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Hypothesis-driven MS-based targeted proteomics has gained great popularity in a relatively short timespan. Next to the widely established selected reaction monitoring (SRM) workflow, data-independent acquisition (DIA), also referred to as sequential window acquisition of all theoretical spectra (SWATH) was introduced as a high-throughput targeted proteomics method. DIA facilitates increased proteome coverage, however, does not yet reach the sensitivity obtained with SRM. Therefore, a well-informed method selection is crucial for designing a successful targeted proteomics experiment. This is especially the case when targeting less conventional peptides such as those that contain PTMs, as these peptides do not always adhere to the optimal fragmentation considerations for targeted assays. Here, we provide insight into the performance of DIA, SRM, and MRM cubed (MRM(3) ) in the analysis of phosphorylation dynamics throughout the phosphoinositide 3-kinase mechanistic target of rapamycin (PI3K-mTOR) and mitogen-activated protein kinase (MAPK) signaling network. We observe indeed that DIA is less sensitive when compared to SRM, however demonstrates increased flexibility, by postanalysis selection of alternative phosphopeptide precursors. Additionally, we demonstrate the added benefit of MRM(3) , allowing the quantification of two poorly accessible phosphosites. In total, targeted proteomics enabled the quantification of 42 PI3K-mTOR and MAPK phosphosites, gaining a so far unachieved in-depth view mTOR signaling events linked to tyrosine kinase inhibitor resistance in non-small cell lung cancer.

Aims To evaluate whether integrated care for atrial fibrillation (AF) can be safely orchestrated in primary care. Methods and results The ALL-IN trial was a cluster randomized, open-label, pragmatic non-inferiority trial performed in primary care practices in the Netherlands. We randomized 26 practices: 15 to the integrated care intervention and 11 to usual care. The integrated care intervention consisted of (i) quarterly AF check-ups by trained nurses in primary care, also focusing on possibly interfering comorbidities, (ii) monitoring of anticoagulation therapy in primary care, and finally (iii) easy-access availability of consultations from cardiologists and anticoagulation clinics. The primary endpoint was all-cause mortality during 2 years of follow-up. In the intervention arm, 527 out of 941 eligible AF patients aged ≥65 years provided informed consent to undergo the intervention. These 527 patients were compared with 713 AF patients in the control arm receiving usual care. Median age was 77 (interquartile range 72–83) years. The all-cause mortality rate was 3.5 per 100 patient-years in the intervention arm vs. 6.7 per 100 patient-years in the control arm [adjusted hazard ratio (HR) 0.55; 95% confidence interval (CI) 0.37–0.82]. For non-cardiovascular mortality, the adjusted HR was 0.47 (95% CI 0.27–0.82). For other adverse events, no statistically significant differences were observed. Conclusion In this cluster randomized trial, integrated care for elderly AF patients in primary care showed a 45% reduction in all-cause mortality when compared with usual care.

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Background The CHA2DS2-VASc decision rule is widely recommended for estimating stroke risk in patients with atrial fibrillation (AF), although validation studies show ambiguous and conflicting results. Objectives To: (i) review existing studies validating CHA2DS2-VASc in AF patients who are not (yet) anticoagulated; (ii) meta-analyze estimates of stroke risk per score; and (iii) explore sources of heterogeneity across the validation studies. Methods We performed a systematic literature review and random effects meta-analysis of studies externally validating CHA2DS2-VASc in AF patients not receiving anticoagulants. To explore between-study heterogeneity in stroke risk, we stratified studies to the clinical setting in which patient enrollment started, and performed meta-regression. Results In total, 19 studies were evaluated, with over two million person-years of follow-up. In studies recruiting AF patients in hospitals, stroke risks for scores of 0, 1 and 2 were 0.4% (approximate 95% prediction interval [PI] 0.2-3.2%), 1.2% (95% PI 0.1-3.8%), and 2.2% (95% PI 0.03-7.8%), respectively. These were consistently higher than those in studies recruiting patients from the open general population, with risks of 0.2% (95% PI 0.0-0.9%), 0.7% (95% PI 0.3-1.2%) and 1.5% (95% PI 0.4-3.3%) for scores of 0, 1, and 2, respectively. Heterogeneity, as reflected by the wide PIs, could not be fully explained by meta-regression. Conclusions Studies validating CHA2DS2-VASc show high heterogeneity in predicted stroke risks for different scores.

IntroductionClinical guidelines recommend non-vitamin K antagonist oral anticoagulants (NOACs) over vitamin K antagonists (VKAs) for stroke prevention in most patients with atrial fibrillation (AF). Frail elderly were under-represented in the landmark NOAC-trials, leaving a knowledge gap on the optimal anticoagulant management (VKA or NOAC) in this increasing population. The aim of the Frail-AF (FRAIL-AF) study is to assess whether switching from international normalised ratio (INR)-guided VKA-management to a NOAC-based treatment strategy compared with continuing VKA-management is safe in frail elderly patients with AF.Methods and analysisThe FRAIL-AF study is a pragmatic, multicentre, open-label, randomised controlled clinical trial. Frail elderly (age ≥75 years plus a Groningen Frailty Indicator score ≥3) who receive VKA-treatment for AF in the absence of a mechanical heart valve or severe mitral valve stenosis will be randomised to switch to a NOAC-based treatment strategy or to continue INR-guided VKA-management. Patients with severe renal impairment (estimated glomerular filtration rate <30 mL/min/1.73 m2) will be excluded from randomisation. Based on existing trial evidence in non-frail patients, we will aim to explore whether NOAC-treatment is superior to VKA-therapy in reducing major or clinically relevant non-major bleeding events. Secondary outcomes include minor bleeding, the composite of ischaemic and haemorrhagic stroke, health-related quality of life and cost-effectiveness. The follow-up period for all subjects is 12 months.Ethics and disseminationThe protocol was approved by the Medical Research Ethics Committee of the University Medical Center Utrecht, the Netherlands and by the Central Committee on Research Involving Human Subjects, the Netherlands. All patients are asked written informed consent. Results are expected in 2022 and will be disseminated through peer-reviewed journals as well as presentations at national and international conferences.Trial registration numberEudraCT: 2017-000393-11; The Netherlands Trial Registry: 6721 (FRAIL-AF study).

Background: Research on prognostic prediction models frequently uses data from routine healthcare. However, potential misclassification of predictors when using such data may strongly affect the studied associations. There is no doubt that such misclassification could lead to the derivation of suboptimal prediction models. The extent to which misclassification affects the validation of existing prediction models is currently unclear. We aimed to quantify the amount of misclassification in routine care data and its effect on the validation of the existing risk prediction model. As an illustrative example, we validated the CHA2DS2-VASc prediction rule for predicting mortality in patients with atrial fibrillation (AF).

Targeted therapies against oncogenic receptor tyrosine kinases (RTK) show promising results in the clinic. Unfortunately, despite the initial positive response, most patients develop therapeutic resistance. Most research has focused on acquired resistance occurring after an extensive time of treatment; however, the question remains as to how cells can survive an initial treatment, as early resistance to apoptosis will enable cells to develop any growth-stimulating mechanism. Here, the non-small cell lung cancer (NSCLC) PC9 cell line was used to systematically profile, by mass spectrometry, changes in the proteome, kinome, and phosphoproteome during early treatment with the EGFR inhibitor afatinib. Regardless of the response, initial drug-sensitive cells rapidly adapt to targeted therapy, and within days, cells regained the capacity to proliferate, despite persisting target inhibition. These data reveal a rapid reactivation of mTOR and MAPK signaling pathways after initial inhibition and an increase in abundance and activity of cytoskeleton and calcium signaling-related proteins. Pharmacologic inhibition of reactivated pathways resulted in increased afatinib efficacy. However more strikingly, cells that were restricted from accessing extracellular calcium were extremely sensitive to afatinib treatment. These findings were validated using three additional inhibitors tested in four different NSCLC cell lines, and the data clearly indicated a role for Ca signaling during the development of adaptive resistance. From a therapeutic point of view, the increased inhibitor efficacy could limit or even prevent further resistance development. Combined targeting of calcium signaling and RTKs may limit drug resistance and improve treatment efficacy. .