The Effect of Metabolic Syndrome on the Occurrence of Restenosis After Carotid Endarterectomy

Objectives: The metabolic syndrome (MetS) is a cluster of risk factors for cardiovascular disease. The effect of MetS on clinical outcome in patients with cerebrovascular disease remains largely unknown because conflicting results have been published. This study aimed to determine the influence of MetS on the occurrence of restenosis after carotid endarterectomy (CEA). Methods: All patients who underwent CEA between June 2003 and December 2014 in two tertiary academic referral centres in The Netherlands were included. MetS was defined if three or more of the following criteria were present: hypertension, obesity, high fasting serum blood glucose, high serum triglycerides, or low serum high density lipoprotein cholesterol. The primary outcome measure was the occurrence of ipsilateral restenosis after index surgery. The secondary outcome measure was (all cause) mortality during follow up. For the primary analysis, missing data were multiply imputed using multivariable imputation by chained equations. A Cox proportional hazards model was used to perform an adjusted analysis on the multiply imputed data sets. Results: A total of 1668 CEA procedures (in 1577 patients) were performed. The presence or absence of MetS could not be determined in 263 patients because of missing data. There was no significant difference in freedom from restenosis in the MetS group vs. the no-MetS group (hazard ratio [HR], 1.10; 95% confidence interval [CI] 0.98-1.23; p = .10) or in all cause mortality (HR 1.20; 95% CI 0.94-1.54; p = .14). Conclusion: This study shows that MetS does not predict restenosis after CEA. Also, the presence of MetS did not influence patient survival negatively

Plasma Testosterone Levels and Atherosclerotic Plaque Gene Expression in Men With Advanced Atherosclerosis

Aims: Low plasma testosterone levels have been shown to predict worse outcome in men with severe atherosclerotic disease. We hypothesized that a low plasma testosterone level affects disease risk through changes in gene expression in atherosclerotic plaques. Therefore, we studied plasma testosterone levels in relation to gene expression levels in atherosclerotic plaque tissue of men with advanced atherosclerotic disease. Methods: Plasma testosterone levels were measured in 203 men undergoing carotid endarterectomy. The corresponding atherosclerotic plaque tissue was used for RNA sequencing. First, we assessed how often the androgen receptor gene was expressed in the plaque. Second, correlations between plasma testosterone levels and pre-selected testosterone-sensitive genes were assessed. Finally, differences within the RNA expression profile of the plaque as a whole, characterized into gene regulatory networks and at individual gene level were assessed in relation to testosterone levels. Results: Testosterone plasma levels were low with a median of 11.6 nmol/L (IQR: 8.6-13.8). RNA-seq of the plaque resulted in reliable expression data for 18,850 genes to be analyzed. Within the RNA seq data, the androgen-receptor gene was expressed in 189 out of 203 (93%) atherosclerotic plaques of men undergoing carotid endarterectomy. The androgen receptor gene expression was not associated with testosterone plasma levels. There were no significant differences in gene expression of atherosclerotic plaques between different endogenous testosterone levels. This remained true for known testosterone-sensitive genes, the complete transcriptomic profile, male-specific gene co-expression modules as well as for individual genes. Conclusion: In men with severe atherosclerotic disease the androgen receptor is highly expressed in plaque tissue. However, plasma testosterone levels were neither associated with pre-selected testosterone sensitive genes, gene expression profiles nor gene regulatory networks in late-stage atherosclerotic plaques. The effect of testosterone on gene expression of the late-stage atherosclerotic plaque appears limited, suggesting that alternate mechanisms explain its effect on clinical outcomes

The age- and sex-specific composition of atherosclerotic plaques in vascular surgery patients

BACKGROUND AND AIMS: The sex- and age-related differences in the composition of iliofemoral atherosclerotic plaques are largely unknown. Therefore, the aim of the current study is to gain insight into plaque composition across strata of age and sex in a large cohort of vascular surgery patients. METHODS: Peripheral atherosclerotic plaques of patients who underwent iliofemoral endarterectomy (n = 790) were harvested between 2002 and 2014. The plaques were semi-quantitatively analyzed for the presence of lipid cores, calcifications, plaque hemorrhages (PH), collagen, macrophage and smooth muscle cell (SMC) content, and quantitatively for microvessel density. Patients were stratified by age tertiles and sex. RESULTS: Ageing was independently associated with rupture-prone iliofemoral plaque characteristics, such as higher prevalence of plaque calcifications (OR 1.52 (95%CI:1.03-2.24) p = 0.035) and PH (OR 1.46 (95%CI:1.01-2.09) p = 0.042), and lower prevalence of collagen (OR 0.52 (95%CI:0.31-0.86) p = 0.012) and SMCs (OR 0.59 (95%CI:0.39-0.90) p = 0.015). Sex-stratified data showed that men had a higher prevalence of lipid cores (OR 1.62 (95%CI:1.06-2.45) p = 0.025) and PH (OR 1.62 (95%CI:1.16-2.54) p = 0.004) compared to women. These sex-differences attenuated with increasing age, with women showing an age-related increase in calcifications (p = 0.002), PH (p = 0.015) and decrease in macrophages (p = 0.005). In contrast, men only showed a decrease in collagen (p = 0.043). CONCLUSIONS: Atherosclerotic iliofemoral plaques derived from men display more rupture-prone characteristics compared to women. Yet, this difference is attenuated with an increase in age, with older women having more rupture-prone characteristics compared to younger women

Transcriptomic-based clustering of human atherosclerotic plaques identifies subgroups with different underlying biology and clinical presentation

Histopathological studies have revealed key processes of atherosclerotic plaque thrombosis. However, the diversity and complexity of lesion types highlight the need for improved sub-phenotyping. Here we analyze the gene expression profiles of 654 advanced human carotid plaques. The unsupervised, transcriptome-driven clustering revealed five dominant plaque types. These plaque phenotypes were associated with clinical presentation and showed differences in cellular compositions. Validation in coronary segments showed that the molecular signature of these plaques was linked to coronary ischemia. One of the plaque types with the most severe clinical symptoms pointed to both inflammatory and fibrotic cell lineages. Further, we did a preliminary analysis of potential circulating biomarkers that mark the different plaques phenotypes. In conclusion, the definition of the plaque at risk for a thrombotic event can be fine-tuned by in-depth transcriptomic-based phenotyping. These differential plaque phenotypes prove clinically relevant for both carotid and coronary artery plaques and point to distinct underlying biology of symptomatic lesions

Mutations in the small GTP-ase late endosomal protein RAB7 cause Charcot-Marie-Tooth type 2B neuropathy

Charcot-Marie-Tooth type 2B (CMT2B) is clinically characterized by marked distal muscle weakness and wasting and a high frequency of foot ulcers, infections, and amputations of the toes because of recurrent infections. CMT2B maps to chromosome 3q13-q22. We refined the CMT2B locus to a 2.5-cM region and report two missense mutations (Leu129Phe and Val162Met) in the small GTP-ase late endosomal protein RAB7 which causes the CMT2B phenotype in three extended families and in three patients with a positive family history. The alignment of RAB7 orthologs shows that both missense mutations target highly conserved amino acid residues. RAB7 is ubiquitously expressed, and we found expression in sensory and motor neurons

Genetic landscape of congenital insensitivity to pain and hereditary sensory and autonomic neuropathies

Congenital insensitivity to pain (CIP) and hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders exclusively or predominantly affecting the sensory and autonomic neurons. Due to the rarity of the diseases and findings based mainly on single case reports or small case series, knowledge about these disorders is limited. Here, we describe the molecular workup of a large international cohort of CIP/HSAN patients including patients from normally under-represented countries. We identify 80 previously unreported pathogenic or likely pathogenic variants in a total of 73 families in the >20 known CIP/HSAN-associated genes. The data expand the spectrum of disease-relevant alterations in CIP/HSAN, including novel variants in previously rarely recognized entities such as ATL3-, FLVCR1- and NGF-associated neuropathies and previously under-recognized mutation types such as larger deletions. In silico predictions, heterologous expression studies, segregation analyses and metabolic tests helped to overcome limitations of current variant classification schemes that often fail to categorize a variant as disease-related or benign. The study sheds light on the genetic causes and disease-relevant changes within individual genes in CIP/HSAN. This is becoming increasingly important with emerging clinical trials investigating subtype or gene-specific treatment strategies

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival