAT as a potential tumor suppressor in myxoid liposarcomas.
doi:10.1186/gb-2013-14-12-r137
PMCID: PMC4054884  PMID: 24345474
7.  Nuclear pore component Nup98 is a potential tumor suppressor and regulates post-transcriptional expression of select p53 target genes 
Molecular cell  2012;48(5):799-810.
Summary
The p53 tumor suppressor utilizes multiple mechanisms to selectively regulate its myriad target genes, which in turn mediate diverse cellular processes. Here, using conventional and single molecule mRNA analyses, we demonstrate that the nucleoporin Nup98 is required for full expression of p21, a key effector of the p53 pathway, but not several other p53 target genes. Nup98 regulates p21 mRNA levels by a post-transcriptional mechanism in which a complex containing Nup98 and the p21 mRNA 3′-UTR protects p21 mRNA from degradation by the exosome. An in silico approach revealed another p53 target (14-3-3σ) to be similarly regulated by Nup98. The expression of Nup98 is reduced in murine and human hepatocellular carcinomas (HCC) and correlates with p21 expression in HCC patients. Our study elucidates a previously unrecognized function of wild-type Nup98 in regulating select p53 target genes that is distinct from the well-characterized oncogenic properties of Nup98 fusion proteins.
doi:10.1016/j.molcel.2012.09.020
PMCID: PMC3525737  PMID: 23102701
9.  Cytosolic and nuclear caspase-8 have opposite impact on survival after liver resection for hepatocellular carcinoma 
BMC Cancer  2013;13:532.
Background
An imbalance between proliferation and apoptosis is one of the main features of carcinogenesis. TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis upon binding to the TRAIL death receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2, whereas binding to TRAIL-R3 and TRAIL-R4 might promote cell survival and proliferation. The anti-tumor activity of TRAIL-R1 and TRAIL-R2 agonists is currently investigated in clinical trials. To gain further insight into the regulation of apoptosis in hepatocellular carcinoma (HCC), we investigated the TRAIL pathway and the regulators of apoptosis caspase-8, Bcl-xL and Mcl-1 in patients with HCC regarding patient survival.
Methods
We analyzed 157 hepatocellular carcinoma patients who underwent partial liver resection or orthotopic liver transplantation and healthy control liver tissue using immunohistochemistry on tissue microarrays for the expression of TRAIL-R1 to TRAIL-R4, caspase-8, Bcl-xL and Mcl-1. Immunohistochemical data were evaluated for potential associations with clinico-pathological parameters and survival.
Results
Whereas TRAIL-R1 was downregulated in HCC in comparison to normal liver tissue, TRAIL-R2 and –R4 were upregulated in HCC, especially in G2 and G3 tumors. TRAIL-R1 downregulation and upregulation of TRAIL-R2 and TRAIL-R4 correlated with tumor dedifferentiation (G2/G3). TRAIL-R3, Bcl-xL and Mcl-1 showed no differential expression in tumor tissue compared to normal tissue. The expression levels of TRAIL receptors did not correlate with patient survival after partial hepatectomy. Interestingly, in tumor tissue, but not in normal hepatocytes, caspase-8 showed a strong nuclear staining. Low cytosolic and high nuclear staining intensity of caspase-8 significantly correlated with impaired survival after partial hepatectomy, which, for cytosolic caspase-8, was independent from tumor grade.
Conclusions
Assessment of TRAIL-receptor expression patterns may have therapeutic implications for the use of TRAIL receptor agonists in HCC therapy. Tumor-specific nuclear localisation of caspase-8 in HCC suggests an apoptosis-independent function of caspase-8 and correlates with patient survival.
doi:10.1186/1471-2407-13-532
PMCID: PMC3834100  PMID: 24209510
HCC; Apoptosis; TRAIL receptors; Nuclear caspase-8
10.  Stathmin Regulates Keratinocyte Proliferation and Migration during Cutaneous Regeneration 
PLoS ONE  2013;8(9):e75075.
Cutaneous regeneration utilizes paracrine feedback mechanisms to fine-tune the regulation of epidermal keratinocyte proliferation and migration. However, it is unknown how fibroblast-derived hepatocyte growth factor (HGF) affects these mutually exclusive processes in distinct cell populations. We here show that HGF stimulates the expression and phosphorylation of the microtubule-destabilizing factor stathmin in primary human keratinocytes. Quantitative single cell- and cell population-based analyses revealed that basal stathmin levels are important for the migratory ability of keratinocytes in vitro; however, its expression is moderately induced in the migration tongue of mouse skin or organotypic multi-layered keratinocyte 3D cultures after full-thickness wounding. In contrast, clearly elevated stathmin expression is detectable in hyperproliferative epidermal areas. In vitro, stathmin silencing significantly reduced keratinocyte proliferation. Automated quantitative and time-resolved analyses in organotypic cocultures demonstrated a high correlation between Stathmin/phospho-Stathmin and Ki67 positivity in epidermal regions with proliferative activity. Thus, activation of stathmin may stimulate keratinocyte proliferation, while basal stathmin levels are sufficient for keratinocyte migration during cutaneous regeneration.
doi:10.1371/journal.pone.0075075
PMCID: PMC3774809  PMID: 24066165
11.  High salt intake causes adverse fetal programming—vascular effects beyond blood pressure 
Nephrology Dialysis Transplantation  2012;27(9):3464-3476.
Do detrimental effects on the vasculature of a high dietary sodium intake precede the development of hypertension?
Background
High salt intake causes hypertension, adverse cardiovascular outcomes and potentially also blood pressure (BP)-independent target organ damage. Excess salt intake in pregnancy is known to affect BP in the offspring. The present study was designed to assess whether high salt intake in pregnancy affects BP and vascular morphology in the offspring.
Methods
Sprague–Dawley rats were fed a standard rodent diet with low–normal (0.15%) or high (8.0%) salt content during pregnancy and lactation. After weaning at 4 weeks of age, offspring were maintained on the same diet or switched to a high- or low-salt diet, respectively. Vascular geometry was assessed in male offspring at 7 and 12 weeks postnatally.
Results
Up to 12 weeks of age, there was no significant difference in telemetrically measured BP between the groups of offspring. At 12 weeks of age, wall thickness of central (aorta, carotid), muscular (mesenteric) and intrapulmonary arteries was significantly higher in offspring of mothers on a high-salt diet irrespective of the post-weaning diet. This correlated with increased fibrosis of the aortic wall, more intense nitrotyrosine staining as well as elevated levels of marinobufagenin (MBG) and asymmetric dimethyl arginine (ADMA).
Conclusions
High salt intake in pregnant rats has long-lasting effects on the modeling of central and muscular arteries in the offspring independent of postnatal salt intake and BP. Circulating MBG and ADMA and local oxidative stress correlate with the adverse vascular modeling.
doi:10.1093/ndt/gfs027
PMCID: PMC3433771  PMID: 22431707
blood pressure; fetal programming; nitric oxide; salt; vessel development
12.  Correction: Automated Universal BRAF State Detection within the Activation Segment in Skin Metastases by Pyrosequencing-Based Assay U-BRAFV600 
PLoS ONE  2013;8(6):10.1371/annotation/b31c5248-91ad-4668-930a-24543b19d6e7.
doi:10.1371/annotation/b31c5248-91ad-4668-930a-24543b19d6e7
PMCID: PMC3692735
13.  Focal Nodular Hyperplasia and Hepatocellular Adenoma around the World Viewed through the Scope of the Immunopathological Classification 
Focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA) are benign hepatocellular tumors. The risk of bleeding and malignant transformation of HCA are strong arguments to differentiate HCA from FNH. Despite great progress that has been made in the differential radiological diagnosis of the 2 types of nodules, liver biopsy is sometimes necessary to separate the 2 entities. Identification of HCA subtypes using immunohistochemical techniques, namely, HNF1A-inactivated HCA (35–40%), inflammatory HCA (IHCA), and beta-catenin-mutated inflammatory HCA (b-IHCA) (50–55%), beta-catenin-activated HCA (5–10%), and unclassified HCA (10%) has greatly improved the diagnostic accuracy of benign hepatocellular nodules. If HCA malignant transformation occurs in all HCA subgroups, the risk is by far the highest in the β-catenin-mutated subgroups (b-HCA, b-IHCA). In the coming decade the management of HCA will be more dependent on the identification of HCA subtypes, particularly for smaller nodules (<5 cm) in terms of imaging, follow-up, and resection.
doi:10.1155/2013/268625
PMCID: PMC3654480  PMID: 23691331
14.  Automated Universal BRAF State Detection within the Activation Segment in Skin Metastases by Pyrosequencing-Based Assay U-BRAFV600 
PLoS ONE  2013;8(3):e59221.
Malignant melanoma is a highly-aggressive type of malignancy with considerable metastatic potential and frequent resistance to cytotoxic agents. BRAF mutant protein was recently recognized as therapeutic target in metastatic melanoma. We present a newly-developed U-BRAFV600 approach – a universal pyrosequencing-based assay for mutation detection within activation segment in exon 15 of human braf. We identified 5 different BRAF mutations in a single assay analyzing 75 different formalin-fixed paraffin-embedded (FFPE) samples of cutaneous melanoma metastases from 29 patients. We found BRAF mutations in 21 of 29 metastases. All mutant variants were quantitatively detectable by the newly-developed U-BRAFV600 assay. These results were confirmed by ultra-deep-sequencing validation (∼60,000-fold coverage). In contrast to all other BRAF state detection methods, the U-BRAFV600 assay is capable of automated quantitative identification of at least 36 previously-published BRAF mutations. Under the precaution of a minimum of 3% mutated cells in front of a background of wild type cells, U-BRAFV600 assay design completely excludes false wild-type results. The corresponding algorithm for classification of BRAF-mutated variants is provided. The single-reaction assay and data analysis automation makes our approach suitable for the assessment of large clinical sample sizes. Therefore, we suggest U-BRAFV600 assay as a most powerful sequencing-based diagnostic tool to automatically identify BRAF state as a prerequisite to targeted therapy.
doi:10.1371/journal.pone.0059221
PMCID: PMC3608589  PMID: 23555633
15.  A Frequent PNPLA3 Variant Is a Sex Specific Disease Modifier in PSC Patients with Bile Duct Stenosis 
PLoS ONE  2013;8(3):e58734.
Background & Aims
Primary sclerosing cholangitis predominantly affects males and is an important indication for liver transplantation. The rs738409 variant (I148M) of the PNPLA3 gene is associated with alcoholic and non-alcoholic liver disease and we evaluated its impact on the disease course of PSC.
Methods
The I148M polymorphism was genotyped in 121 German PSC patients of a long-term prospective cohort and 347 Norwegian PSC patients.
Results
In the prospective German cohort, actuarial survival free of liver transplantation was significantly reduced for I148M carriers (p = 0.011) compared to wildtype patients. This effect was restricted to patients with severe disease, as defined by development of dominant stenosis (DS) requiring endoscopic intervention. DS patients showed markedly decreased survival (p = 0.004) when carrying the I148M variant (I148M: mean 13.8 years; 95% confidence interval: 11.6–16.0 vs. wildtype: mean 18.6 years; 95% confidence interval: 16.3–20.9) while there was no impact on survival in patients without a DS (p = 0.87). In line with previous observations of sex specific effects of the I148M polymorphism, the effect on survival was further restricted to male patients (mean survival 11.9 years; 95% confidence interval: 10.0–14.0 in I148M carriers vs. 18.8 years; 95% confidence interval: 16.2–21.5 in wildtype; p<0.001) while female patients were unaffected by the polymorphism (p = 0.65). These sex specific findings were validated in the Norwegian cohort (p = 0.013).
Conclusions
In male PSC patients with severe disease with bile duct stenosis requiring intervention, the common I148M variant of the PNPLA3 gene is a risk factor for reduced survival.
doi:10.1371/journal.pone.0058734
PMCID: PMC3591368  PMID: 23505555
16.  Chemoradiation in patients with isolated recurrent pancreatic cancer - therapeutical efficacy and probability of re-resection 
Background
In the present retrospective analysis we analysed the therapeutic outcome of a set of patients, who were treated with chemoradiation (CRT) for recurrent pancreatic cancer (RPC) in a single institution.
Patients and Methods
Forty-one patients had a history of primary resection for pancreatic cancer. In case of an unresectable recurrency patients were treated with CRT at our institution between 2002 and 2010 with a median dose of 48.4 Gy (range 39.6–54 Gy). Concurrent chemotherapy regimes included Gemcitabine (GEM) in 37/41 patients (90%) and Fluorouracil (FU) or Capecitabine (CAP) in 4/41 patients (10%). Patients were re-evaluated after CRT with computed tomography and/or explorative laparotomy. During re-resection or laparotomy 15 patients received an additional intraoperative radiotherapy (IORT) with a median dose of 15 Gy (range 12–15 Gy). Median age was 65 years (range 39–76 years) and there were 26 male and 15 female patients.
Results
The median overall survival (mOS), local control (LC) and progression-free survival (PFS) were 16.1, 13.8 and 6.9 months respectively for all patients after the first day of CRT. Re-resection was possible in five patients (12%) and a complete remission (CR) as defined by tumor-free biopsy was seen in 6 patients (15%). When re-resection could be achieved after CRT mOS was improved to 28.3 months (n = 5 patients, 95%-CI 10.2 – 46.3 months). Patients receiving IORT had a significantly improved mOS compared to no IORT (p = 0.034). Fifteen patients (37%) experienced a local tumour progression and main site of distant metastasis was the liver (11 patients, 27%).Overall treatment-related toxicity was mild, grade III hematologic toxicity was observed in 11 patients (27%).
Conclusion
In summary we observed a good therapeutic response with mild to moderate toxicity levels for CRT in RPC. Overall survival and PFS were clearly improved in case of induction of a complete remission (tumor-free biopsies) or after achieving a re-resection, thus providing a curative intended therapy even in case of disease recurrence.
doi:10.1186/1748-717X-8-27
PMCID: PMC3570445  PMID: 23369246
17.  A Systems Biology Study on NFκB Signaling in Primary Mouse Hepatocytes 
The cytokine tumor necrosis factor-alpha (TNFα) is one of the key factors during the priming phase of liver regeneration as well as in hepatocarcinogenesis. TNFα activates the nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) signaling pathway and contributes to the conversion of quiescent hepatocytes to activated hepatocytes that are able to proliferate in response to growth factor stimulation. Different mathematical models have been previously established for TNFα/NFκB signaling in the context of tumor cells. Combining these mathematical models with time-resolved measurements of expression and phosphorylation of TNFα/NFκB pathway constituents in primary mouse hepatocytes revealed that an additional phosphorylation step of the NFκB isoform p65 has to be considered in the mathematical model in order to sufficiently describe the dynamics of pathway activation in the primary cells. Also, we addressed the role of basal protein turnover by experimentally measuring the degradation rate of pivotal players in the absence of TNFα and including this information in the model. To elucidate the impact of variations in the protein degradation rates on TNFα/NFκB signaling on the overall dynamic behavior we used global sensitivity analysis that accounts for parameter uncertainties and showed that degradation and translation of p65 had a major impact on the amplitude and the integral of p65 phosphorylation. Finally, our mathematical model of TNFα/NFκB signaling was able to predict the time-course of the complex formation of p65 and of the inhibitor of NFκB (IκB) in primary mouse hepatocytes, which was experimentally verified. Hence, we here present a mathematical model for TNFα/NFκB signaling in primary mouse hepatocytes that provides an important basis to quantitatively disentangle the complex interplay of multiple factors in liver regeneration and tumorigenesis.
doi:10.3389/fphys.2012.00466
PMCID: PMC3533138  PMID: 23293603
mathematical modeling; p65; IκB; protein degradation; hepatocytes; signaling
18.  Epithelial-to-Mesenchymal Transition in Pancreatic Ductal Adenocarcinoma and Pancreatic Tumor Cell Lines: The Role of Neutrophils and Neutrophil-Derived Elastase 
Pancreatic ductal adenocarcinoma (PDAC) is frequently associated with fibrosis and a prominent inflammatory infiltrate in the desmoplastic stroma. Moreover, in PDAC, an epithelial-to-mesenchymal transition (EMT) is observed. To explore a possible connection between the infiltrating cells, particularly the polymorphonuclear neutrophils (PMN) and the tumor cell transition, biopsies of patients with PDAC (n = 115) were analysed with regard to PMN infiltration and nuclear expression of β-catenin and of ZEB1, well-established indicators of EMT. In biopsies with a dense PMN infiltrate, a nuclear accumulation of β-catenin and of ZEB1 was observed. To address the question whether PMN could induce EMT, they were isolated from healthy donors and were cocultivated with pancreatic tumor cells grown as monolayers. Rapid dyshesion of the tumor cells was seen, most likely due to an elastase-mediated degradation of E-cadherin. In parallel, the transcription factor TWIST was upregulated, β-catenin translocated into the nucleus, ZEB1 appeared in the nucleus, and keratins were downregulated. EMT was also induced when the tumor cells were grown under conditions preventing attachment to the culture plates. Here, also in the absence of elastase, E-cadherin was downmodulated. PMN as well as prevention of adhesion induced EMT also in liver cancer cell line. In conclusion, PMN via elastase induce EMT in vitro, most likely due to the loss of cell-to-cell contact. Because in pancreatic cancers the transition to a mesenchymal phenotype coincides with the PMN infiltrate, a contribution of the inflammatory response to the induction of EMT and—by implication—to tumor progression is possible.
doi:10.1155/2012/720768
PMCID: PMC3514849  PMID: 23227088
19.  Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment 
Cell host & microbe  2011;9(1):32-45.
SUMMARY
Hepatitis C virus (HCV) is a major causative agent of chronic liver disease in humans. To gain insight into host factor requirements for HCV replication we performed a siRNA screen of the human kinome and identified 13 different kinases, including phosphatidylinositol-4 kinase III alpha (PI4KIIIα) as required for HCV replication. Consistent with elevated levels of the PI4KIIIα product phosphatidylinositol-4-phosphate (PI4P) detected in HCV infected cultured hepatocytes and liver tissue from chronic hepatitis C patients, the enzymatic activity of PI4KIIIα was critical for HCV replication. Viral nonstructural protein 5A (NS5A) was found to interact with PI4KIIIα and stimulate its kinase activity. The absence of PI4KIIIα activity induced a dramatic change in the ultrastructural morphology of the membranous HCV replication complex. Our analysis suggests that the direct activation of a lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex.
doi:10.1016/j.chom.2010.12.002
PMCID: PMC3433060  PMID: 21238945
20.  Loss of the abundant nuclear non-coding RNA MALAT1 is compatible with life and development 
RNA Biology  2012;9(8):1076-1087.
The metastasis-associated lung adenocarcinoma transcript 1, MALAT1, is a long non-coding RNA (lncRNA) that has been discovered as a marker for lung cancer metastasis. It is highly abundant, its expression is strongly regulated in many tumor entities including lung adenocarcinoma and hepatocellular carcinoma as well as physiological processes, and it is associated with many RNA binding proteins and highly conserved throughout evolution. The nuclear transcript MALAT-1 has been functionally associated with gene regulation and alternative splicing and its regulation has been shown to impact proliferation, apoptosis, migration and invasion.
 
Here, we have developed a human and a mouse knockout system to study the loss-of-function phenotypes of this important ncRNA. In human tumor cells, MALAT1 expression was abrogated using Zinc Finger Nucleases. Unexpectedly, the quantitative loss of MALAT1 did neither affect proliferation nor cell cycle progression nor nuclear architecture in human lung or liver cancer cells. Moreover, genetic loss of Malat1 in a knockout mouse model did not give rise to any obvious phenotype or histological abnormalities in Malat1-null compared with wild-type animals. Thus, loss of the abundant nuclear long ncRNA MALAT1 is compatible with cell viability and normal development.
doi:10.4161/rna.21089
PMCID: PMC3551862  PMID: 22858678
MALAT1; human knockout model; knockout mouse; long non-coding RNA
21.  E–N-cadherin heterodimers define novel adherens junctions connecting endoderm-derived cells 
The Journal of Cell Biology  2011;195(5):873-887.
Contradicting the “cadherin switch” model, mixed E-cadherin–N-cadherin heterodimeric adherens junctions are prevalent in a variety of endodermal cells and endoderm-derived tumors.
Intercellular junctions play a pivotal role in tissue development and function and also in tumorigenesis. In epithelial cells, decrease or loss of E-cadherin, the hallmark molecule of adherens junctions (AJs), and increase of N-cadherin are widely thought to promote carcinoma progression and metastasis. In this paper, we show that this “cadherin switch” hypothesis does not hold for diverse endoderm-derived cells and cells of tumors derived from them. We show that the cadherins in a major portion of AJs in these cells can be chemically cross-linked in E–N heterodimers. We also show that cells possessing E–N heterodimer AJs can form semistable hemihomotypic AJs with purely N-cadherin–based AJs of mesenchymally derived cells, including stroma cells. We conclude that these heterodimers are the major AJ constituents of several endoderm-derived tissues and tumors and that the prevailing concept of antagonistic roles of these two cadherins in developmental and tumor biology has to be reconsidered.
doi:10.1083/jcb.201106023
PMCID: PMC3257573  PMID: 22105347
22.  Transient telomere dysfunction induces chromosomal instability and promotes carcinogenesis 
The Journal of Clinical Investigation  2012;122(6):2283-2288.
Telomere shortening limits the proliferative capacity of a cell, but perhaps surprisingly, shortening is also known to be associated with increased rates of tumor initiation. A current hypothesis suggests that telomere dysfunction increases tumor initiation by induction of chromosomal instability, but that initiated tumors need to reactivate telomerase for genome stabilization and tumor progression. This concept has not been tested in vivo, since appropriate mouse models were lacking. Here, we analyzed hepatocarcinogenesis in a mouse model of inducible telomere dysfunction on a telomerase-proficient background, in telomerase knockout mice with chronic telomere dysfunction (G3 mTerc–/–), and in WT mice with functional telomeres and telomerase. Transient or chronic telomere dysfunction enhanced the rates of chromosomal aberrations during hepatocarcinogenesis, but only telomerase-proficient mice exhibited significantly increased rates of macroscopic tumor formation in response to telomere dysfunction. In contrast, telomere dysfunction resulted in pronounced accumulation of DNA damage, cell-cycle arrest, and apoptosis in telomerase-deficient liver tumors. Together, these data provide in vivo evidence that transient telomere dysfunction during early or late stages of tumorigenesis promotes chromosomal instability and carcinogenesis in telomerase-proficient mice.
doi:10.1172/JCI61745
PMCID: PMC3366409  PMID: 22622037
23.  Unique Cell Type-Specific Junctional Complexes in Vascular Endothelium of Human and Rat Liver Sinusoids 
PLoS ONE  2012;7(4):e34206.
Liver sinusoidal endothelium is strategically positioned to control access of fluids, macromolecules and cells to the liver parenchyma and to serve clearance functions upstream of the hepatocytes. While clearance of macromolecular debris from the peripheral blood is performed by liver sinusoidal endothelial cells (LSECs) using a delicate endocytic receptor system featuring stabilin-1 and -2, the mannose receptor and CD32b, vascular permeability and cell trafficking are controlled by transcellular pores, i.e. the fenestrae, and by intercellular junctional complexes. In contrast to blood vascular and lymphatic endothelial cells in other organs, the junctional complexes of LSECs have not yet been consistently characterized in molecular terms. In a comprehensive analysis, we here show that LSECs express the typical proteins found in endothelial adherens junctions (AJ), i.e. VE-cadherin as well as α-, β-, p120-catenin and plakoglobin. Tight junction (TJ) transmembrane proteins typical of endothelial cells, i.e. claudin-5 and occludin, were not expressed by rat LSECs while heterogenous immunreactivity for claudin-5 was detected in human LSECs. In contrast, junctional molecules preferentially associating with TJ such as JAM-A, B and C and zonula occludens proteins ZO-1 and ZO-2 were readily detected in LSECs. Remarkably, among the JAMs JAM-C was considerably over-expressed in LSECs as compared to lung microvascular endothelial cells. In conclusion, we show here that LSECs form a special kind of mixed-type intercellular junctions characterized by co-occurrence of endothelial AJ proteins, and of ZO-1 and -2, and JAMs. The distinct molecular architecture of the intercellular junctional complexes of LSECs corroborates previous ultrastructural findings and provides the molecular basis for further analyses of the endothelial barrier function of liver sinusoids under pathologic conditions ranging from hepatic inflammation to formation of liver metastasis.
doi:10.1371/journal.pone.0034206
PMCID: PMC3317944  PMID: 22509281
24.  Neoadjuvant chemoradiation with Gemcitabine for locally advanced pancreatic cancer 
Introduction
To evaluate efficacy and secondary resectability in patients with locally advanced pancreatic cancer (LAPC) treated with neoadjuvant chemoradiotherapy (CRT).
Patients and methods
A total of 215 patients with locally advanced pancreatic cancer were treated with chemoradiation at a single institution. Radiotherapy was delivered with a median dose of 52.2 Gy in single fractions of 1.8 Gy. Chemotherapy was applied concomitantly as gemcitabine (GEM) at a dose of 300 mg/m2 weekly, followed by adjuvant cycles of full-dose GEM (1000 mg/m2). After neoadjuvant CRT restaging was done to evaluate secondary resectability. Overall and disease-free survival were calculated and prognostic factors were estimated.
Results
After CRT a total of 26% of all patients with primary unresectable LAPC were chosen to undergo secondary resection. Tumour free resection margins could be achieved in 39.2% (R0-resection), R1-resections were seen in 41.2%, residual macroscopic tumour in 11.8% (R2) and in 7.8% resection were classified as Rx. Patients with complete resection after CRT showed a significantly increased median overall survival (OS) with 22.1 compared to 11.9 months in non-resected patients. Median OS and disease-free survival (DFS) of all patients were 12.3 and 8.1 months respectively. In most cases the first site of disease progression was systemic with hepatic (52%) and peritoneal (36%) metastases.
Discussion
A high percentage of patients with locally advanced pancreatic cancer can undergo secondary resection after gemcitabine-based chemoradiation and has a relative long-term prognosis after complete resection.
doi:10.1186/1748-717X-7-28
PMCID: PMC3338091  PMID: 22385572
25.  Decentral gene expression analysis for ER+/Her2− breast cancer: results of a proficiency testing program for the EndoPredict assay 
Virchows Archiv  2012;460(3):251-259.
Gene expression profiles provide important information about the biology of breast tumors and can be used to develop prognostic tests. However, the implementation of quantitative RNA-based testing in routine molecular pathology has not been accomplished, so far. The EndoPredict assay has recently been described as a quantitative RT-PCR-based multigene expression test to identify a subgroup of hormone–receptor-positive tumors that have an excellent prognosis with endocrine therapy only. To transfer this test from bench to bedside, it is essential to evaluate the test–performance in a multicenter setting in different molecular pathology laboratories. In this study, we have evaluated the EndoPredict (EP) assay in seven different molecular pathology laboratories in Germany, Austria, and Switzerland. A set of ten formalin-fixed paraffin-embedded tumors was tested in the different labs, and the variance and accuracy of the EndoPredict assays were determined using predefined reference values. Extraction of a sufficient amount of RNA and generation of a valid EP score was possible for all 70 study samples (100%). The EP scores measured by the individual participants showed an excellent correlation with the reference values, respectively, as reflected by Pearson correlation coefficients ranging from 0.987 to 0.999. The Pearson correlation coefficient of all values compared to the reference value was 0.994. All laboratories determined EP scores for all samples differing not more than 1.0 score units from the pre-defined references. All samples were assigned to the correct EP risk group, resulting in a sensitivity and specificity of 100%, a concordance of 100%, and a kappa of 1.0. Taken together, the EndoPredict test could be successfully implemented in all seven participating laboratories and is feasible for reliable decentralized assessment of gene expression in luminal breast cancer.
doi:10.1007/s00428-012-1204-4
PMCID: PMC3306560  PMID: 22371223
Breast cancer; Prognosis; mRNA; Quality control

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