Cohort profile: 'Biomarkers of Personalised Medicine' (BioPersMed): a single-centre prospective observational cohort study in Graz/Austria to evaluate novel biomarkers in cardiovascular and metabolic diseases.

PURPOSE: Accumulating evidence points towards a close relationship between cardiovascular, endocrine and metabolic diseases. The BioPersMed Study (Biomarkers of Personalised Medicine) is a single-centre prospective observational cohort study with repetitive examination of participants in 2-year intervals. The aim is to evaluate the predictive impact of various traditional and novel biomarkers of cardiovascular, endocrine and metabolic pathways in asymptomatic individuals at risk for cardiovascular and/or metabolic disease. PARTICIPANTS: Between 2010 and 2016, we recruited 1022 regional individuals into the study. Subjects aged 45 years or older presenting with at least one traditional cardiovascular risk factor or manifest type 2 diabetes mellitus (T2DM) were enrolled. The mean age of the participants was 57±8 years, 55% were female, 18% had T2DM, 33% suffered from arterial hypertension, 15% were smokers, 42% had hyperlipidaemia, and only 26% were at low cardiovascular risk according to the Framingham 'Systematic COronary Risk Evaluation'. FINDINGS TO DATE: Study procedures during screening and follow-up visits included a physical examination and comprehensive cardiovascular, endocrine, metabolic, ocular and laboratory workup with biobanking of blood and urine samples. The variety of assessed biomarkers allows a full phenotyping of individuals at cardiovascular and metabolic risk. Preliminary data from the cohort and relevant biomarker analyses were already used as control population for genomic studies in local and international research cooperation. FUTURE PLANS: Participants will undergo comprehensive cardiovascular, endocrine and metabolic examinations for the next decades and clinical outcomes will be adjudicated prospectively.

Two-Tier Care Pathways for Liver Fibrosis Associated to Non-Alcoholic Fatty Liver Disease in HIV Mono-Infected Patients.

(1) Background: Developing strategies to identify significant liver fibrosis in people with HIV (PWH) is crucial to prevent complications of non-alcoholic fatty liver disease (NAFLD). We aim to investigate if five simple serum biomarkers applied to PWH can optimize a care pathway to identify significant liver fibrosis defined by transient elastography (TE). (2) Methods: A two-tier fibrosis pathway was applied to three prospective cohorts of PWH undergoing TE with CAP. NAFLD was diagnosed as a controlled attenuation parameter ≥ 248 dB/m. Five simple fibrosis biomarkers (FIB-4 < 1.3, BARD score 0-1, NAFLD fibrosis score < -1.455, AST:ALT ratio < 0.8 and APRI < 0.5) were applied as first-tiers to exclude significant liver fibrosis. We determined the decrease in referral for TE that would have occurred based on biomarker assessment and the discordance between low simple fibrosis biomarkers and high TE (≥7.1 kPa), indicating significant liver fibrosis. (3) Results: Of the 1749 consecutive PWH, 15.1% had significant liver fibrosis by TE and 39.1% had NAFLD. The application of the fibrosis biomarkers as first tiers would have resulted in a decrease in TE referrals between 24.9% (BARD score) and 86.3% (APRI). The lowest discordance rate was with NAFLD fibrosis score (8.5%). After adjustments, BMI (odds ratio (OR) 1.12, 95% CI: 1.08-1.17) and triglycerides (OR 1.26, 95% CI: 1.11-1.44) were independent predictors of discordance for APRI < 0.5 and TE ≥ 7.1. The performance of the two-tier pathways was similar in PWH with and without NAFLD. (4) Conclusions: Implementing a two-tier pathway could save a substantial proportion up of TE examinations, reducing costs and helping resource optimization in HIV care. Patients with metabolic risk factors for NAFLD and low fibrosis biomarker may still be considered for TE referral.

"Dangerous liaisons: NAFLD and liver fibrosis increase cardiovascular risk in HIV".

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is strongly associated with cardiovascular disease in the general population. We aimed to assess the impact of NAFLD and liver fibrosis on intermediate-high cardiovascular risk in people living with HIV. METHODS: We included people living with HIV from three cohorts. NAFLD and significant liver fibrosis were defined using transient elastography: controlled attenuation parameter ≥288 dB/m and liver stiffness measurement ≥7.1 kPa, respectively. Cardiovascular risk was assessed with the atherosclerotic cardiovascular disease (ASCVD) risk estimator in patients aged between 40 and 75 years and categorised as low if <5%, borderline if 5%-7.4%, intermediate if 7.5%-19.9% and high if ≥20% or with the presence of a previous cardiovascular event. Patients with hepatitis B and/or hepatitis C virus co-infection, alcohol abuse and unreliable transient elastography measurements were excluded. Predictors of intermediate-high cardiovascular risk were investigated in multivariable analysis by logistic regression and also by stratifying according to body mass index (BMI; cut-offs of 25 and 30 kg/m2 ) and age (cut-off of 60 years). RESULTS: Of 941 patients with HIV alone included, 423 (45%), 128 (13.6%), 260 (27.6%) and 130 (13.8%) were categorised as at low, borderline, intermediate and high ASCVD risk, respectively. Predictors of intermediate-high ASCVD risk were NAFLD (adjusted odds ratio [aOR] 2.11; 95% confidence interval [CI] 1.40-3.18; p < 0.001), liver fibrosis (aOR 1.64; 95% CI 1.03-2.59; p = 0.034), duration of HIV (aOR 1.04; 95% CI 1.02-1.06; p < 0.001), and previous exposure to thymidine analogues and/or didanosine (aOR 1.54; 95% CI 1.09-2.18; p = 0.014). NAFLD was also associated with higher cardiovascular risk in normoweight patients (aOR 2.97; 95% CI 1.43-6.16; p = 0.003), in those with BMI <30 kg/m2 (aOR 2.30; 95% CI 1.46-3.61; p < 0.001) and in those aged <60 years (aOR 2.19; 95% CI 1.36-3.54; p = 0.001). CONCLUSION: Assessment of cardiovascular disease should be targeted in people living with HIV with NAFLD and/or significant liver fibrosis, even if they are normoweight and young.

Cyclic GMP modulating drugs in cardiovascular diseases: mechanism-based network pharmacology.

Mechanism-based therapy centred on the molecular understanding of disease-causing pathways in a given patient is still the exception rather than the rule in medicine, even in cardiology. However, recent successful drug developments centred around the second messenger cyclic guanosine-3'-5'-monophosphate (cGMP), which is regulating a number of cardiovascular disease modulating pathways, are about to provide novel targets for such a personalized cardiovascular therapy. Whether cGMP breakdown is inhibited or cGMP synthesis is stimulated via guanylyl cyclases or their upstream regulators in different cardiovascular disease phenotypes, the outcomes seem to be so far uniformly protective. Thus, a network of cGMP-modulating drugs has evolved that act in a mechanism-based, possibly causal manner in a number of cardiac conditions. What remains a challenge is the detection of cGMPopathy endotypes amongst cardiovascular disease phenotypes. Here, we review the growing clinical relevance of cGMP and provide a glimpse into the future on how drugs interfering with this pathway may change how we treat and diagnose cardiovascular diseases altogether.

High-Sensitivity Cardiac Troponin T for Risk Stratification in Patients With Embolic Stroke of Undetermined Source.

BACKGROUND AND PURPOSE: Optimal secondary prevention for patients with embolic stroke of undetermined source (ESUS) remains unknown. We aimed to assess whether high-sensitivity cardiac troponin T (hs-cTnT) levels are associated with major vascular events and whether hs-cTnT may identify patients who benefit from anticoagulation following ESUS. METHODS: Data were obtained from the biomarker substudy of the NAVIGATE ESUS trial, a randomized controlled trial testing the efficacy of rivaroxaban versus aspirin for secondary stroke prevention in ESUS. Patients were dichotomized at the hs-cTnT upper reference limit (14 ng/L, Gen V, Roche Diagnostics). Cox proportional hazard models were computed to explore the association between hs-cTnT, the combined cardiovascular end point (recurrent stroke, myocardial infarction, systemic embolism, cardiovascular death), and recurrent ischemic stroke. RESULTS: Among 1337 patients enrolled at 111 participating centers in 18 countries (mean age 67±9 years, 61% male), hs-cTnT was detectable in 95% and at/above the upper reference limit in 21%. During a median follow-up of 11 months, the combined cardiovascular end point occurred in 68 patients (5.0%/y, rivaroxaban 28 events, aspirin 40 events; hazard ratio, 0.67 [95% CI, 0.41-1.1]), and recurrent ischemic stroke occurred in 50 patients (4.0%/y, rivaroxaban 16 events, aspirin 34 events, hazard ratio 0.45 [95% CI, 0.25-0.81]). Annualized combined cardiovascular end point rates were 8.2% (9.5% rivaroxaban, 7.0% aspirin) for those above hs-cTnT upper reference limit and 4.8% (3.1% rivaroxaban, 6.6% aspirin) below with a significant treatment modification (P=0.04). Annualized ischemic stroke rates were 4.7% above hs-cTnT upper reference limit and 3.9% below, with no suggestion of an interaction between hs-cTnT and treatment (P=0.3). CONCLUSIONS: In patients with ESUS, hs-cTnT was associated with increased cardiovascular event rates. While fewer recurrent strokes occurred in patients receiving rivaroxaban, outcomes were not stratified by hs-cTn results. Our findings support using hs-cTnT for cardiovascular risk stratification but not for decision-making regarding anticoagulation therapy in patients with ESUS. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02313909.

Multicenter Evaluation of Independent High-Throughput and RT-qPCR Technologies for the Development of Analytical Workflows for Circulating miRNA Analysis.

BACKGROUND: Among emerging circulating biomarkers, miRNA has the potential to detect lung cancer and follow the course of the disease. However, miRNA analysis deserves further standardization before implementation into clinical trials or practice. Here, we performed international ring experiments to explore (pre)-analytical factors relevant to the outcome of miRNA blood tests in the context of the EU network CANCER-ID. METHODS: Cell-free (cfmiRNA) and extracellular vesicle-derived miRNA (EVmiRNA) were extracted using the miRNeasy Serum/Plasma Advanced, and the ExoRNeasy Maxi kit, respectively, in a plasma cohort of 27 NSCLC patients and 20 healthy individuals. Extracted miRNA was investigated using small RNA sequencing and hybridization platforms. Validation of the identified miRNA candidates was performed using quantitative PCR. RESULTS: We demonstrate the highest read counts in healthy individuals and NSCLC patients using QIAseq. Moreover, QIAseq showed 15.9% and 162.9% more cfmiRNA and EVmiRNA miRNA counts, respectively, in NSCLC patients compared to healthy control samples. However, a systematic comparison of selected miRNAs revealed little agreement between high-throughput platforms, thus some miRNAs are detected with one technology, but not with the other. Adding to this, 35% (9 of 26) of selected miRNAs in the cfmiRNA and 42% (11 of 26) in the EVmiRNA fraction were differentially expressed by at least one qPCR platform; about half of the miRNAs (54%) were concordant for both platforms. CONCLUSIONS: Changing of (pre)-analytical methods of miRNA analysis has a significant impact on blood test results and is therefore a major confounding factor. In addition, to confirm miRNA biomarker candidates screening studies should be followed by targeted validation using an independent platform or technology.

Prevalence, Predictors, and Severity of Lean Nonalcoholic Fatty Liver Disease in Patients Living With Human Immunodeficiency Virus.

BACKGROUND: The burden of nonalcoholic fatty liver disease (NAFLD) is growing in people living with human immunodeficiency virus (HIV). NAFLD is associated with obesity; however, it can occur in normoweight (lean) patients. We aimed to investigate lean NAFLD in patients living with HIV. METHODS: We included patients living with HIV mono-infection from 3 prospective cohorts. NAFLD was diagnosed by transient elastography (TE) and defined as controlled attenuation parameter ≥248 dB/m, in absence of alcohol abuse. Lean NAFLD was defined when a body mass index was <25 kg/m2. Significant liver fibrosis was defined as TE ≥7.1 kPa. The presence of diabetes, hypertension, or hyperlipidemia defined metabolically abnormal patients. RESULTS: We included 1511 patients, of whom 57.4% were lean. The prevalence of lean NAFLD patients in the whole cohort was 13.9%. NAFLD affected 24.2% of lean patients. The proportions of lean NAFLD patients who were metabolically abnormal or had elevated alanine aminotransferase (ALT) were higher than among those who were lean patients without NAFLD (61.9% vs 48.9% and 36.7% vs 24.2%, respectively). Lean NAFLD patients had a higher prevalence of significant liver fibrosis than lean patients without NAFLD (15.7% vs 7.6%, respectively). After adjusting for sex, ethnicity, hypertension, CD4 cell count, nadir CD4 <200µ/L, and time since HIV diagnosis, predictors of NAFLD in lean patients were age (adjusted OR [aOR], 1.29; 95% confidence interval [CI], 1.04-1.59), high triglycerides (aOR, 1.34; 95% CI, 1.11-1.63), and high ALT (aOR, 1.15; 95% CI, 1.05-1.26), while a high level of high-density lipoprotein cholesterol was protective (aOR, 0.45; 95% CI, .26-.77). CONCLUSIONS: NAFLD affects 1 in 4 lean patients living with HIV mono-infection. Investigations for NAFLD should be proposed in older patients with dyslipidemia and elevated ALT, even if normoweight.

Nonalcoholic Fatty Liver Disease and the Development of Metabolic Comorbid Conditions in Patients With Human Immunodeficiency Virus Infection.

BACKGROUND: Cardiovascular and liver disease are main causes of death in people with human immunodeficiency virus (HIV) (PWH). In HIV-uninfected patients, nonalcoholic fatty liver disease (NAFLD) is associated with incident metabolic complications. We investigated the effect of NAFLD on development of metabolic comorbid conditions in PWH. METHODS: We included PWH undergoing a screening program for NAFLD using transient elastography. NAFLD was defined as a controlled attenuation parameter ≥248 dB/m with exclusion of other liver diseases. Incident diabetes, hypertension, dyslipidemia, and chronic kidney disease were investigated using survival analysis and Cox proportional hazards. RESULTS: The study included 485 HIV-monoinfected patients. During a median follow-up of 40.1 months (interquartile range, 26.5-50.7 months), patients with NAFLD had higher incidences of diabetes (4.74 [95% confidence interval, 3.09-7.27] vs 0.87 [.42-1.83] per 100 person-years) and dyslipidemia (8.16 [5.42-12.27] vs 3.99 [2.67-5.95] per 100 person-years) than those without NAFLD. With multivariable analysis, NAFLD was an independent predictor of diabetes (adjusted hazard ratio, 5.13; 95% confidence interval, 2.14-12.31) and dyslipidemia (2.35; 1.34-4.14) development. CONCLUSIONS: HIV-monoinfected patients with NAFLD are at higher risk of incident diabetes and dyslipidemia. Early referral strategies and timely management of metabolic risk may improve outcomes.

Integrating circulating miRNA analysis in the clinical management of lung cancer: Present or future?

Liquid biopsy holds great promise to complement traditional analysis on cancerous tissue during clinical management of cancer: screening of patients, (early) disease diagnosis, prognosis, therapy selection as well as early response to treatment and disease monitoring. Among emerging circulating biomarkers, cell-free miRNA (cfmiRNA) may have potential in detecting lung cancer and following the course of the disease. Furthermore, several studies highlighted the possibility to utilize these regulatory RNAs to obtain prognostic information as well as to verify patient's response towards treatment. However, despite these findings, cfmiRNA is not used in the clinical practice as biomarkers to date, since their clinical utility and validity has not been confirmed in prospective clinical studies yet. In addition, there is no consensus on standardized (pre)analytical procedures. In this review, we present an overview of cfmiRNA biomarker candidates for clinical management of lung cancer and we discuss the issue of assay standardization.

Hepatocyte Growth Factor Receptor overexpression predicts reduced survival but its targeting is not effective in unselected HNSCC patients.

BACKGROUND: MET has emerged as target in head and neck squamous cell carcinoma (HNSCC). However, clinical data on MET inhibition in HNSCC are limited. METHODS: HNSCC biopsies and cell lines were tested for MET activity. The response of cell lines to BAY-853474 was tested in proliferation assays. The prognostic value of MET expression was also analyzed. RESULTS: HNSCC cell lines do not respond to MET inhibition. MET-dependent gastric cancer cell lines have much higher levels of MET expression and phosphorylation than HNSCC cell lines. Clinical samples of HNSCC contain much less MET than responsive models. CONCLUSIONS: No clinical response to MET inhibitors in monotherapy may be expected in unselected cases of HNSCC. Only selected patients with MET amplifications should be treated with MET inhibitors. Patients with increased MET immunoreactivity have shorter overall survival. MET might be useful as marker for the detection of patients with more aggressive types of HNSCC.

Characteristics of circulating endothelial cells obtained from non-ST-segment elevation myocardial infarction patients with additional diastolic dysfunction of left ventricle observed in echocardiography.

BACKGROUND: Circulating endothelial cells (CEC) may be used to find new strategies for the early di-agnosis of cardiovascular diseases. The major objective of the project is to broaden knowledge of CEC biology by determining their phenotypic characteristics. The additional aim is to clarify whether on the basis of these information it is possible to identify the origin of CEC release (from various cardiovascular compartments). METHODS: Circulating endothelial cells were collected from arterial blood prior to angiography, as well as from arterial and venous blood obtained after angiography/coronary angioplasty, from 18 patients with non-ST-segment elevation myocardial infarction (NSTEMI). CECs were quantified by flow cytometry and defined as Syto16 (dye)+, CD45dim/neg, CD31+ and CD146+. The additional CD36+ was establish as a marker of endothelial cells released from small vessels of the microcirculation. RESULTS: The total number of CECs increased significantly after the percutaneous transluminal coronary angioplasty (PTCA) in the arterial system. Number of CECs isolated at similar time points (after invasive procedure) did not differ significantly between arteries and veins, but the number of CD36+ CECs after coronary angioplasty was significantly higher in the venous system, than in the arterial system. CONCLUSIONS: The number of CD36+ in artery samples obtained after coronary angioplasty (PTCA) had tendency to be decreased (in comparison to the sample obtained before angiography). It was major difference between those who had PTCA performed vs. those who had not.

Circulating Tumor Cell PD-L1 Expression as Biomarker for Therapeutic Efficacy of Immune Checkpoint Inhibition in NSCLC.

Over the last decade, the immune checkpoint blockade targeting the programmed death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) axis has improved progression-free and overall survival of advanced non-small cell lung cancer (NSCLC) patients. PD-L1 tumor expression, along with tumor mutational burden, is currently being explored as a predictive biomarker for responses to immune checkpoint inhibitors (ICIs). However, lung cancer patients may have insufficient tumor tissue samples and the high bleeding risk often prevents additional biopsies and, as a consequence, immunohistological evaluation of PD-L1 expression. In addition, PD-L1 shows a dynamic expression profile and can be influenced by intratumoral heterogeneity as well as the immune cell infiltrate in the tumor and its microenvironment, influencing the response rate to PD-1/PD-L1 axis ICIs. Therefore, to identify subgroups of patients with advanced NSCLC that will most likely benefit from ICI therapies, molecular characterization of PD-L1 expression in circulating tumor cells (CTCs) might be supportive. In this review, we highlight the use of CTCs as a complementary diagnostic tool for PD-L1 expression analysis in advanced NSCLC patients. In addition, we examine technical issues of PD-L1 measurement in tissue as well as in CTCs.

Test-Retest Reliability of Non-Invasive Cardiac Output Measurement during Exercise in Healthy Volunteers in Daily Clinical Routine / Confiabilidade Teste-Reteste de Medição Não-Invasiva de Débito Cardíaco durante Exercício em Voluntários Saudáveis sob Condições Clínicas de Rotina

Abstract Background: Thoracic bioreactance (TB), a noninvasive method for the measurement of cardiac output (CO), shows good test-retest reliability in healthy adults examined under research and resting conditions. Objective: In this study, we evaluate the test-retest reliability of CO and cardiac power (CPO) output assessment during exercise assessed by TB in healthy adults under routine clinical conditions. Methods: 25 test persons performed a symptom-limited graded cycling test in an outpatient office on two different days separated by one week. Cardiorespiratory (power output, VO2peak) and hemodynamic parameters (heart rate, stroke volume, CO, mean arterial pressure, CPO) were measured at rest and continuously under exercise using a spiroergometric system and bioreactance cardiograph (NICOM, Cheetah Medical). Results: After 8 participants were excluded due to measurement errors (outliers), there was no systematic bias in all parameters under all conditions (effect size: 0.2-0.6). We found that all noninvasively measured CO showed acceptable test-retest-reliability (intraclass correlation coefficient: 0.59-0.98; typical error: 0.3-1.8). Moreover, peak CPO showed better reliability (intraclass correlation coefficient: 0.80-0.85; effect size: 0.9-1.1) then the TB CO, thanks only to the superior reliability of MAP (intraclass correlation coefficient: 0.59-0.98; effect size: 0.3-1.8). Conclusion: Our findings preclude the clinical use of TB in healthy subject population when outliers are not identified.

Resumo Fundamento: A biorreatância torácica (BT), um método não invasivo destinado à medição do débito cardíaco (DC), mostra boa confiabilidade teste-reteste em adultos saudáveis examinados em condições de pesquisa e repouso. Objetivo: No presente estudo, avaliamos a confiabilidade teste-reteste da avaliação do DC e trabalho cardíaco (TC) durante exercício, avaliado por BT em adultos saudáveis sob condições clínicas de rotina. Métodos: 25 indivíduos realizaram teste ergométrico gradual sintoma-limitante em ambiente ambulatorial em dois dias diferentes, com intervalo de uma semana. Parâmetros cardiorrespiratórios (trabalho cardíaco, VO2máx) e hemodinâmicos (frequência cardíaca, volume sistólico, DC, pressão arterial média, TC) foram medidos em repouso e continuamente sob exercício utilizando sistema espiroergométrico e cardiógrafo de biorreatância (NICOM, Cheetah Medical). Resultados: Após 8 participantes terem sido excluídos devido a erros de medição (outliers), não houve viés sistemático em nenhum dos parâmetros em todas as condições (tamanho do efeito: 0,2-0,6). Observamos que todos os débitos cardíacos medidos de forma não invasiva apresentaram níveis aceitáveis de confiabilidade teste-reteste (coeficiente de correlação intraclasse: 0,59-0,98; erro típico: 0,3-1,8). Além disso, TC máximo apresentou melhor confiabilidade (coeficiente de correlação intraclasse: 0,80-0,85; tamanho do efeito: 0,9-1,1), seguido do DC pela BT, graças apenas à confiabilidade superior da PAM (coeficiente de correlação intraclasse: 0,59-0,98; tamanho do efeito: 0,3-1,8). Conclusão: Nossos achados impedem o uso clínico da BT em indivíduos saudáveis quando outliers não forem identificados.

Test-Retest Reliability of Non-Invasive Cardiac Output Measurement during Exercise in Healthy Volunteers in Daily Clinical Routine.

BACKGROUND: Thoracic bioreactance (TB), a noninvasive method for the measurement of cardiac output (CO), shows good test-retest reliability in healthy adults examined under research and resting conditions. OBJECTIVE: In this study, we evaluate the test-retest reliability of CO and cardiac power (CPO) output assessment during exercise assessed by TB in healthy adults under routine clinical conditions. METHODS: 25 test persons performed a symptom-limited graded cycling test in an outpatient office on two different days separated by one week. Cardiorespiratory (power output, VO2peak) and hemodynamic parameters (heart rate, stroke volume, CO, mean arterial pressure, CPO) were measured at rest and continuously under exercise using a spiroergometric system and bioreactance cardiograph (NICOM, Cheetah Medical). RESULTS: After 8 participants were excluded due to measurement errors (outliers), there was no systematic bias in all parameters under all conditions (effect size: 0.2-0.6). We found that all noninvasively measured CO showed acceptable test-retest-reliability (intraclass correlation coefficient: 0.59-0.98; typical error: 0.3-1.8). Moreover, peak CPO showed better reliability (intraclass correlation coefficient: 0.80-0.85; effect size: 0.9-1.1) then the TB CO, thanks only to the superior reliability of MAP (intraclass correlation coefficient: 0.59-0.98; effect size: 0.3-1.8). CONCLUSION: Our findings preclude the clinical use of TB in healthy subject population when outliers are not identified.

Multicenter Evaluation of Circulating Plasma MicroRNA Extraction Technologies for the Development of Clinically Feasible Reverse Transcription Quantitative PCR and Next-Generation Sequencing Analytical Work Flows.

BACKGROUND: In human body fluids, microRNA (miRNA) can be found as circulating cell-free miRNA (cfmiRNA), as well as secreted into extracellular vesicles (EVmiRNA). miRNAs are being intensively evaluated as minimally invasive liquid biopsy biomarkers in patients with cancer. The growing interest in developing clinical assays for circulating miRNA necessitates careful consideration of confounding effects of preanalytical and analytical parameters. METHODS: By using reverse transcription quantitative real-time PCR and next-generation sequencing (NGS), we compared extraction efficiencies of 5 different protocols for cfmiRNA and 2 protocols for EVmiRNA isolation in a multicentric manner. The efficiency of the different extraction methods was evaluated by measuring exogenously spiked cel-miR-39 and 6 targeted miRNAs in plasma from 20 healthy individuals. RESULTS: There were significant differences between the tested methods. Although column-based extraction methods were highly effective for the isolation of endogenous miRNA, phenol extraction combined with column-based miRNA purification and ultracentrifugation resulted in lower quality and quantity of isolated miRNA. Among all extraction methods, the ubiquitously expressed miR-16 was represented with high abundance when compared with other targeted miRNAs. In addition, the use of miR-16 as an endogenous control for normalization of quantification cycle values resulted in a decreased variability of column-based cfmiRNA extraction methods. Cluster analysis of normalized NGS counts clearly indicated a method-dependent bias. CONCLUSIONS: The choice of plasma miRNA extraction methods affects the selection of potential miRNA marker candidates and mechanistic interpretation of results, which should be done with caution, particularly across studies using different protocols.

Circulating endothelial cells as biomarker for cardiovascular diseases.

BACKGROUND: Endothelial dysfunction is involved in several cardiovascular diseases. Elevated levels of circulating endothelial cells (CECs) and low levels of endothelial progenitor cells (EPCs) have been described in different cardiovascular conditions, suggesting their potential use as diagnostic biomarkers for endothelial dysfunction. Compared to typical peripheral blood leukocyte subsets, CECs and EPCs occur at very low frequency. The reliable identification and characterization of CECs and EPCs is a prerequisite for their clinical use, however, a validated method to this purpose is still missing but a key for rare cell events. OBJECTIVES: To establish a validated flow cytometric procedure in order to quantify CECs and EPCs in human whole blood. METHODS: In the establishment phase, the assay sensitivity, robustness, and the sample storage conditions were optimized as prerequisite for clinical use. In a second phase, CECs and EPCs were analyzed in heart failure with preserved (HFpEF) and reduced (HFrEF) ejection fraction, in arterial hypertension (aHT), and in diabetic nephropathy (DN) in comparison to age-matched healthy controls. RESULTS: The quantification procedure for CECs and EPCs showed high sensitivity and reproducibility. CEC values resulted significantly increased in patients with DN and HFpEF in comparison to healthy controls. CEC quantification showed a diagnostic sensitivity of 90% and a sensitivity of 68.0%, 70.4%, and 66.7% for DN, HFpEF, and aHT, respectively. CONCLUSION: A robust and precise assay to quantify CECs and EPCs in pre-clinical and clinical studies has been established. CEC counts resulted to be a good diagnostic biomarker for DN and HFpEF.

ctDNA and CTCs in Liquid Biopsy - Current Status and Where We Need to Progress.

We discuss the current status of liquid biopsy and its advantages and challenges with a focus on pre-analytical sample handling, technologies and workflows. The potential of circulating tumor cells and circulating tumor DNA is pointed out and an overview of corresponding technologies is given.

EGFR overexpression is not common in patients with head and neck cancer. Cell lines are not representative for the clinical situation in this indication.

BACKGROUND: Based on expression data, Epidermal Growth Factor Receptor (EGFR) emerged as therapeutic target in Head and Neck Cancer but clinical efficacy of EGFR inhibitors was very limited. We reinvestigated the EGFR expression and activation status necessary for response in cell lines and compared that to clinical samples. METHODS: Clinical samples of head and neck squamous cell carcinoma (HNSCC, n=63), mostly from late stage (IV) and poorly or undifferentiated character and cultured cell lines (n=14) were tested by immunohistochemistry (IHC) (n=55) and sandwich immunoassays (n=63) for expression and phosphorylation of EGFR (Tyrosine-1173). Response of 14 different HNSCC cell lines to Erlotinib was tested in proliferation assays. RESULTS: Most HNSCC cell lines respond to Erlotinib. EGFR is phosphorylated in these cell lines. Resistant cell lines display very low level EGFR expression and phosphorylation. EGFR activity in clinical samples is significantly below that observed in cell lines. In clinical samples, EGFR is not overexpressed on the single cellular level. We show similar levels of EGFR expression in growing keratinocytes and tumor cells. CONCLUSIONS: Cell lines are not representative of the clinical situation in HNSCC. Larger studies should investigate whether patient subgroups with activating EGFR mutations or overexpression can be identified.

Peritoneal Dialysis for AKI in Cameroon: Commercial vs Locally-Made Solutions.

BACKGROUND: Acute kidney injury (AKI) is common in low- and middle-income countries, and is associated with a high mortality. The high mortality rate is in large part due to the inability to perform dialysis in resource-limited settings. Due to significant cost advantages, peritoneal dialysis (PD) has been used to treat AKI in these settings. The costs, however, remain high when commercial solutions are used. METHODS: This is a retrospective cohort study of the outcome, and of the peritonitis rates, of patients with AKI treated with either commercially manufactured PD solutions or locally-made PD solutions. A program to treat AKI with PD was started at Mbingo Baptist Hospital in Cameroon. Between May 2013 and January 2015, solutions and connection sets were provided by the Saving Young Lives Program. From January 2015 through March 2017, solutions were locally produced and available tubing was used. RESULTS: Mortality in hospitalized AKI patients was 28% during the period when commercial solutions and tubing were utilized, and 33% when locally produced solutions and available tubing were utilized. In both groups, peritonitis occurred in 16% of treatment courses. CONCLUSIONS: Locally produced PD solutions, used with locally available tubing, were used to treat AKI with PD. The mortality and peritonitis rates were similar whether locally produced or commercial supplies were used.