Hierarchical clustering of flow cytometry data for the study of conventional central chondrosarcoma

We have investigated the use of hierarchical clustering of flow cytometry data to classify samples of conventional central chondrosarcoma, a malignant cartilage forming tumor of uncertain cellular origin, according to similarities with surface marker profiles of several known cell types. Human primary chondrosarcoma cells, articular chondrocytes, mesenchymal stem cells, fibroblasts, and a panel of tumor cell lines from chondrocytic or epithelial origin were clustered based on the expression profile of eleven surface markers. For clustering, eight hierarchical clustering algorithms, three distance metrics, as well as several approaches for data preprocessing, including multivariate outlier detection, logarithmic transformation, and z-score normalization, were systematically evaluated. By selecting clustering approaches shown to give reproducible results for cluster recovery of known cell types, primary conventional central chondrosacoma cells could be grouped in two main clusters with distinctive marker expression signatures: one group clustering together with mesenchymal stem cells (CD49b-high/CD10-low/CD221-high) and a second group clustering close to fibroblasts (CD49b-low/CD10-high/CD221-low). Hierarchical clustering also revealed substantial differences between primary conventional central chondrosarcoma cells and established chondrosarcoma cell lines, with the latter not only segregating apart from primary tumor cells and normal tissue cells, but clustering together with cell lines from epithelial lineage. Our study provides a foundation for the use of hierarchical clustering applied to flow cytometry data as a powerful tool to classify samples according to marker expression patterns, which could lead to uncover new cancer subtypes.

Premature Osteoblast Clustering by Enamel Matrix Proteins Induces Osteoblast Differentiation through Up-Regulation of Connexin 43 and N-Cadherin

In recent years, enamel matrix derivative (EMD) has garnered much interest in the dental field for its apparent bioactivity that stimulates regeneration of periodontal tissues including periodontal ligament, cementum and alveolar bone. Despite its widespread use, the underlying cellular mechanisms remain unclear and an understanding of its biological interactions could identify new strategies for tissue engineering. Previous in vitro research has demonstrated that EMD promotes premature osteoblast clustering at early time points. The aim of the present study was to evaluate the influence of cell clustering on vital osteoblast cell-cell communication and adhesion molecules, connexin 43 (cx43) and N-cadherin (N-cad) as assessed by immunofluorescence imaging, real-time PCR and Western blot analysis. In addition, differentiation markers of osteoblasts were quantified using alkaline phosphatase, osteocalcin and von Kossa staining. EMD significantly increased the expression of connexin 43 and N-cadherin at early time points ranging from 2 to 5 days. Protein expression was localized to cell membranes when compared to control groups. Alkaline phosphatase activity was also significantly increased on EMD-coated samples at 3, 5 and 7 days post seeding. Interestingly, higher activity was localized to cell cluster regions. There was a 3 fold increase in osteocalcin and bone sialoprotein mRNA levels for osteoblasts cultured on EMD-coated culture dishes. Moreover, EMD significantly increased extracellular mineral deposition in cell clusters as assessed through von Kossa staining at 5, 7, 10 and 14 days post seeding. We conclude that EMD up-regulates the expression of vital osteoblast cell-cell communication and adhesion molecules, which enhances the differentiation and mineralization activity of osteoblasts. These findings provide further support for the clinical evidence that EMD increases the speed and quality of new bone formation in vivo.

Does published orthodontic research account for clustering effects during statistical data analysis?

In orthodontics, multiple site observations within patients or multiple observations collected at consecutive time points are often encountered. Clustered designs require larger sample sizes compared to individual randomized trials and special statistical analyses that account for the fact that observations within clusters are correlated. It is the purpose of this study to assess to what degree clustering effects are considered during design and data analysis in the three major orthodontic journals. The contents of the most recent 24 issues of the American Journal of Orthodontics and Dentofacial Orthopedics (AJODO), Angle Orthodontist (AO), and European Journal of Orthodontics (EJO) from December 2010 backwards were hand searched. Articles with clustering effects and whether the authors accounted for clustering effects were identified. Additionally, information was collected on: involvement of a statistician, single or multicenter study, number of authors in the publication, geographical area, and statistical significance. From the 1584 articles, after exclusions, 1062 were assessed for clustering effects from which 250 (23.5 per cent) were considered to have clustering effects in the design (kappa = 0.92, 95 per cent CI: 0.67-0.99 for inter rater agreement). From the studies with clustering effects only, 63 (25.20 per cent) had indicated accounting for clustering effects. There was evidence that the studies published in the AO have higher odds of accounting for clustering effects [AO versus AJODO: odds ratio (OR) = 2.17, 95 per cent confidence interval (CI): 1.06-4.43, P = 0.03; EJO versus AJODO: OR = 1.90, 95 per cent CI: 0.84-4.24, non-significant; and EJO versus AO: OR = 1.15, 95 per cent CI: 0.57-2.33, non-significant). The results of this study indicate that only about a quarter of the studies with clustering effects account for this in statistical data analysis.

Clustering of health behaviours in adult survivors of childhood cancer and the general population

Little is known about engagement in multiple health behaviours in childhood cancer survivors.

His-Purkinje system re-entry in patients with clustering ventricular tachycardia episodes

AIMS: Multiple arrhythmia re-inductions were recently shown in His-Purkinje system (HPS) ventricular tachycardia (VT). We hypothesized that HPS VT was a frequent mechanism of repetitive or incessant VT and assessed diagnostic criteria to select patients likely to have HPS VT. METHODS AND RESULTS: Consecutive patients with clustering VT episodes (>3 sustained monomorphic VT within 2 weeks) were included in the analysis. HPS VT was considered plausible in patients with (i) impaired left ventricular function associated with dilated cardiomyopathy or valvular heart disease; or (ii) ECG during VT similar to sinus rhythm QRS or to bundle-branch block QRS. HPS VT was plausible in 12 of 48 patients and HPS VT was demonstrated in 6 of 12 patients (50%, or 13% of the whole study group). Median VT cycle length was 318 ms (250-550). Catheter ablation was successful in all six patients. CONCLUSION: His-Purkinje system VT is found in a significant number of patients with repetitive or incessant VT episodes, and in a large proportion of patients with predefined clinical or electrocardiographic characteristics. Since it is easily amenable to catheter ablation, our data support the screening of all patients with repetitive VT in this regard and an invasive approach in a selected group of patients.