Heterozygous loss-of-function variants in DOCK4 cause neurodevelopmental delay and microcephaly.

Neurons form the basic anatomical and functional structure of the nervous system, and defects in neuronal differentiation or formation of neurites are associated with various psychiatric and neurodevelopmental disorders. Dynamic changes in the cytoskeleton are essential for this process, which is, inter alia, controlled by the dedicator of cytokinesis 4 (DOCK4) through the activation of RAC1. Here, we clinically describe 7 individuals (6 males and one female) with variants in DOCK4 and overlapping phenotype of mild to severe global developmental delay. Additional symptoms include coordination or gait abnormalities, microcephaly, nonspecific brain malformations, hypotonia and seizures. Four individuals carry missense variants (three of them detected de novo) and three individuals carry null variants (two of them maternally inherited). Molecular modeling of the heterozygous missense variants suggests that the majority of them affect the globular structure of DOCK4. In vitro functional expression studies in transfected Neuro-2A cells showed that all missense variants impaired neurite outgrowth. Furthermore, Dock4 knockout Neuro-2A cells also exhibited defects in promoting neurite outgrowth. Our results, including clinical, molecular and functional data, suggest that loss-of-function variants in DOCK4 probable cause a variable spectrum of a novel neurodevelopmental disorder with microcephaly.

Myelin insulation as a risk factor for axonal degeneration in autoimmune demyelinating disease.

Axonal degeneration determines the clinical outcome of multiple sclerosis and is thought to result from exposure of denuded axons to immune-mediated damage. Therefore, myelin is widely considered to be a protective structure for axons in multiple sclerosis. Myelinated axons also depend on oligodendrocytes, which provide metabolic and structural support to the axonal compartment. Given that axonal pathology in multiple sclerosis is already visible at early disease stages, before overt demyelination, we reasoned that autoimmune inflammation may disrupt oligodendroglial support mechanisms and hence primarily affect axons insulated by myelin. Here, we studied axonal pathology as a function of myelination in human multiple sclerosis and mouse models of autoimmune encephalomyelitis with genetically altered myelination. We demonstrate that myelin ensheathment itself becomes detrimental for axonal survival and increases the risk of axons degenerating in an autoimmune environment. This challenges the view of myelin as a solely protective structure and suggests that axonal dependence on oligodendroglial support can become fatal when myelin is under inflammatory attack.

Dual Targeting of the EGFR/HER2 Pathway in Combination with Systemic Chemotherapy in Refractory Pancreatic Cancer-The CONKO-008 Phase I Investigation.

BACKGROUND: Primary objective of this present trial was to define the maximum tolerable dose of lapatinib in combination with oxaliplatin, 5-fluorouracil, and folinic acid (OFF) in refractory pancreatic cancer. The secondary objective was to assess the safety and efficacy of lapatinib plus OFF. METHODS: We conducted a phase I trial using an accelerated dose escalation design in patients with refractory pancreatic cancer. Lapatinib was given on days 1 to 42 in combination with folinic acid 200 mg/m2 day + 5-fluorouracil 2000 mg/m2 (24 h) on days 1, 8, 15, and 22, and oxaliplatin 85 mg/m2 days 8 and 22 of a 43-day cycle (OFF). Toxicity and efficacy were evaluated. RESULTS: In total, eighteen patients were enrolled: dose level 1 (1000 mg) was assigned to seven patients, dose level 2 (1250 mg), five patients; and dose level 3 (1500 mg), six patients. Dose-limiting toxicities were diarrhea and/or neutropenic enterocolitis observed in two of six patients: one diarrhea III°, one diarrhea IV°, as well as neutropenic enterocolitis. The maximum tolerable dose of lapatinib was 1250 mg OD. CONCLUSIONS: The combination of lapatinib 1250 mg OD with platinum-containing chemotherapy is safe and feasible in patients with refractory pancreatic cancer and warrants further investigation.

Alpha synuclein processing by MMP-3 - implications for synucleinopathies.

α-Synuclein (aSyn) is a protein implicated in physiological functions such as neurotransmitter release at the synapse and the regulation of gene expression in the nucleus. In addition, pathological aSyn assemblies are characteristic for a class of protein aggregation disorders referred to as synucleinopathies, where aSyn aggregates appear as Lewy bodies and Lewy neurites or as glial cytoplasmic inclusions. We recently discovered a novel post-translational pyroglutamate (pGlu) modification at Gln79 of N-truncated aSyn that promotes oligomer formation and neurotoxicity in human synucleinopathies. A priori, the appearance of pGlu79-aSyn in vivo involves a two-step process of free N-terminal Gln79 residue generation and subsequent cyclization of Gln79 into pGlu79. Prime candidate enzymes for these processes are matrix metalloproteinase-3 (MMP-3) and glutaminyl cyclase (QC). Here, we analyzed the expression of aSyn, MMP-3, QC and pGlu79-aSyn in brains of two transgenic mouse models for synucleinopathies (BAC-SNCA and ASO) by triple immunofluorescent labellings and confocal laser scanning microscopy. We report a co-localization of these proteins in brain structures typically affected by aSyn pathology, namely hippocampus in BAC-SNCA mice and substantia nigra in ASO mice. In addition, Western blot analyses revealed a high abundance of QC, MMP-3 and transgenic human aSyn in brain stem and thalamus but lower levels in cortex/hippocampus, whereas endogenous mouse aSyn was found to be most abundant in cortex/hippocampus, followed by thalamus and brain stem. During aging of ASO mice, we observed no differences between controls and transgenic mice in MMP-3 levels but higher QC content in thalamus of 6-month-old transgenic mice. Transgenic human aSyn abundance transiently increased and then showed decrease in oldest ASO mice analyzed. Immunohistochemistry revealed a successive increase in intraneuronal and extracellular formation of pGlu79-aSyn in substantia nigra during aging of ASO mice. Together, our data are supportive for a role of MMP-3 and QC in the generation of pGlu79-aSyn in brains affected by aSyn pathology.

Finding the best clearing approach - Towards 3D wide-scale multimodal imaging of aged human brain tissue.

The accessibility of new wide-scale multimodal imaging techniques led to numerous clearing techniques emerging over the last decade. However, clearing mesoscopic-sized blocks of aged human brain tissue remains an extremely challenging task. Homogenizing refractive indices and reducing light absorption and scattering are the foundation of tissue clearing. Due to its dense and highly myelinated nature, especially in white matter, the human brain poses particular challenges to clearing techniques. Here, we present a comparative study of seven tissue clearing approaches and their impact on aged human brain tissue blocks (> 5 mm). The goal was to identify the most practical and efficient method in regards to macroscopic transparency, brief clearing time, compatibility with immunohistochemical processing and wide-scale multimodal microscopic imaging. We successfully cleared 26 × 26 × 5 mm3-sized human brain samples with two hydrophilic and two hydrophobic clearing techniques. Optical properties as well as light and antibody penetration depths highly vary between these methods. In addition to finding the best clearing approach, we compared three microscopic imaging setups (the Zeiss Laser Scanning Microscope (LSM) 880 , the Miltenyi Biotec Ultramicroscope ll (UM ll) and the 3i Marianas LightSheet microscope) regarding optimal imaging of large-scale tissue samples. We demonstrate that combining the CLARITY technique (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) with the Zeiss LSM 880 and combining the iDISCO technique (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) with the Miltenyi Biotec UM ll are the most practical and efficient approaches to sufficiently clear aged human brain tissue and generate 3D microscopic images. Our results point out challenges that arise from seven clearing and three imaging techniques applied to non-standardized tissue samples such as aged human brain tissue.

Hepatitis B subviral envelope particles use the COPII machinery for intracellular transport via selective exploitation of Sec24A and Sec23B.

Hepatitis B virus (HBV) is a leading cause of liver disease. Its success as a human pathogen is related to the immense production of subviral envelope particles (SVPs) contributing to viral persistence by interfering with immune functions. To explore cellular pathways involved in SVP formation and egress, we investigated host-pathogen interactions. Yeast-based proteomics revealed Sec24A, a component of the coat protein complex II (COPII), as an interaction partner of the HBV envelope S domain. To understand how HBV co-opts COPII as a proviral machinery, we studied roles of key Sec proteins in HBV-expressing liver cells. Silencing of Sar1, Sec23, and Sec24, which promote COPII assembly concomitant with cargo loading, strongly diminished endoplasmic reticulum (ER) envelope export and SVP secretion. By analysing Sec paralog specificities, we unexpectedly found that the HBV envelope is a selective interaction partner of Sec24A and Sec23B whose functions could not be substituted by their related isoforms. In support, we found that HBV replication upregulated Sec24A and Sec23B transcription. Furthermore, HBV encountered the Sec24A/Sec23B complex via an interaction that involved the N-terminal half of Sec24A and a di-arginine motif of its S domain, mirroring a novel ER export code. Accordingly, an interference with the COPII/HBV cross-talk might display a tool to effectively inhibit SVP release.

Novel Protein Kinase Inhibitors Related to Tau Pathology Modulate Tau Protein-Self Interaction Using a Luciferase Complementation Assay.

The current number of drugs available for the treatment of Alzheimer's disease (AD) is strongly limited and their benefit for therapy is given only in the early state of the disease. An effective therapy should affect those processes which mainly contribute to the neuronal decay. There have been many approaches for a reduction of toxic Aß peptides which mostly failed to halt cognitive deterioration in patients. The formation of neurofibrillary tangles (NFT) and its precursor tau oligomers have been suggested as main cause of neuronal degeneration because of a direct correlation of their density to the degree of dementia. Reducing of tau aggregation may be a viable approach for the treatment of AD. NFT consist of hyperphosphorylated tau protein and tau hyperphosphorylation reduces microtubule binding. Several protein kinases are discussed to be involved in tau hyperphosphorylation. We developed novel inhibitors of three protein kinases (gsk-3ß, cdk5, and cdk1) and discussed their activity in relation to tau phosphorylation and on tau⁻tau interaction as a nucleation stage of a tau aggregation in cells. Strongest effects were observed for those inhibitors with effects on all the three kinases with emphasis on gsk-3ß in nanomolar ranges.

TDP-43 self-interaction is modulated by redox-active compounds Auranofin, Chelerythrine and Riluzole.

Amyotrophic lateral sclerosis (ALS) represents a fatal neurodegenerative disease, which is characterized by a rapid loss of lower and upper motor neurons. As a major neuropathological hallmark, protein aggregates containing the Transactivating Response Region (TAR) DNA Binding Protein (TDP-43) are detectable in about 95% of sporadic ALS patients. TDP-43 interacts with itself physiologically to form liquid droplets, which may progress to pathological aggregates. In this study, we established the NanoBit luciferase complementation assay to measure TDP-43 self-interaction and found the fusion of the split luciferase subunits to the N-terminus of the protein as the strongest interacting partners. A screen of pharmacologically active compounds from the LOPAC®1280 library identified auranofin, chelerythrine and riluzole as dose-dependent inhibitors of TDP-43 self-interaction. Further analysis of drug action of the gold-containing thioredoxin reductase inhibitor auranofin revealed a redistribution from insoluble TDP-43 protein pool to PBS-soluble protein pool in N2a cells. In addition, auranofin treatment diminished reduced glutathione as a sign for oxidative modulation.

CONKO-005: Adjuvant Chemotherapy With Gemcitabine Plus Erlotinib Versus Gemcitabine Alone in Patients After R0 Resection of Pancreatic Cancer: A Multicenter Randomized Phase III Trial.

Purpose Gemcitabine is standard of care in the adjuvant treatment of resectable pancreatic ductal adenocarcinoma (PDAC). The epidermal growth factor receptor tyrosine kinase inhibitor erlotinib in combination with gemcitabine has shown efficacy in the treatment of advanced PDAC and was considered to improve survival in patients with primarily resectable PDAC after R0 resection. Patients and Methods In an open-label, multicenter trial, patients were randomly assigned to one of two study arms: gemcitabine 1,000 mg/m2 days 1, 8, 15, every 4 weeks plus erlotinib 100 mg once per day (GemErlo) or gemcitabine (Gem) alone for six cycles. The primary end point of the study was to improve disease-free survival (DFS) from 14 to 18 months by adding erlotinib to gemcitabine. Results In all, 436 patients were randomly assigned at 57 study centers between April 2008 and July 2013. A total of 361 instances (83%) of disease recurrence were observed after a median follow-up of 54 months. Median treatment duration was 22 weeks in both arms. There was no difference in median DFS (GemErlo 11.4 months; Gem 11.4 months) or median overall survival (GemErlo 24.5 months; Gem 26.5 months). There was a trend toward long-term survival in favor of GemErlo (estimated survival after 1, 2, and 5 years for GemErlo was 77%, 53%, and 25% v 79%, 54%, and 20% for Gem, respectively). The occurrence or the grade of rash was not associated with a better survival in the GemErlo arm. Conclusion To the best of our knowledge, CONKO-005 is the first study to investigate the combination of chemotherapy and a targeted therapy in the adjuvant treatment of PDAC. GemErlo for 24 weeks did not improve DFS or overall survival over Gem.

Is sporadic Alzheimer's disease a developmental disorder?

Alzheimer's disease (AD) is a neurodegenerative disorder of higher age that specifically occurs in human. Its clinical phase, characterized by a decline in physiological, psychological, and social functioning, is preceded by a long clinically silent phase of at least several decades that might perhaps even start very early in life. Overall, key functional abilities in AD patients decline in reverse order of the development of these abilities during normal childhood and adolescence. Early symptoms of AD, thus, typically affect mental functions that have been acquired only during very recent hominid evolution and as such are specific to human. Neurofibrillar degeneration, a typical neuropathological lesion of the disease and one of the most robust pathological correlates of cognitive impairment, is rarely seen in non-primate mammals and even non-human primates hardly develop a pathology comparable to those seen in AD patients. Neurofibrillar degeneration is not randomly distributed throughout the AD brain. It preferentially affects brain areas that become increasingly predominant during the evolutionary process of encephalization. During progression of the disease, it affects cortical areas in a stereotypic sequence that inversely recapitulates ontogenetic brain development. The specific distribution of cortical pathology in AD, moreover, appears to be determined by the modular organization of the cerebral cortex which basically is a structural reflection of its ontogeny. Here, we summarize recent evidence that phylogenetic and ontogenetic dimensions of brain structure and function provide the key to our understanding of AD. More recent molecular biological studies of the potential pathogenetic role of a genomic mosaic in the brains of patients with AD might even provide arguments for a developmental origin of AD. This article is part of a series "Beyond Amyloid".

Tau and tauopathies.

Most neurodegenerative diseases are characterized by intracellular aggregates of insoluble proteins. As for the majority of these disorders, aetiology and pathogenesis are only poorly understood; current nosological concepts are largely based on these molecular signatures of protein aggregates which also provide valuable tools for neuropathological differential diagnosis. The microtubule associated protein tau is one of these proteins that form intracellular fibrillary deposits in neurons and glial cells of a large variety of disorders today collectively referred to as tauopathies. While dysfunction of tau has unequivocally been shown to be able to cause neurodegeneration, the precise mechanisms of how tau is involved in neurodegenerative disorders is still poorly understood. After research has focused for several decades on the axonal function of tau and on the fibrillar tau aggregation, more recent cell biological studies have opened up new insights into the role of tau at the synapse and in the nucleus. According to currently emerging cell biological concepts, tau might play a role in the regulation of neuronal plasticity in a wide array of neuronal networks. In addition, it might be involved in regulating genome stability. The most intriguing question relevant both to physiological and pathophysiological function of tau is the biological meaning of the large heterogeneity of isoforms of tau which apparently is a rather promiscuous molecule. The present review is divided into two parts. First, we give an overview on the molecular biology and cell biology of tau and its physiological functions. The second part deals with the pathophysiology of tau and description of tauopathies which comprise more than 20 disorders including Alzheimer's disease, progressive supranuclear palsy, cortico basal degeneration, Pick's disease and others.

Tau phosphorylation-associated spine regression does not impair hippocampal-dependent memory in hibernating golden hamsters.

The microtubule-associated protein tau, in its hyperphosphorylated form, is the major component of paired helical filaments and other aggregates in neurodegenerative disorders commonly referred to as "tauopathies". Recent evidence, however, indicates that mislocalization of hyperphosphorylated tau to subsynaptic sites leads to synaptic impairment and cognitive decline even long before formation of tau aggregates and neurodegeneration occur. A similar, but reversible hyperphosphorylation of tau occurs under physiologically controlled conditions during hibernation. Here, we study the hibernating Golden hamster (Syrian hamster, Mesocricetus auratus). A transient spine reduction was observed in the hippocampus, especially on apical dendrites of hippocampal CA3 pyramidal cells, but not on their basal dendrites. This distribution of structural synaptic regression was correlated to the distribution of phosphorylated tau, which was highly abundant in apical dendrites but hardly detectable in basal dendrites. Surprisingly, hippocampal memory assessed by a labyrinth maze was not affected by hibernation. The present study suggests a role for soluble hyperphosphorylated tau in the process of reversible synaptic regression, which does not lead to memory impairment during hibernation. We hypothesize that tau phosphorylation associated spine regression might mainly affect unstable/dynamic spines while sparing established/stable spines.

Second-Line Treatment in Pancreatic Cancer Patients: Who Profits?--Results From the CONKO Study Group.

OBJECTIVES: With increasing numbers of therapeutic options in inoperable pancreatic cancer (PAC), patients tend to receive more than just a first line (FL) therapy. METHODS: All patients who started FL for PAC at our institution (1997-2012) were retrospectively studied to identify patient's and treatment characteristics. Significant parameters in regard to second-line (SL) related survival were looked for as the basis for a prognostic model. This score was validated in a patient cohort from the CONKO-003 study. RESULTS: Two hundred eighty of 521 (53.7%) patients received SL therapy, median overall survival (OS) from the beginning of SL (OS2) was 5.1 months. Significant more SL patients had undergone surgery, a higher Karnofsky performance state (KPS) and a duration of FL longer than 4 months.Prognostic factors impacting OS2 were KPS, carbohydrate antigen 19-9 levels at start of SL and the duration of FL. These 3 factors establish a prognostic score--validated in CONKO-003--for SL patients with 3 subgroups: "good" (median OS2, 9.3 months), "intermediate" (median OS2, 7.1 months), "poor" prognosis (median OS2, 3.8 months; P < 0.001). CONCLUSIONS: Among patients with PAC, more than 50% receive SL therapy. Our prognostic model identifies 3 subgroups and can identify patients with a maximum benefit of SL therapy.

Efficacy of Prophylactic Low-Molecular Weight Heparin for Ambulatory Patients With Advanced Pancreatic Cancer: Outcomes From the CONKO-004 Trial.

PURPOSE: Advanced pancreatic cancer (APC), in addition to its high mortality, accounts for the highest rates of venous thromboembolic events (VTEs). Enoxaparin, a low-molecular weight heparin, is effective in prevention and treatment of VTEs. Some small studies have indicated that this benefit might extend to patients with cancer. PATIENTS AND METHODS: Patients with histologically proven APC were randomly assigned to ambulant first-line chemotherapy and prophylactic use of enoxaparin or chemotherapy alone to investigate the probable reduction in symptomatic VTEs and the impact on survival. RESULTS: A total of 312 patients were recruited as one of the protocol end points was reached. Within the first 3 months, the numbers of symptomatic VTEs were as follows: 15 of 152 patients in the observation group and two of 160 patients in the enoxaparin group (hazard ratio [HR], 0.12; 95% CI, 0.03 to 0.52; χ(2) P = .001). The numbers of major bleeding events were as follows: five of 152 patients in the observation arm and seven of 160 patients in the enoxaparin arm (HR, 1.4; 95% CI, 0.35 to 3.72; χ(2) P = 1.0). Overall cumulative incidence rates of symptomatic VTEs were 15.1% (observation) and 6.4% (enoxaparin; HR, 0.40; 95% CI, 0.19 to 0.83; P = .01). Progression-free (HR, 1.06; 95% CI, 0.84 to 1.32; P = .64) and overall survival (HR, 1.01; 95% CI, 0.87 to 1.38; P = .44) did not differ between groups. CONCLUSION: This study demonstrates the high efficacy and feasibility of primary pharmacologic prevention of symptomatic VTEs in outpatients with APC. Treatment efficacy was not affected by simultaneous treatment with enoxaparin in this trial setting.

Brain hypometabolism triggers PHF-like phosphorylation of tau, a major hallmark of Alzheimer's disease pathology.

Sporadic Alzheimer's disease (AD) is a chronic progressive neurodegenerative disorder of unknown cause characterized by fibrillar accumulation of the Aß-peptide and aggregates of the microtubule-associated protein tau in a hyperphosphorylated form. Already at preclinical stages, AD is characterized by hypometabolic states which are a good predictor of cognitive decline. Here, we summarize recent evidence derived from the study of hibernating animals that brain hypometabolism can trigger PHF-like hyperphosphorylation of tau. We put forward the concept that particular types of neurons respond to a hypometabolic state with an elevated phosphorylation of tau protein which represents a physiological mechanism involved in regulating synaptic gain. If, in contrast to hibernation, the hypometabolic state is not terminated after a definite time but rather persists and progresses, the elevated phosphorylation of tau protein endures and the protective reaction associated with it might turn into a pathological cascade leading to neurodegeneration.

Second-line oxaliplatin, folinic acid, and fluorouracil versus folinic acid and fluorouracil alone for gemcitabine-refractory pancreatic cancer: outcomes from the CONKO-003 trial.

PURPOSE: To assess the efficacy of a second-line regimen of oxaliplatin and folinic acid-modulated fluorouracil in patients with advanced pancreatic cancer who have experienced progression while receiving gemcitabine monotherapy. PATIENTS AND METHODS: A randomized, open-label, phase III study was conducted in 16 institutions throughout Germany. Recruitment ran from January 2004 until May 2007, and the last follow-up concluded in December 2012. Overall, 168 patients age 18 years or older who experienced disease progression during first-line gemcitabine therapy were randomly assigned to folinic acid and fluorouracil (FF) or oxaliplatin and FF (OFF). Patients were stratified according to the presence of metastases, duration of first-line therapy, and Karnofsky performance status. RESULTS: Median follow-up was 54.1 months, and 160 patients were eligible for the primary analysis. The median overall survival in the OFF group (5.9 months; 95% CI, 4.1 to 7.4) versus the FF group (3.3 months; 95% CI, 2.7 to 4.0) was significantly improved (hazard ratio [HR], 0.66; 95% CI, 0.48 to 0.91; log-rank P = .010). Time to progression with OFF (2.9 months; 95% CI, 2.4 to 3.2) versus FF (2.0 months; 95% CI, 1.6 to 2.3) was significantly extended also (HR, 0.68; 95% CI, 0.50 to 0.94; log-rank P = .019). Rates of adverse events were similar between treatment arms, with the exception of grades 1 to 2 neurotoxicity, which were reported in 29 patients (38.2%) and six patients (7.1%) in the OFF and FF groups, respectively (P < .001). CONCLUSION: Second-line OFF significantly extended the duration of overall survival when compared with FF alone in patients with advanced gemcitabine-refractory pancreatic cancer.

KRAS mutations in codon 12 or 13 are associated with worse prognosis in pancreatic ductal adenocarcinoma.

OBJECTIVE: Mutations in the KRAS and P53 genes belong to the most frequently observed genetic alterations in pancreatic ductal adenocarcinoma. The aim of this study was to examine the frequency and prognostic impact of KRAS mutations. In addition, we attempted to define molecular subgroups with distinct biologic behavior by combination of KRAS sequencing data with p53 protein expression data. METHODS: KRAS mutational analyses were performed in a study group of 153 patients by Sanger sequencing. Immunohistochemistry for p53 was performed on tissue microarrays. RESULTS: KRAS mutations in codon 12 or 13 were found in 68% of cases. Nuclear staining for p53 was detectable in 110 (68%) of 162 evaluable cases. We found no correlation between KRAS mutational status and p53 expression. KRAS mutational status but not p53 immunohistochemistry was an independent prognostic factor in the study group (P = 0.02). In a stratified analysis according to KRAS mutational status, p53 expression failed to define prognostic groups beyond the impact of KRAS mutational status. CONCLUSIONS: Our results support the crucial role of KRAS mutational status in pancreatic cancer biology. KRAS mutational status may serve as a prognostic marker. However, its predictive role for targeted therapies remains to be evaluated.

Involvement of ESCRT-II in hepatitis B virus morphogenesis.

The hepatitis B virus (HBV) is an enveloped DNA virus that replicates via reverse transcription of its pregenomic RNA (pgRNA). Budding of HBV is supposed to occur at intracellular membranes and requires scission functions of the endosomal sorting complex required for transport (ESCRT) provided by ESCRT-III and VPS4. Here, we have investigated the impact of the upstream-acting ESCRT-I and ESCRT-II complexes in HBV morphogenesis. RNA interference knockdown of the ESCRT-I subunits TSG101 and VPS28 did not block, but rather stimulate virus release. In contrast, RNAi-mediated depletion of the ESCRT-II components EAP20, EAP30 and EAP45 greatly reduced virus egress. By analyzing different steps of the HBV maturation pathway, we find that the knockdown of ESCRT-II not only inhibited the production and/or release of enveloped virions, but also impaired intracellular nucleocapsid formation. Transcription/translation studies revealed that the depletion of ESCRT-II neither affected the synthesis and nuclear export of HBV-specific RNAs nor the expression of the viral core and envelope proteins. Moreover, the absence of ESCRT-II had no effects on the assembly capability and integrity of HBV core/capsids. However, the level of encapsidated pgRNA was significantly reduced in ESCRT-II-depleted cells, implicating that ESCRT-II directs steps accompanying the formation of replication-competent nucleocapsids, like e.g. assisting in RNA trafficking and encapsidation. In support of this, the capsid protein was found to interact and colocalize with ESCRT-II subunits in virus-producing cells. Together, these results indicate an essential role for ESCRT-II in the HBV life cycle and suggest that ESCRT-II functions prior to the final HBV budding reaction.

Intensified chemotherapy and simultaneous treatment with heparin in outpatients with pancreatic cancer - the CONKO 004 pilot trial.

BACKGROUND: Advanced pancreatic cancer (APC), beside its high mortality, causes the highest rates of venous thromboembolic events (VTE). Enoxaparin, a low molecular weight heparin (LMWH), is effective in prevention and treatment of VTE. Some small studies indicated that this benefit might extend to patients with cancer and probably prolong survival due to independent mechanisms. We initiated this safety investigation to get feasibility information on intensified chemotherapy combined with LMWH in outpatients with APC treated in 1st line. METHODS: The trial was a prospective, open-label, single center investigation in outpatients with inoperable pancreatic cancer who were treated with intensified first-line chemotherapy along with concomitant application of subcutaneous LMWH. The combined chemotherapy consisted of gemcitabine 1 g/m2 (30 min), 5-FU 750 mg/m2 (24 h), folinic acid 200 mg/m2 (30 min), and Cisplatin 30 mg/m2 (90 min) on day 1 and 8; q3w for the first 12 weeks (GFFC) followed by gemcitabine alone in patients without cancer progression. The simultaneous application of prophylactic enoxaparin started on day 1 of chemotherapy with a fixed dose of 40 mg daily. Statistical analyses were performed using R 3.01 with software package CMPRSK and SPSS software v19.0. RESULTS: The investigation was stopped after recruitment of 19 patients. At this time 15 patients had completed the required 12 weeks of treatment. Based on 71 cycles of GFFC + enoxaparin (median 4/pt [range: 2-4]) and 108 cycles of single-agent gemcitabine + enoxaparin (median 4/pt [range: 0-18]) the cumulative frequency of NCI-CTC toxicities grade 3/4 was below 10%. One case (5%) of a symptomatic non-lethal thromboembolic event was observed while receiving LMWH treatment. No severe bleeding event as defined in the protocol has been observed. The median overall survival was 10.05 [95% CI: 8.67-18.14] months. CONCLUSIONS: The addition of enoxaparin to GFFC chemotherapy is feasible, safe and does not appear to affect the efficacy or the toxicity profile of the chemotherapy regimen in patients with advanced pancreatic adenocarcinoma. Based on these findings we have initiated the randomized CONKO-004 trial to examine whether enoxaparin reduces the incidence of thromboembolic events or increases overall outcome. TRIAL REGISTRATION: Clinical Trials NCT01945879.

Does long-term survival in patients with pancreatic cancer really exist? Results from the CONKO-001 study.

BACKGROUND: Long-term survival (LTS) in patients (pts) with pancreatic cancer is still uncommon, little data is available to identify long-term survivors. The CONKO-001 study, which established gemcitabine after resection as adjuvant therapy, may provide data to answer this question. METHODS: CONKO-001 pts with an overall survival ≥5 years were compared to those who survived <5 years. Central re-evaluation of primary histology was performed. Univariate analysis with the χ(2) -test identified qualifying factors. Logistic regression was used to investigate the influence of these covariates on LTS. RESULTS: Of the evaluable 354 CONKO-001 pts, 54 (15%) with an overall survival ≥5 years were identified. It was possible to obtain tumor specimens of 39 pts (72%). Histological re-evaluation confirmed adenocarcinoma in 38 pts, 1 showed a high-grade neuroendocrine tumor. Univariate analysis for all 53 LTS pts with adenocarcinoma compared to the remaining 300 non-LTS pts revealed as relevant active treatment, tumor grading, tumor size, lymph nodes. No significance could be demonstrated for resection margin, sex, age, Karnofsky performance status, CA 19-9 at study entry. In multivariate analysis, tumor grading, active treatment, tumor size, lymph node involvement were independent prognostic factors for LTS. CONCLUSION: Long-term survival can be achieved in adenocarcinoma of the pancreas.