Aminothiazolone Inhibitors Disrupt the Protein-RNA Interaction of METTL16 and Modulate the m6A RNA Modification.

Targeting RNA-binding and modifying proteins via small molecules to modulate post-transcriptional modifications have emerged as a new frontier for chemical biology and therapeutic research. One such RNA-binding protein that regulates the most prevalent eukaryotic RNA modification, N6-methyladenosine (m6A), is the methyltransferase-like protein 16 (METTL16), which plays an oncogenic role in cancers by cofunctioning with other nucleic acid-binding proteins. To date, no potent small-molecule inhibitor of METTL16 or modulator interfering with the METTL16-RNA interaction has been reported and validated, highlighting the unmet need to develop such small molecules to investigate the METTL16-involved regulatory network. Herein, we described the identification of a series of first-in-class aminothiazolone METTL16 inhibitors via a discovery pipeline that started with a fluorescence-polarization (FP)-based screening. Structural optimization of the initial hit yielded inhibitors, such as compound 45, that showed potent single-digit micromolar inhibition activity against the METTL16-RNA binding. The identified aminothiazolone inhibitors can be useful probes to elucidate the biological function of METTL16 upon perturbation and evaluate the therapeutic potential of METTL16 inhibition via small molecules at the post-transcriptional level.

N-Biphenyl Pyrrolinones and Dibenzofurans as RNA-Binding Protein LIN28 Inhibitors Disrupting the LIN28-Let-7 Interaction.

The RNA-binding protein LIN28 is a regulator of miRNA let-7 biogenesis. Inhibitors of LIN28 are highly sought after given the central role that LIN28 plays in tumorigenesis and development of cancer stem cells as well as LIN28's association with poor clinical prognosis. Although LIN28 inhibitors of different scaffolds have been reported, the potential of most LIN28 inhibiting small molecules was not fully explored since very limited structure-activity relationship (SAR) studies have been performed. We previously identified trisubstituted pyrrolinones as a new class of LIN28 inhibitors disrupting the LIN28-let-7 interaction. Here, we performed extensive SAR by evaluating 95 small molecules and identified new trisubstituted pyrrolinones featuring either an N-biphenyl or N-dibenzofuran substituent, overthrowing the existing conclusion that a salicylic acid moiety is indispensable for activity. Exchange of the negatively charged salicylic acid moiety in LIN28 inhibitors with a heterocyclic substituent is beneficial for membrane permeability, leading to increased activity in a cellular assay, and will potentially reduce toxicity.

Spirocyclic Chromenopyrazole Inhibitors Disrupting the Interaction between the RNA-Binding Protein LIN28 and Let-7.

Small-molecule inhibitors of the RNA-binding and regulating protein LIN28 have the potential to be developed as chemical probes for biological perturbation and as therapeutic candidates. Reported small molecules disrupting the interaction between LIN28 and let-7 miRNA suffer from moderate to weak inhibitory activity and flat structure-activity relationship, which hindered the development of next-generation LIN28 inhibitors that warrant further evaluations. We report herein the identification of new LIN28 inhibitors utilizing a spirocyclization strategy based on a chromenopyrazole scaffold. Representative compounds 2-5 showed potent in vitro inhibitory activity against LIN28-let-7 interaction and single-digit micromolar potency in inhibiting the proliferation of LIN28-expressing JAR cancer cells. The spirocyclic compound 5 incorporated a position that is amenable for functional group appendage and further structural modifications. The binding mode of compound 5 with the LIN28 cold shock domain was rationalized via a molecular docking analysis.

ADC textural features in patients with single brain metastases improve clinical risk models.

AIMS: In this retrospective study we performed a quantitative textural analysis of apparant diffusion coefficient (ADC) images derived from diffusion weighted MRI (DW-MRI) of single brain metastases (BM) patients from different primary tumors and tested whether these imaging parameters may improve established clinical risk models. METHODS: We identified 87 patients with single BM who had a DW-MRI at initial diagnosis. Applying image segmentation, volumes of contrast-enhanced lesions in T1 sequences, hyperintense T2 lesions (peritumoral border zone (T2PZ)) and tumor-free gray and white matter compartment (GMWMC) were generated and registered to corresponding ADC maps. ADC textural parameters were generated and a linear backward regression model was applied selecting imaging features in association with survival. A cox proportional hazard model with backward regression was fitted for the clinical prognostic models (diagnosis-specific graded prognostic assessment score (DS-GPA) and the recursive partitioning analysis (RPA)) including these imaging features. RESULTS: Thirty ADC textural parameters were generated and linear backward regression identified eight independent imaging parameters which in combination predicted survival. Five ADC texture features derived from T2PZ, the volume of the T2PZ, the normalized mean ADC of the GMWMC as well as the mean ADC slope of T2PZ. A cox backward regression including the DS-GPA, RPA and these eight parameters identified two MRI features which improved the two risk scores (HR = 1.14 [1.05;1.24] for normalized mean ADC GMWMC and HR = 0.87 [0.77;0.97]) for ADC 3D kurtosis of the T2PZ.) CONCLUSIONS: Textural analysis of ADC maps in patients with single brain metastases improved established clinical risk models. These findings may aid to better understand the pathogenesis of BM and may allow selection of patients for new treatment options.

MRI Response Assessment in Glioblastoma Patients Treated with Dendritic-Cell-Based Immunotherapy.

Introduction: In this post hoc analysis we compared various response-assessment criteria in newly diagnosed glioblastoma (GB) patients treated with tumor lysate-charged autologous dendritic cells (Audencel) and determined the differences in prediction of progression-free survival (PFS) and overall survival (OS). Methods: 76 patients enrolled in a multicenter phase II trial receiving standard of care (SOC, n = 40) or SOC + Audencel vaccine (n = 36) were included. MRI scans were evaluated using MacDonald, RANO, Vol-RANO, mRANO, Vol-mRANO and iRANO criteria. Tumor volumes (T1 contrast-enhancing as well as T2/FLAIR volumes) were calculated by semiautomatic segmentation. The Kruskal-Wallis-test was used to detect differences in PFS among the assessment criteria; for correlation analysis the Spearman test was used. Results: There was a significant difference in median PFS between mRANO (8.6 months) and Vol-mRANO (8.6 months) compared to MacDonald (4.0 months), RANO (4.2 months) and Vol-RANO (5.4 months). For the vaccination arm, median PFS by iRANO was 6.2 months. There was no difference in PFS between SOC and SOC + Audencel. The best correlation between PFS/OS was detected for mRANO (r = 0.65) and Vol-mRANO (r = 0.69, each p < 0.001). A total of 16/76 patients developed a pure T2/FLAIR progressing disease, and 4/36 patients treated with Audencel developed pseudoprogression. Conclusion: When comparing different response-assessment criteria in GB patients treated with dendritic cell-based immunotherapy, the best correlation between PFS and OS was observed for mRANO and Vol-mRANO. Interestingly, iRANO was not superior for predicting OS in patients treated with Audencel.

Small molecules with tetrahydroquinoline-containing Povarov scaffolds as inhibitors disrupting the Protein-RNA interaction of LIN28-let-7.

Inhibition of the RNA-binding protein LIN28 and disruption of the protein-RNA interaction of LIN28-let-7 with small molecules holds great potential to develop new anticancer therapeutics. Herein, we report the LIN28 inhibitory activities of a series of 30 small molecules with a tricyclic tetrahydroquinoline (THQ)-containing scaffold obtained from a Povarov reaction. The THQ molecules were structurally optimized by varying the 2-benzoic acid substituent, the fused ring at 3- and 4-positions, and the substituents at the phenyl moiety of the tetrahydroquinoline core. Among the tested compounds, GG-43 showed dose-dependent inhibition in an EMSA validation assay and low micromolar inhibitory activity in a fluorescence polarization-based assay measuring disruption of LIN28-let-7 interaction. Binding mode between GG-43 and the cold shock domain of LIN28 was proposed via a molecular docking analysis. The study provides one of the first systematic analyses on structural features that are required for LIN28 inhibition, and indicates the necessity to develop small molecules with new scaffolds as LIN28-targeting probes and therapeutic candidates. In parallel, this study demonstrates the polypharmacological nature of tricyclic THQ-containing scaffolds accessible through Povarov reactions.

Changing the dietary composition improves inflammation but not adipocyte thermogenesis in diet-induced obese mice.

Pronounced weight loss was shown to improve adipocyte dysfunction and insulin sensitivity in obese subjects. While bariatric surgery is frequently accompanied by adverse side effects, weight loss due to caloric restriction is often followed by weight regain. Here we aimed to determine whether switching the diet from a metabolically harmful Western type diet to a balanced standard diet is sufficient to reverse adipocyte dysfunction in diet-induced obese mice. Male C57BL/6 mice were fed a Western diet for 10 weeks and afterwards switched to a standard diet for eight more weeks (WD/SD mice) or continued to be fed a Western diet (WD/WD mice) ad libitum. Mice fed SD for 18 weeks served as control group (SD/SD). Insulin sensitivity was similar in WD/SD and SD/SD mice despite increased body weight in WD/SD mice. Beiging markers Ucp-1, Cidea and Cox8b were drastically reduced in subcutaneous adipose tissue of WD/SD mice when compared with SD/SD mice. Also, in brown adipose tissue morphologic features and markers of thermogenesis were still altered in both WD/SD and WD/WD mice. However, adipocyte size, Hif1α and macrophage infiltration were significantly lower in both, brown and white adipose tissues of WD/SD compared to WD/WD mice and additionally, a shift toward anti-inflammatory M2 phenotype was found in WD/SD mice only. In conclusion our data suggest that switching the diet is sufficient to improve adipose tissue inflammation, while western diet negatively affects thermogenic capacity of brown adipose tissue, and inhibits beiging of white adipose tissue in the long-term.

Trisubstituted Pyrrolinones as Small-Molecule Inhibitors Disrupting the Protein-RNA Interaction of LIN28 and Let-7.

Modulation of protein-RNA interaction (PRI) using small molecules is a promising strategy to develop therapeutics. LIN28 is an RNA-binding protein that blocks the maturation of the tumor suppressor let-7 microRNAs. Herein, we performed a fluorescence polarization-based screening and identified trisubstituted pyrrolinones as small-molecule inhibitors disrupting the LIN28-let-7 interaction. The most potent compound C902 showed dose-dependent inhibition in an EMSA validation assay, enhanced thermal stability of the cold shock domain of LIN28, and increased mature let-7 levels in JAR cells. The structure-activity relationship study revealed key structural features contributing to either PRI inhibition or stabilization of protein-protein interaction (PPI). The pyrrolinones identified in this study not only represent a new class of LIN28-binding molecules that diversify the limited available LIN28 inhibitors but also represent the first examples of small molecules that showed substituent-dependent PRI inhibitory and PPI activating activities.

Intrafractional 6D head movement increases with time of mask fixation during stereotactic intracranial RT-sessions.

BACKGROUND: The present study investigates the intrafractional accuracy of a frameless thermoplastic mask used for head immobilization during stereotactic radiotherapy. Non-invasive masks cannot completely prohibit head movements. Previous studies attempted to estimate the magnitude of intrafractional inaccuracy by means of pre- and postfractional measurements only. However, this might not be sufficient to accurately map also intrafractional head movements. MATERIALS AND METHODS: Intrafractional deviation of mask-fixed head positions was measured in five patients during a total of 94 fractions by means of close-meshed repeated ExacTrac measurements (every 1.4 min) conducted during the entire treatment session. A median of six (range: 4 to 11) measurements were recorded per fraction, delivering a dataset of 453 measurements. RESULTS: Random errors (SD) for the x, y and z axes were 0.27 mm, 0.29 mm and 0.29 mm, respectively. Median 3D deviation was 0.29 mm. Of all 3D intrafractional motions, 5.5 and 0.4% exceeded 1 mm and 2 mm, respectively. A moderate correlation between treatment duration and mean 3D displacement was determined (rs = 0.45). Mean 3D deviation increased from 0.21 mm (SD = 0.26 mm) in the first 2 min to a maximum of 0.53 mm (SD = 0.31 mm) after 10 min of treatment time. CONCLUSION: Pre- and post-treatment measurement is not sufficient to adequately determine the range of intrafractional head motion. Thermoplastic masks provide both reliable interfractional and intrafractional immobilization for image-guided stereotactic hypofractionated radiotherapy. Greater positioning accuracy may be obtained by reducing treatment duration (< 6 min) and applying intrafractional correction. TRIAL REGISTRATION: Clinicaltrials.gov, NCT03896555, Registered 01 April 2019 - retrospectively registered.

Evaluation of mitochondrial function in chronic myofascial trigger points - a prospective cohort pilot study using high-resolution respirometry.

BACKGROUND: Myofascial trigger points (MTrPs) are hyperirritable areas in the fascia of the affected muscle, possibly related to mitochondrial impairment. They can result in pain and hypoxic areas within the muscle. This pilot study established a minimally invasive biopsy technique to obtain high-quality MTrP tissue samples to evaluate mitochondrial function via high-resolution respirometry. Secondary objectives included the feasibility and safety of the biopsy procedure. METHODS: Twenty healthy males participated in this study, 10 with a diagnosis of myofascial pain in the musculus (m.) trapezius MTrP (TTP group) and 10 with a diagnosis of myofascial pain in the m. gluteus medius (GTP group). Each participant had 2 muscle biopsies taken in one session. The affected muscle was biopsied followed by a biopsy from the m. vastus lateralis to be used as a control. Measurements of oxygen consumption were carried out using high-resolution respirometry. RESULTS: Mitochondrial respiration was highest in the GTP group compared to the TTP group and the control muscle whereas no differences were observed between the GTP and the control muscle. When normalizing respiration to an internal reference state, there were no differences between muscle groups. None of the participants had hematomas or reported surgical complications. Patient-reported pain was minimal for all 3 groups. All participants reported a low procedural burden. CONCLUSIONS: This pilot study used a safe and minimally invasive technique for obtaining biopsies from MTrPs suitable for high-resolution respirometry analysis of mitochondrial function. The results suggest that there are no qualitative differences in mitochondrial function of MTrPs of the trapezius and gluteus medius muscles compared to the vastus lateralis control muscle, implying that alterations of mitochondrial function do not appear to have a role in the development of MTrPs. TRIAL REGISTRATION: Registered as No. 20131128-850 at the Coordinating Center for Clinical Studies of the Medical University of Innsbruck, trial registration date: 28th November 2013 and retrospectively registered on 11th of October 2018 at ClinicalTrials.gov with the ID NCT03704311 .

Circulating Wnt inhibitory factor 1 levels are associated with development of cardiovascular disease.

BACKGROUND AND AIMS: Wnt signaling is involved in atherosclerotic plaque formation directly and indirectly by modulating cardiovascular risk factors. We investigated whether circulating concentrations of Wnt inhibitors are associated with cardiovascular events in subjects with intermediate cardiovascular risk. METHODS: 904 non-diabetic subjects participating in the SAPHIR study were assessed. In the SAPHIR study, middle-aged women without overt atherosclerotic disease at study entry were followed up for 10 years. 88 patients of our study cohort developed cardiovascular disease at follow-up (CVD group). Subjects of the CVD group were 1:2 case-control matched for age, sex, BMI and smoking behavior with subjects without overt cardiovascular disease after a 10 year-follow-up (control group). 18 patients of the CVD group and 19 subjects of the control group were retrospectively excluded due to fulfilling exclusion criteria. Baseline circulating sclerostin, dickkopf (DKK)-1, secreted frizzled-related protein (SFRP)-1 and Wnt inhibitory factor (WIF)-1 levels were assessed by ELISA. RESULTS: Baseline systemic SFRP-1 and WIF-1 levels were significantly higher in patients with cardiovascular events (n = 70) when compared to healthy controls (n = 157) while DKK-1 and sclerostin levels were similar in both groups. Logistic regression analysis revealed WIF-1 as a significant predictor of future cardiovascular events. CONCLUSIONS: Our data suggest that increased SFRP-1 and WIF-1 levels precede the development of symptomatic atherosclerotic disease. Assessment of systemic WIF-1 levels, which turned out to be independently associated with CVD, might help to early identify patients at intermediate cardiovascular risk.

Development of a novel prognostic score for breast cancer patients using mRNA expression of CHAC1.

AIM: To develop a prognostic score for primary breast cancer patients integrating conventional predictors and the novel biomarker CHAC1 to aid adjuvant chemotherapy decisions. PATIENTS & METHODS: A prognostic score for overall survival was developed using: conventional predictors from a dataset of 1777 patients and the weight of CHAC1 mRNA expression from an independent dataset of 106 patients using multivariate Cox regression. RESULTS: The new score includes: CHAC1 mRNA expression, age, tumor size, HER2 neu status, lymph node status and degree of malignancy. Using a cut-off value of 11 score points, 10-year survival was 82% in low-risk (n = 34) and 43% in high-risk patients (n = 72). The addition of CHAC1 resulted in 16% reclassification. CONCLUSION: Including CHAC1 in prognostic prediction may aid (and change) personalized treatment selection.

Reduced mRNA expression levels of NFE2L2 are associated with poor outcome in breast cancer patients.

BACKGROUND: The transcription factor nuclear factor erythroid 2-related factor 2 (NFE2L2; previously known as NRF2) is a crucial regulator of the intracellular antioxidant response. It controls the expression of genes involved in the detoxification and elimination of reactive oxidants and electrophilic agents. The role of NFE2L2 in cancer is subject of controversial discussion, as it has been reported to have both pro-and anti-tumourigenic functions. To shed some light on this paradox, we analysed the NFE2L2 mRNA expression levels in breast cancer and its association with clinicopathological features and survival. METHODS: We retrospectively evaluated the NFE2L2 mRNA expression levels in tumour tissue of two independent breast cancer patient cohorts. In the training set we analysed data from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). In the test set we measured the NFE2L2 mRNA expression levels in 176 breast tumour tissues by quantitative real-time reverse transcription PCR (qRT-PCR). Group differences were analysed using Mann-Whitney U-test, and associations between NFE2L2 mRNA expression levels and clinicopathological features were examined by means of univariate and multivariate survival analyses. Furthermore, we compared NFE2L2 mRNA expression levels between tumour and normal breast tissue samples by means of 108 paired samples from the The Cancer Genome Atlas (TCGA) dataset. RESULTS: In the training set we identified an independent predictive value for high NFE2L2 mRNA expression levels [HRdisease specific death 0.8 (0.6-1.0), P = 0.041; HRdeath 0.8 (0.6-1.0), P = 0.023] especially in the subgroup of oestrogen receptor (ER) positive tumours [HRdisease specific death 0.6 (0.4-0.9), P = 0.008; HRdeath 0.6 (0.4-0.8), P = 0.001]. Similarly, we found this association also in the test set [HRrelapse 0.4 (0.2-0.9), P = 0.031] and again, more pronounced in patients with ER positive tumours [HRrelapse 0.2 (0.1-0.7), P = 0.012]. In addition, we observed generally lower NFE2L2 expression levels in tumour tissues than in normal breast tissues. CONCLUSION: We concluded that reduced NFE2L2 mRNA expression in tumour tissues is an independent predictor of shortened survival in breast cancer patients.

Association of TNFRSF10D DNA-methylation with the survival of melanoma patients.

In this retrospective pilot study, the DNA-methylation status of genes that have been demonstrated to be involved in melanoma carcinogenesis was analyzed in order to identify novel biomarkers for the risk assessment of melanoma patients. We analyzed DNA extracted from punch-biopsies from 68 formalin-fixed paraffin-embedded (FFPE) melanoma specimens. Using MethyLight PCR, we examined 20 genes in specimens from a training set comprising 36 melanoma patients. Selected candidate genes were validated in a test set using FFPE tissue samples from 32 melanoma patients. First, we identified the TNFRSF10D DNA-methylation status (TNFRSF10D methylated vs. unmethylated) as a prognostic marker for overall (p = 0.001) and for relapse-free survival (p = 0.008) in the training set. This finding was confirmed in the independent test set (n = 32; overall survival p = 0.041; relapse-free survival p = 0.012). In a multivariate Cox-regression analysis including all patients, the TNFRSF10D DNA-methylation status remained as the most significant prognostic parameter for overall and relapse-free survival (relative-risk (RR) of death, 4.6 (95% CI: 2.0-11.0; p < 0.001), RR of relapse, 7.2 (95% CI: 2.8-18.3; p < 0.001)). In this study, we demonstrate that TNFRSF10D DNA-methylation analysis of a small tissue-punch from archival FFPE melanoma tissue is a promising approach to provide prognostic information in patients with melanoma.

Progression types after antiangiogenic therapy are related to outcome in recurrent glioblastoma.

OBJECTIVE: This retrospective study analyzed whether the type of radiologic progression, classified according to contrast enhancement on MRI T1-weighted sequences and changes in T2-hyperintense signal, is relevant for outcome in patients with progressive glioblastoma (pGB) treated with bevacizumab. METHODS: MRI scans of 83 patients with pGB treated with bevacizumab were evaluated prior to and at disease progression. Based on initial decrease in and subsequent flare-up of contrast enhancement in T1 and 2 patterns of T2-hyperintense tumor progression, progression types (PTs) were categorized as cT1 flare-up, T2-diffuse, T2-circumscribed, or primary nonresponder. Overall survival (OS), survival from start of bevacizumab therapy (OS_Bev), survival after bevacizumab failure (OS_PostBev), time from initial diagnosis until initiation of bevacizumab therapy (StartBevT), and time to bevacizumab progression were evaluated using Kaplan-Meier curves, log-rank test, and Cox regression analyses. RESULTS: The time observed for development of a T2-diffuse (n = 15) or a cT1 flare-up (n = 35) progression was longer than for progression in primary nonresponders (n = 16) or T2-circumscribed progression (n = 17). The T2-diffuse PT showed longer OS, OS_Bev, OS_PostBev, and StartBevT compared to the other PTs. Postprogression therapy tended to be relevant only for patients with a T2-circumscribed PT. CONCLUSIONS: Radiologic PTs following bevacizumab treatment failure show differences in time to development and are related to outcome. We therefore hypothesize that these PTs reflect a different glioma biology, including differential resistance mechanisms to bevacizumab, and may be associated with different responses to postprogression therapy.

Ischemia and functional status of the left arm and quality of life after left subclavian artery coverage during stent grafting of thoracic aortic diseases.

BACKGROUND: The objective of this study was to report on the incidence of left arm ischemia, left arm function, and quality of life after thoracic endovascular aortic repair (TEVAR) by stent grafting with and without coverage of the left subclavian artery (LSA). METHODS: All patients who underwent TEVAR since 1996 in our institution were included. Basic demographic parameters, underlying disease, details of TEVAR, long-term left arm function (Disabilities of the Arm, Shoulder, and Hand [DASH] questionnaire), and quality of life (12-Item Short Form Health Survey) were analyzed. End points were left arm ischemia, need for LSA revascularization (before or after TEVAR), long-term functional impairment, and quality of life. RESULTS: A total of 138 patients underwent TEVAR for degenerative aneurysm (n = 64), traumatic aortic injury (TAI; n = 38), or Stanford type B dissection (n = 36). Seventy-three patients (52.9%) had LSA coverage, which led to partial or complete LSA occlusion in 49 (35.5%). Selectively, nine patients (6.5%) had primary LSA revascularization. After TEVAR, left arm ischemia was observed in only one patient, who consecutively needed a left carotid to subclavian bypass. During a mean follow-up period of 4.1 ± 3.7 years, no additional patient needed secondary LSA revascularization. In comparing patients with occluded vs patent LSA, the Physical Component Summary (PCS) and Mental Component Summary (MCS) health scores (12-Item Short Form Health Survey) as well as DASH scores were similar. However, subgroup analysis showed better PCS scores for TAI patients with patent LSA, whereas MCS and DASH scores were similar in TAI patients, and scores were indifferent within thoracic aortic aneurysm and Stanford type B dissection subgroups. In comparing different subgroups, TAI patients had significantly better PCS, MCS, and DASH scores. CONCLUSIONS: TEVAR is associated with a low risk of peri-interventional left arm ischemia. During long-term follow-up, secondary LSA revascularization is uncommon. Coverage of the LSA has no impact on left arm function and quality of life, probably with the exception of physical health scores in patients with TAI.

The risk of carotid artery stenting compared with carotid endarterectomy is greatest in patients treated within 7 days of symptoms.

OBJECTIVE: Among patients with symptomatic carotid artery stenosis, carotid artery stenting (CAS) is associated with a higher risk of periprocedural stroke or death than carotid endarterectomy (CEA). Uncertainty remains whether the balance of risk changes with time since the most recent ischemic event. METHODS: We investigated the association of time between the qualifying ischemic event and treatment (0-7 days, 8-14 days, and >14 days) with the risk of stroke or death within 30 days after CAS or CEA in a pooled analysis of data from individual patients randomized in the Endarterectomy vs Angioplasty in Patients with Symptomatic Severe Carotid Stenosis (EVA-3S) trial, the Stent-Protected Angioplasty versus Carotid Endarterectomy (SPACE) trial, and the International Carotid Stenting Study (ICSS). Data were analyzed with a fixed-effect binomial regression model adjusted for source trial. RESULTS: Information on time of qualifying event was available for 2839 patients. In the first 30 days after intervention, any stroke or death occurred significantly more often in the CAS group (110/1434 [7.7%]) compared with the CEA group (54/1405 [3.8%]; crude risk ratio, 2.0; 95% confidence interval, 1.5-2.7). Patients undergoing CEA within the first 7 days of the qualifying event had the lowest periprocedural stroke or death rate (3/106 [2.8%]). Patients treated with CAS in the same period had a 9.4% risk of periprocedural stroke or death (13/138; risk ratio CAS vs CEA: 3.4; 95% confidence interval, 1.01-11.8; adjusted for age, sex, and type of qualifying event). Patients treated between 8 and 14 days showed a periprocedural stroke or death rate of 3.4% (7/208) and 8.1% (19/234), respectively, for CEA and CAS. The latest treatment group had 4% complications in the CEA group (44/1091) and 7.3% in the CAS group (78/1062). CONCLUSIONS: The increase in risk of CAS compared with CEA appears to be greatest in patients treated within 7 days of symptoms. Early surgery might remain most effective in stroke prevention in patients with symptomatic carotid artery stenosis.

Clinical relevance of CDH1 and CDH13 DNA-methylation in serum of cervical cancer patients.

This study was designed to investigate the DNA-methylation status of E-cadherin (CDH1) and H-cadherin (CDH13) in serum samples of cervical cancer patients and control patients with no malignant diseases and to evaluate the clinical utility of these markers. DNA-methylation status of CDH1 and CDH13 was analyzed by means of MethyLight-technology in serum samples from 49 cervical cancer patients and 40 patients with diseases other than cancer. To compare this methylation analysis with another technique, we analyzed the samples with a denaturing high performance liquid chromatography (DHPLC) PCR-method. The specificity and sensitivity of CDH1 DNA-methylation measured by MethyLight was 75% and 55%, and for CDH13 DNA-methylation 95% and 10%. We identified a specificity of 92.5% and a sensitivity of only 27% for the CDH1 DHPLC-PCR analysis. Multivariate analysis showed that serum CDH1 methylation-positive patients had a 7.8-fold risk for death (95% CI: 2.2-27.7; p = 0.001) and a 92.8-fold risk for relapse (95% CI: 3.9-2207.1; p = 0.005). We concluded that the serological detection of CDH1 and CDH13 DNA-hypermethylation is not an ideal diagnostic tool due to low diagnostic specificity and sensitivity. However, it was validated that CDH1 methylation analysis in serum samples may be of potential use as a prognostic marker for cervical cancer patients.