CSL112 Infusion Rapidly Increases APOA1 Exchange Rate via Specific Serum Amyloid-Poor HDL Subpopulations When Administered to Patients Post-Myocardial Infarction.

BACKGROUND: To characterize the effects of CSL112 (human APOA1 [apolipoprotein A1]) on the APOA1 exchange rate (AER) and the relationships with specific HDL (high-density lipoprotein) subpopulations when administered in the 90-day high-risk period post-acute myocardial infarction. METHODS: A subset of patients (n=50) from the AEGIS-I (ApoA-I Event Reducing in Ischemic Syndromes I) study received either placebo or CSL112 post-acute myocardial infarction. AER was measured in AEGIS-I plasma samples incubated with lipid-sensitive fluorescent APOA1 reporter. HDL particle size distribution was assessed by native gel electrophoresis followed by fluorescent imaging and detection of APOA1 and SAA (serum amyloid A) by immunoblotting. RESULTS: CSL112 infusion increased AER peaking at 2 hours and returning to baseline 24 hours post-infusion. AER correlated with cholesterol efflux capacity (r=0.49), HDL-cholesterol (r=0.30), APOA1 (r=0.48), and phospholipids (r=0.48; all P<0.001) over all time points. Mechanistically, changes in cholesterol efflux capacity and AER induced by CSL112 reflected HDL particle remodeling resulting in increased small HDL species that are highly active in mediating ABCA1 (ATP-binding cassette transporter 1)-dependent efflux, and large HDL species with high capacity for APOA1 exchange. The lipid-sensitive APOA1 reporter predominantly exchanged into SAA-poor HDL particles and weakly incorporated into SAA-enriched HDL species. CONCLUSIONS: Infusion of CSL112 enhances metrics of HDL functionality in patients with acute myocardial infarction. This study demonstrates that in post-acute myocardial infarction patients, HDL-APOA1 exchange involves specific SAA-poor HDL populations. Our data suggest that progressive enrichment of HDL with SAA may generate dysfunctional particles with impaired HDL-APOA1 exchange capacity, and that infusion of CSL112 improves the functional status of HDL with respect to HDL-APOA1 exchange. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT02108262.

Effect of Kinase Inhibitors on the Technetium-99m Uptake into Thyroid Carcinoma Cells In Vitro.

BACKGROUND/AIM: We evaluated the potential of the kinase inhibitors sorafenib, lenvatinib and selumetinib on increasing the uptake of technetium-99m into thyroid cancer cells. MATERIALS AND METHODS: Four established cell lines and three patient's cell cultures were treated with 0.1, 1 and 5 µM of sorafenib, lenvatinib and selumetinib for 72 hours. After incubation with 1 MBq of technetium-99m, the radioactivity uptake was measured. RESULTS: The experiments showed heterogeneous results. Maximum technetium-99m uptake increases of 312% (sorafenib), 326% (lenvatinib) and 759% (selumetinib) were obtained using the highest applied concentrations. In some tests, an uptake reduction or no effect was observed. CONCLUSION: Kinase inhibitors have a positive effect on technetium-99m uptake. Due to study limitations, a redifferentiating effect of the drugs could not be definitely proven. Unspecific cytotoxicity might have a confounding effect.

Different Effects of Two Protocols for Pre-Procedural Analgosedation on Vital Signs in Neonates during and after Endotracheal Intubation.

BACKGROUND: Analgosedation is often used for endotracheal intubation in neonates, but no consensus exists on the optimal pre-procedural medication. AIMS: To compare the time to intubation and vital signs during and after intubation in 2 NICUs using different premedication protocols. METHODS: Prospective observational study in 2 tertiary NICUs, comparing fentanyl and optional vecuronium for elective neonatal endotracheal intubation (NICU-1) with atropine, morphine, midazolam and optional pancuronium (NICU-2). Primary endpoints were: time to intubate and number of intubation attempts; secondary endpoints were: deviations of heart rate, oxygen saturation and blood pressure from baseline until 20 min post intubation. RESULTS: 45 and 30 intubations were analyzed in NICU-1 and NICU-2. Time to intubation was longer in NICU-1 (7 min) than in NICU-2 (4 min; p=0.029), but the mean number of intubation attempts did not differ significantly. Bradycardias (34 vs. 1, p<0.001) and hypoxemias (136 vs. 48, p<0.001) were more frequent in NICU-1, and tachycardias (59 vs. 72, p<0.001) more frequent in NICU-2. Mean arterial blood pressure (MAP) increased in NICU-1 (+6.18 mmHg) and decreased in NICU-2 (-5.83 mmHg), whereas mean heart rates (HR) decreased in NICU-1 (-19.29 bpm) and increased in NICU-2 (+15.93 bpm). MAP and HR returned to baseline 6-10 min after intubation in NICU-1 and after 11-15 min and 16-20 min in NICU-2, respectively. CONCLUSIONS: The two protocols yielded significant differences in the time to intubation and in the extent and duration of physiologic changes during and post-intubation. Short acting drugs should be preferred and vital signs should be closely monitored at least 20 min post intubation. More studies are required to identify analgosedation protocols that minimize potentially harmful events during endotracheal intubation.

Vascularization of Primary, Peripheral Lung Carcinoma in CEUS - A Retrospective Study (n = 89 Patients).

PURPOSE: To describe the vascularization of peripheral lung carcinoma in CEUS and to compare with B-mode ultrasound (US) and clinical data. MATERIALS AND METHODS: From April 2004 until September 2015, n = 89 patients with peripheral lung carcinoma were investigated by B-mode US and CEUS. The extent (EE: hypoechoic, hyperechoic), homogeneity (HE: homogeneous, inhomogeneous) and time of enhancement (TE) have been defined. Early pulmonary-arterial enhancement (PA) before contrast floating to the thoracic wall was differentiated from simultaneous or delayed bronchial-arterial enhancement (BA). CEUS parameters were compared by B-mode US and histology. RESULTS: n = 25 patients had early PA enhancement (TE: 8 ±â€Š3.7 s), and n = 64 (72 %) had simultaneous/delayed BA enhancement (TE: 17.6 ±â€Š6.2 s) (p < 0.001). PA enhancement (EE/HE) was hyperechoic (n = 11/25), homogeneous (n = 11/25) and showed an air bronchogram more often (n = 11/17, p < 0.001). BA enhancement (EE/HE) was frequently hypoechoic (n = 34/64) and inhomogeneous (n = 54/64). BA enhancement was associated with necrosis (n = 36/42, p = 0.009). PA and BA enhancement distributed to different histologies: n = 42 adenocarcinomas (18 PA, 24 BA), n = 30 squamous cell carcinomas (4 PA, 26 BA), n = 13 other types of NSCLC (3 PA, 10 BA), and n = 4 SCLC (0 PA, 4 BA) (p = 0.016). CONCLUSION: The vascularization of peripheral lung carcinomas is heterogeneous and is influenced by histology. In this study, lung carcinomas are predominantly supplied by bronchial arteries, whereas a part of adenocarcinomas and non-adenocarcinomas show PA enhancement.

Selumetinib Activity in Thyroid Cancer Cells: Modulation of Sodium Iodide Symporter and Associated miRNAs.

BACKGROUND: The MEK (mitogen-activated protein kinase)⁻inhibitor selumetinib led to increased radioiodine uptake and retention in a subgroup of patients suffering from radioiodine refractory differentiated thyroid cancer (RR-DTC). We aimed to analyse the effect of selumetinib on the expression of sodium iodide symporter (NIS; SLC5A5) and associated miRNAs in thyroid cancer cells. METHODS: Cytotoxicity was assessed by viability assay in TPC1, BCPAP, C643 and 8505C thyroid cancer cell lines. NIS, hsa-let-7f-5p, hsa-miR-146b-5p, and hsa-miR-146b-3p expression was determined by quantitative RT-PCR. NIS protein was detected by Western blot. Radioiodine uptake was performed with a Gamma counter. RESULTS: Selumetinib caused a significant reduction of cell viability in all thyroid cancer cell lines. NIS transcript was restored by selumetinib in all cell lines. Its protein level was found up-regulated in TPC1 and BCPAP cells and down-regulated in C643 and 8505C cells after treatment with selumetinib. Treatment with selumetinib caused a down-regulation of hsa-let-7f-5p, hsa-miR-146b-5p and hsa-miR-146b-3p in TPC1 and BCPAP cells. In 8505C cells, a stable or down-regulated hsa-miR-146b-5p was detected after 1h and 48h of treatment. C643 cells showed stable or up-regulated hsa-let-7f-5p, hsa-miR-146b-5p and hsa-miR-146b-3p. Selumetinib treatment caused an increase of radioiodine uptake, which was significant in TPC1 cells. CONCLUSIONS: The study shows for the first time that selumetinib restores NIS by the inhibition of its related targeting miRNAs. Further studies are needed to clarify the exact mechanism activated by hsa-miR-146b-5p, hsa-miR-146b-3p and hsa-let7f-5p to stabilise NIS. Restoration of NIS could represent a milestone for the treatment of advanced RR-DTC.

Maintenance of cellular respiration indicates drug resistance in acute myeloid leukemia.

Primary resistance to induction therapy is an unsolved clinical problem in acute myeloid leukemia (AML). Here we investigated drug resistance in AML at the level of cellular metabolism in order to identify early predictors of therapeutic response. Using extracellular flux analysis, we compared metabolic drug responses in AML cell lines sensitive or resistant to cytarabine or sorafenib after 24h of drug treatment to a small cell lung cancer (SCLC) cell line exposed to etoposide. Only drug-resistant AML cells maintained oxidative metabolism upon drug exposure while SCLC cells displayed an overall metabolic shift towards glycolysis, i.e. a Warburg effect to escape drug toxicity. Moreover, primary AML blasts displayed very low glycolytic activity, while oxygen consumption was readily detectable, indicating an essential role of oxidative pathways in the bioenergetics of AML blasts. In line with these observations, analysis of the mitochondrial membrane potential using tetramethylrhodamine ethyl ester staining and flow cytometry allowed for clear discrimination between drug sensitive and resistant AML cell line clones and primary blasts after 24h of treatment with cytarabine or sorafenib. Our data reveal a distinct metabolic phenotype of resistant AML cells and suggest that disrupting oxidative metabolism rather than glycolysis may enhance the cytotoxic effects of chemotherapy in AML.

Expression of hedgehog signalling pathway in anaplastic thyroid cancer.

The purpose of this work is to study the activation of the hedgehog signalling pathway is associated with tumour progression in various types of cancer, hence the development of specific antagonists raises hope for new therapeutic strategies. Therefore, the expression of hedgehog pathway components in anaplastic thyroid cancer (ATC) and effects of the hedgehog inhibitor Cyclopamine on ATC cells were investigated in this study. Expression of the ligand Sonic Hedgehog (SHh), the transmembrane protein Smoothened (Smo), the receptor Patched (Ptc) and the target gene Gli-1 was evaluated in two ATC cell lines (Hth 74, C643) by RT-PCR and in tumour specimens by immunohistochemistry. The corresponding gene products were examined by western blotting analysis. After treatment with different concentrations of Cyclopamine the time-dependent course of cell viability in ATC cell lines was evaluated by MTT assay. SHh, Smo, Ptc and Gli were clearly expressed on mRNA and protein levels in both cell lines and in tumour samples (41 %SHh, 65 %Smo, 65 %Ptc and 65 %Gli). Treatment with Cyclopamine showed a time- and dose-dependent inhibition of cell numbers with IC50 values between 1 and 4 µM in both cell lines, comparable to other types of cancer. In conclusion, we believe that the hedgehog pathway is expressed in anaplastic thyroid carcinoma specimens and proliferation of ATC cell lines can be influenced by the Hh inhibitor Cyclopamine. Aberrant activation of this pathway might be involved in the aggressive biology of anaplastic cancer and further evaluation regarding a possible clinical impact of pathway inhibition is warranted.

Pretherapeutic drug evaluation by tumor xenografting in anaplastic thyroid cancer.

BACKGROUND: Despite various attempts at modifying usual treatment modalities, anaplastic thyroid cancer (ATC) is still associated with unfavorable prognosis. Results of preclinical investigations are often of limited transferability to clinical tumor biology. Individualized multimodal treatment regimens, including novel growth-inhibiting drugs, might be a future option. METHODS: Tumor tissue, freshly prepared from a patient operated for ATC, was xenotransplanted to nude mice. While the patient obtained a hyperfractionated external beam radiation, mice carrying xenotransplanted tumors were randomized (n = 6) and treated by multikinase inhibitors (sorafenib [S]: vascular endothelial growth factor receptor [VEGF-R], platelet derived growth factor receptor, RET; vandetanib [V]: VEGF-R, endothelial growth factor receptor [EGF-R]; and MLN8054 [M]: Aurora kinases [AK]). Antiproliferative, antiangiogenic, and proapoptotic effects were evaluated. RESULTS: Treatment of successfully xenotransplanted fresh ATC tumor tissue by multikinase inhibitors and aurora kinase inhibitor reduced the tumor volume up to 61% depending on the drug and time of application (3 wk of treatment: 46% [M], 34% [V], 30% [S]; 5 wk of treatment: 61% [S]). Tumor cell proliferation (BrdU) was reduced between 34% and 58% [S] and [V]. Reduction of tumor vascularity was between 67% [V] and 33% [S] and was accompanied by decreased EGF-R/VEGF-R2 receptor activity [V/V,S]. Tumor cell apoptosis (caspase 3 activity) increased up to 2.4-fold [S]. CONCLUSIONS: Successful in vivo evaluation of novel drugs in xenotransplanted fresh tumor tissue allows in-time (while patient receives standard treatment) prospective analysis for possible additional clinical application. However, technical specifications have to be taken into account to obtain stable in vivo tumor growth. Based on the individual results, a tailored clinical drug application seems possible.

Fat mass and obesity-associated gene (FTO) in eating disorders: evidence for association of the rs9939609 obesity risk allele with bulimia nervosa and anorexia nervosa.

OBJECTIVE: The common single nucleotide polymorphism (SNP) rs9939609 in the fat mass and obesity-associated gene (FTO) is associated with obesity. As genetic variants associated with weight regulation might also be implicated in the etiology of eating disorders, we evaluated whether SNP rs9939609 is associated with bulimia nervosa (BN) and anorexia nervosa (AN). METHODS: Association of rs9939609 with BN and AN was assessed in 689 patients with AN, 477 patients with BN, 984 healthy non-population-based controls, and 3,951 population-based controls (KORA-S4). Based on the familial and premorbid occurrence of obesity in patients with BN, we hypothesized an association of the obesity risk A-allele with BN. RESULTS: In accordance with our hypothesis, we observed evidence for association of the rs9939609 A-allele with BN when compared to the non-population-based controls (unadjusted odds ratio (OR) = 1.142, one-sided 95% confidence interval (CI) 1.001-∞; one-sided p = 0.049) and a trend in the population-based controls (OR = 1.124, one-sided 95% CI 0.932-∞; one-sided p = 0.056). Interestingly, compared to both control groups, we further detected a nominal association of the rs9939609 A-allele to AN (OR = 1.181, 95% CI 1.027-1.359, two-sided p = 0.020 or OR = 1.673, 95% CI 1.101-2.541, two-sided p = 0.015,). CONCLUSION: Our data suggest that the obesity-predisposing FTO allele might be relevant in both AN and BN.

Combined inhibition of cellular pathways as a future therapeutic option in fatal anaplastic thyroid cancer.

Conventional treatment by surgery, radioiodine, and thyroxin-suppressive therapy often fails to cure anaplastic thyroid cancer (ATC). Therefore several attempts have been made to evaluate new therapy options by use of "small molecule inhibitors". ATC was shown to respond to monotherapeutic proteasome and Aurora kinase inhibition in vitro as well as in xenotransplanted tumor cells. Aim of this study was to evaluate the effect of combined treatment targeting the ubiquitin-proteasome system by bortezomib and Aurora kinases by use of MLN8054. Three ATC cell lines (Hth74, C643, and Kat4.1) were used. The antiproliferative effect of combined treatment with bortezomib and MLN8054 was assessed by MTT-assay and cell cycle analysis (FACS). Proapoptotic effects were evaluated by measurement of Caspase-3 activity, and effects on VEGF secretion were analyzed by ELISA. Compared to mono-application combined treatment with bortezomib and MLN8054 resulted in a further decrease of cell density, whereas antagonizing effects were found regarding cell cycle progression. Caspase-3 activity was increased up to 2.7- and 14-fold by mono-application of MLN8054 and bortezomib, respectively. When the two drugs were used in combination, a further enhancement of Caspase-3 activity was achieved, depending on the cell line. VEGF secretion was decreased following bortezomib treatment and remained unchanged by MLN8054. Only in C643 cells, the bortezomib-induced down-regulation was enhanced when MLN8054 was applied simultaneously. In conclusion, our data demonstrate that targeting the proteasome and Aurora kinases simultaneously results in additional antitumoral effects in vitro, especially regarding cell growth and induction of apoptosis. The efficacy of this therapeutic approach remains to be revised by in vivo and clinical application.

Targeting the proteasome as a promising therapeutic strategy in thyroid cancer.

BACKGROUND AND OBJECTIVES: Targeting the ubiquitin-proteasome system by using proteasome inhibitors represents a novel approach for cancer therapy. Anaplastic thyroid cancer (ATC), a subtype of thyroid cancer (TC), fails to respond to conventional TC treatment. Here we investigated the effects of bortezomib on TC in vitro. Further, the study aimed to evaluate its potential for TC treatment in vivo. METHODS: Three anaplastic (Hth74, C643, Kat4), one follicular (FTC133), and one papillary (TPC1) TC cell lines were used. Antiproliferative, proapoptotic, and transcriptional effects of bortezomib treatment were analyzed in vitro and growth inhibition of ATC xenografts in vivo. Tumor samples were analyzed by Ki67, CD31, caspase-3, and NF-κB immunohistochemistry. RESULTS: In vitro, bortezomib inhibited proliferation of TC cells (IC(50) 4-10 nM), increased caspase-3 activity and induced cell cycle arrest. NF-κB activity was affected differently. In vivo, bortezomib treatment was effective in reducing tumor volume (up to 74%), accompanied by reduced proliferation (Ki67) and 57% reduced tumor vascularity. CONCLUSION: Proteasome inhibition is effective in reducing cell growth and inducing apoptosis of ATC in vitro and inhibiting tumor growth and vascularity in vivo. However, the impact on nuclear transcription remains controversial. Clinical evaluation of bortezomib treatment in ATC is warranted.

A meta-analysis of the randomized controlled trials on elective neck dissection versus therapeutic neck dissection in oral cavity cancers with clinically node-negative neck.

There is still no consensus on the optimal treatment of the neck in oral cavity cancer patients with clinical N0 neck. The aim of this study was to assess a possible benefit of elective neck dissection in oral cancers with clinical N0 neck. A comprehensive search and systematic review of electronic databases was carried out for randomized trials comparing elective neck dissection to therapeutic neck dissection (observation) in oral cancer patients with clinical N0 neck. A meta-analysis of the studies which met our defined selection criteria was performed using disease-specific death as the primary outcome, and the relative risk (RR) of disease-specific death was calculated for each of the identified studies. Both fixed-effects (Mantel-Haenszel method) and random-effects models were applied to obtain a combined RR estimate, although between-study heterogeneity was not found to be significant as indicated by an I(2) of 8.5% (p=0.350). Four studies with a total of 283 patients met our inclusion criteria. The results of the meta-analysis showed that elective neck dissection reduced the risk of disease-specific death (fixed-effects model RR=0.57, 95% CI 0.36-0.89, p=0.014; random-effects model RR=0.59, 95% CI 0.37-0.96, p=0.034) compared to observation. This reduction in disease-specific death rate supports the need to perform elective neck dissection in oral cancers with clinical N0 neck.

Evaluation of Aurora kinase inhibition as a new therapeutic strategy in anaplastic and poorly differentiated follicular thyroid cancer.

Due to an unfavorable prognosis using the usual therapy, patients with anaplastic thyroid cancer (ATC) are in desperate need of new therapeutic strategies. The objective of this study was to evaluate the effects of MLN8054, an inhibitor of the Aurora serine/threonine kinases, on ATC cells in vitro and on ATC xenografts as a new therapeutic strategy for ATC. Three anaplastic (Hth74, C643, Kat4) and one follicular (FTC133) thyroid cancer cell lines were evaluated in vitro and Kat4 xenografts in vivo. The antiproliferative effect of MLN8054 (0.1-10 µM) on thyroid cancer cells was quantified by sulphorhodamine B-assay. The proapoptotic effect and the effects on the cell cycle were evaluated by flow cytometry after Annexin-V-FITC staining. Further Histone H3 phosphorylation was analysed. In vivo, antiproliferative and antiangiogenic effects were assessed by tumor volume and morphometric analysis following immunohistochemical staining (Ki-67, pHisH3, CD31). Treatment of the different TC cells with MLN8054 inhibited proliferation in a time- and dose-dependent manner, with IC(50) values between 0.1 and 10 µM. Administration of MLN8054 resulted in an increase of apoptotic cells, decreased Histone H3 phosphorylation and induced cell cycle arrest. In vivo, treatment of ATC by MLN8054 resulted in an up to 86% reduced tumor volume and 89% reduced tumor vascularity. In conclusion, our data demonstrated that Aurora kinase inhibition is effective in reducing cell growth and inducing apoptosis of ATC in vitro and tumor growth and vascularity in vivo. Controlled clinical studies on MLN8054 or comparable compounds would be worthwhile to evaluate its potential therapeutic value for treatment of ATC.

Mutation screen and association studies for the fatty acid amide hydrolase (FAAH) gene and early onset and adult obesity.

BACKGROUND: The orexigenic effects of cannabinoids are limited by activation of the endocannabinoid degrading enzyme fatty acid amide hydrolase (FAAH). The aim of this study was to analyse whether FAAH alleles are associated with early and late onset obesity. METHODS: We initially assessed association of five single nucleotide polymorphisms (SNPs) in FAAH with early onset extreme obesity in up to 521 German obese children and both parents. SNPs with nominal p-values 0.05). CONCLUSIONS: As we observed some evidence for an association of the FAAH variants rs2295632 rs324420 with early onset but not adult obesity, we conclude that the FAAH variants analyzed here at least do not seem to play a major role in the etiology of obesity within our samples.