J. James B. Edelman

Haemolysis during Sample Preparation Alters microRNA Content of Plasma

The presence of cell-free microRNAs (miRNAs) has been detected in a range of body fluids. The miRNA content of plasma/serum in particular has been proposed as a potential source of novel biomarkers for a number of diseases. Nevertheless, the quantification of miRNAs from plasma or serum is made difficult due to inefficient isolation and lack of consensus regarding the optimal reference miRNA. The effect of haemolysis on the quantification and normalisation of miRNAs in plasma has not been investigated in great detail. We found that levels of miR-16, a commonly used reference gene, showed little variation when measured in plasma samples from healthy volunteers or patients with malignant mesothelioma or coronary artery disease. Including samples with evidence of haemolysis led to variation in miR-16 levels and consequently decreased its ability to serve as a reference. The levels of miR-16 and miR-451, both present in significant levels in red blood cells, were proportional to the degree of haemolysis. Measurements of the level of these miRNAs in whole blood, plasma, red blood cells and peripheral blood mononuclear cells revealed that the miRNA content of red blood cells represents the major source of variation in miR-16 and miR-451 levels measured in plasma. Adding lysed red blood cells to non-haemolysed plasma allowed a cut-off level of free haemoglobin to be determined, below which miR-16 and miR-451 levels displayed little variation between individuals. In conclusion, increases in plasma miR-16 and miR-451 are caused by haemolysis. In the absence of haemolysis the levels of both miR-16 and miR-451 are sufficiently constant to serve as normalisers.

The Impact of Hemolysis on Cell-Free microRNA Biomarkers

Cell-free microRNAs in plasma and serum have become a promising source of biomarkers for various diseases. Despite rapid progress in this field, there remains a lack of consensus regarding optimal quantification methods, reference genes, and quality control of samples. Recent studies have shown that hemolysis occurring during blood collection has substantial impact on the microRNA content in plasma/serum. To date, the impact of hemolysis has only been investigated for a limited number of microRNAs, mainly the red blood cell (RBC)-enriched miRs-16 and -451. In contrast, the effect of hemolysis on other microRNAs – in particular those proposed as biomarkers – has not been addressed. In this study we profiled the microRNA content of hemolyzed and non-hemolyzed plasma as well as RBCs to obtain a profile of microRNAs in the circulation affected or unaffected by hemolysis. Profiling by TaqMan Array Microfluidic Cards was used to compare three pairs of hemolyzed and non-hemolyzed plasma (with varying degrees of hemolysis) and one RBC sample. A total of 136 microRNAs were detectable in at least two of the samples, and of those 15 were at least twofold elevated in all three hemolyzed samples. This number increased to 88 microRNAs for the sample with the highest level of hemolysis, with all of these also detected in the RBC profile. Thus these microRNAs represent a large proportion of detectable microRNAs and those most likely to be affected by hemolysis. Several of the hemolysis-susceptible microRNAs (e.g., miRs-21, -106a, -92a, -17, -16) have also been previously proposed as plasma/serum biomarkers of disease, highlighting the importance of rigorous quality control of plasma/serum samples used for measurement of circulating microRNAs. As low-level hemolysis is a frequent occurrence during plasma/serum collection it is critical that this is taken into account in the measurement of any candidate circulating microRNA.

Restoring expression of miR-16: a novel approach to therapy for malignant pleural mesothelioma

BACKGROUND Malignant pleural mesothelioma (MPM) is recalcitrant to treatment and new approaches to therapy are needed. Reduced expression of miR-15/16 in a range of cancer types has suggested a tumour suppressor function for these microRNAs, and re-expression has been shown to inhibit tumour cell proliferation. The miR-15/16 status in MPM is largely unknown. MATERIALS AND METHODS MicroRNA expression was analysed by TaqMan-based RT-qPCR in MPM tumour specimens and cell lines. MicroRNA expression was restored in vitro using microRNA mimics, and effects on proliferation, drug sensitivity and target gene expression were assessed. Xenograft-bearing mice were treated with miR-16 mimic packaged in minicells targeted with epidermal growth factor receptor (EGFR)-specific antibodies. RESULTS Expression of the miR-15 family was consistently downregulated in MPM tumour specimens and cell lines. A decrease of 4- to 22-fold was found when tumour specimens were compared with normal pleura. When MPM cell lines were compared with the normal mesothelial cell line MeT-5A, the downregulation of miR-15/16 was 2- to 10-fold. Using synthetic mimics to restore miR-15/16 expression led to growth inhibition in MPM cell lines but not in MeT-5A cells. Growth inhibition caused by miR-16 correlated with downregulation of target genes including Bcl-2 and CCND1, and miR-16 re-expression sensitised MPM cells to pemetrexed and gemcitabine. In xenograft-bearing nude mice, intravenous administration of miR-16 mimics packaged in minicells led to consistent and dose-dependent inhibition of MPM tumour growth. CONCLUSIONS The miR-15/16 family is downregulated and has tumour suppressor function in MPM. Restoring miR-16 expression represents a novel therapeutic approach for MPM.

Increased Circulating miR-625-3p: A Potential Biomarker for Patients With Malignant Pleural Mesothelioma

Introduction: We investigated the ability of cell-free microRNAs (miRNAs) in plasma and serum to serve as a biomarker for malignant mesothelioma (MM). Methods: Using miRNA microarrays, we profiled plasma samples from MM patients and healthy controls. miRNAs with significantly different abundance between cases and controls were validated in a larger series of MM patients and in an independent series of MM patients using quantitative real-time polymerase chain reaction. Levels of candidate miRNAs were also quantified in MM tumor samples. Results: We compared cell-free miRNA profiles in plasma from MM patients with healthy controls. Reviewing 90 miRNAs previously reported to be associated with MM, we found that the levels of two miRNAs, miR-29c* and miR-92a, were elevated in plasma samples from MM patients. In addition, we identified 15 novel miRNAs present at significantly higher levels in the plasma of MM patients. Further analysis of candidate miRNAs by real time-quantitative polymerase chain reaction confirmed that one of them, miR-625-3p, was present in significantly higher concentration in plasma/serum from MM patients and was able to discriminate between cases and controls, in both the original and the independent series of patients. MiR-625-3p was also found to be up-regulated in tumor specimens from a group of 18 MM patients, who underwent extrapleural pneumonectomy. Conclusion: Our data confirm the potential of miR-29c* and miR-92a as candidate tumor markers and reveal that miR-625-3p is a promising novel diagnostic marker for MM.

Coronary Artery Bypass Grafting With and Without Manipulation of the Ascending Aorta – A Meta-Analysis

BACKGROUND The main criticism of surgery in the SYNTAX trial was increased rate of stroke when compared to percutaneous coronary intervention. We aimed to determine whether avoiding aortic manipulation would decrease the rate of stroke. METHOD We performed a meta-analysis of seven studies comparing coronary artery bypass grafting (CABG) with and without manipulation of the ascending aorta. RESULTS When anaortic off-pump coronary artery bypass grafting (OPCAB) was compared with conventional CABG, the rate of stroke was 0.38% vs. 1.87% (p<0.0001). When anaortic OPCAB was compared with OPCAB using a side-clamp or proximal graft anastomosis device the rate of stroke was 0.31% vs. 1.35% (p=0.003). CONCLUSION Avoiding aortic manipulation during CABG may decrease the rate of peri-operative stroke.

Custodiol for myocardial protection and preservation: a systematic review

INTRODUCTION Custodiol cardioplegia is attractive for minimally invasive cardiac surgery, as a single dose provides a long period of myocardial protection. Despite widespread use in Europe, there is little data confirming its efficacy compared with conventional (blood or crystalloid) cardioplegia. There is similar enthusiasm for its use in organ preservation for transplant, but also a lack of data. This systematic review aimed to assess the evidence for the efficacy of Custodiol in myocardial protection and as a preservation solution in heart transplant. METHODS Electronic searches were performed of six databases from inception to October 2013. Reviewers independently identified studies that compared Custodiol with conventional cardioplegia (blood or extracellular crystalloid) in adult patients for meta-analysis; large case series that reported results using Custodiol were analyzed. Next, we identified studies that compared Custodiol with other organ preservation solutions for organ preservation in heart transplant. RESULTS Fourteen studies compared Custodiol with conventional cardioplegia for myocardial protection in adult cardiac surgery. No difference was identified in mortality; there was a trend for increased incidence of ventricular fibrillation in the Custodiol group that did not reach statistical significance. No difference was identified in studies that compared Custodiol with other solutions for heart transplant. CONCLUSIONS Despite widespread clinical use, the evidence supporting the superiority of Custodiol over other solutions for myocardial protection or organ preservation is limited. Large randomised trials are required.

Fibulin-3 levels in malignant pleural mesothelioma are associated with prognosis but not diagnosis.

Background:Fibulin-3 (FBLN3) was recently presented as a promising novel biomarker for malignant pleural mesothelioma (MPM), warranting independent validation studies.Methods:ELISA was used to measure cellular and secreted FBLN3 in cell lines, in plasma of xenograft tumour-bearing mice, in plasma from two independent series of MPM and non-MPM patients and in pleural fluid from a third series. Diagnostic and prognostic potential of FBLN3 was assessed by receiver operating characteristics curve analysis and Kaplan–Meier method, respectively.Results:FBLN3 was expressed in all MPM and benign mesothelial cell lines tested, and a correlation was observed between cellular protein expression and secreted levels. Human FBLN3 was detectable in plasma of tumour-bearing mice, suggesting that MPM cells contribute to levels of circulating FBLN3. Plasma FBLN3 was significantly elevated in MPM patients from the Sydney cohort, but not the Vienna cohort, but the diagnostic accuracy was low (63%, (95% CI: 50.1–76.4) and 56% (95% CI: 41.5–71.0), respectively). Although FBLN3 levels in pleural effusions were not significantly different between cases and controls, FBLN3 levels in pleural effusion fluid were found to be independently associated with prognosis (hazard ratio of 9.92 (95% CI: 2.14–45.93)).Conclusions:These data confirm the potential prognostic value of pleural effusion FBLN3, but question the diagnostic value of this protein in MPM patients.

Off-Pump Coronary Artery Bypass Surgery Versus Percutaneous Coronary Intervention: A Meta-Analysis of Randomized and Nonrandomized Studies

We performed a meta-analysis of all studies comparing off-pump coronary artery bypass graft surgery (OPCABG) and percutaneous coronary intervention (PCI) for patients with coronary artery disease. Ten studies were included in the meta-analysis and 4,821 patients were compared, of whom 3,450 patients underwent PCI and 1,371 patients underwent OPCABG. The rates of stroke, myocardial infarction, cardiac mortality, and all-cause mortality were similar. The 12-month rate of major adverse cardiac or cerebrovascular events and need for repeat revascularization was significantly lower in the OPCABG group when compared with the PCI group.

Serum Biomarkers of Neurologic Injury in Cardiac Operations

Assessment of subtle neurocognitive decline after surgical procedures has been hampered by heterogeneous testing techniques and a lack of reproducibility. This review summarizes the sensitivity and specificity of biomarkers of neurologic injury to determine whether they can be applied in the postoperative period to accurately predict neurocognitive decline. Creatine kinase-brain type, neuron-specific enolase, and S100B can be released into serum during operations by extracranial sources. Glial fibrillary acidic protein is a sensitive marker, and there are extracranial sources that are antigenically different from the brain-derived form. Serum levels of tau protein after acute neurologic injury do not reliability correlate with incidence.

KCa1.1, a calcium-activated potassium channel subunit alpha 1, is targeted by miR-17-5p and modulates cell migration in malignant pleural mesothelioma

BackgroundMalignant pleural mesothelioma (MPM) is an aggressive, locally invasive, cancer elicited by asbestos exposure and almost invariably a fatal diagnosis. To date, we are one of the leading laboratory that compared microRNA expression profiles in MPM and normal mesothelium samples in order to identify dysregulated microRNAs with functional roles in mesothelioma. We interrogated a significant collection of MPM tumors and normal pleural samples in our biobank in search for novel therapeutic targets.MethodsUtilizing mRNA-microRNA correlations based on differential gene expression using Gene Set Enrichment Analysis (GSEA), we systematically combined publicly available gene expression datasets with our own MPM data in order to identify candidate targets for MPM therapy.ResultsWe identified enrichment of target binding sites for the miR-17 and miR-30 families in both MPM tumors and cell lines. RT-qPCR revealed that members of both families were significantly downregulated in MPM tumors and cell lines. Interestingly, lower expression of miR-17-5p (P = 0.022) and miR-20a-5p (P = 0.026) was clearly associated with epithelioid histology. We interrogated the predicted targets of these differentially expressed microRNA families in MPM cell lines, and identified KCa1.1, a calcium-activated potassium channel subunit alpha 1 encoded by the KCNMA1 gene, as a target of miR-17-5p. KCa1.1 was overexpressed in MPM cells compared to the (normal) mesothelial line MeT-5A, and was also upregulated in patient tumor samples compared to normal mesothelium. Transfection of MPM cells with a miR-17-5p mimic or KCNMA1-specific siRNAs reduced mRNA expression of KCa1.1 and inhibited MPM cell migration. Similarly, treatment with paxilline, a small molecule inhibitor of KCa1.1, resulted in suppression of MPM cell migration.ConclusionThese functional data implicating KCa1.1 in MPM cell migration support our integrative approach using MPM gene expression datasets to identify novel and potentially druggable targets.