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For non-small cell lung cancer (NSCLC) patients with ALK-rearranged tumors, treatment with ALK inhibitors can improve outcomes. However, clinical resistance typically develops over time, and in the majority of cases resistance mechanisms are ALK-independent. We generated tumor cell cultures from multiple regions of an ALK-rearranged clinical tumor specimen, and deployed functional drug screens to identify modulators of resistance to ALK inhibitors. This identified a role for PI3Kβ and EGFR in regulating resistance to ALK inhibition. Furthermore, inhibition of ALK elicited activation of EGFR, and inhibition of PI3Kβ rescued EGFR-mediated ALK inhibitor resistance. In ALK-rearranged primary cultures, cell lines and in vivo xenograft models, combined inhibition of ALK and PI3Kβ prevented compensatory MAPK and PI3K-AKT pathway reactivation and selectively targeted the cancer cells. The combinatorial effect was seen even in the background of TP53 mutations and in epithelial-mesenchymal transformed cells. In conclusion, combinatorial ALK and PI3Kβ inhibitor treatment carries promise as a treatment for ALK-rearranged NSCLC.

SUMMARYFunctional profiling of a cancer patient’s tumor cells holds potential to tailor personalized cancer treatment. Here we report the utility of Fresh Uncultured Tumor-derived EpCAM+ epithelial Cells (FUTC) for ex vivo drug response interrogation. Analysis of murine Kras mutant FUTCs demonstrated pharmacological and adaptive signaling profiles comparable to subtype-matched cultured cells. Applying FUTC profiling on non-small cell lung cancer patient samples, we generated robust drug response data in 18 of 19 cases, where the cells exhibited targeted drug sensitivities corresponding to their oncogenic drivers. In one of these cases, an EGFR mutant lung adenocarcinoma patient refractory to osimertinib, FUTC profiling was used to guide compassionate treatment. FUTC profiling identified selective sensitivity to disulfiram and the combination of carboplatin plus etoposide and the patient received substantial clinical benefit from the treatment with these agents. We conclude that FUTC profiling provides a robust, rapid, and actionable assessment of personalized cancer treatment options.

A key question in precision medicine is how functional heterogeneity in solid tumours informs therapeutic sensitivity. We demonstrate that spatial characteristics of oncogenic signalling and therapy response can be modelled in precision-cut slices from Kras-driven non-small-cell lung cancer with varying histopathologies. Unexpectedly, profiling of in situ tumours demonstrated that signalling stratifies mostly according to histopathology, showing enhanced AKT and SRC activity in adenosquamous carcinoma, and mitogen-activated protein kinase (MAPK) activity in adenocarcinoma. In addition, high intertumour and intratumour variability was detected, particularly of MAPK and mammalian target of rapamycin (mTOR) complex 1 activity. Using short-term treatment of slice explants, we showed that cytotoxic responses to combination MAPK and phosphoinositide 3-kinase-mTOR inhibition correlate with the spatially defined activities of both pathways. Thus, whereas genetic drivers determine histopathology spectra, histopathology-associated and spatially variable signalling activities determine drug sensitivity. Our study is in support of spatial aspects of signalling heterogeneity being considered in clinical diagnostic settings, particularly to guide the selection of drug combinations. (c) 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Although the death rates from cardiovascular diseases (CVD) have declined over the last decades, the burden of disease remains high and CVD remains one of the leading causes of morbidity and mortality in Western developed countries [1, 2] including the Netherlands. [3–6] Tremendous efforts worldwide are put into research and development of novel treatment modalities for CVD to further halt the disease burden, and frequent updates of primary and secondary prevention guidelines should result in the most optimal treatment of each patient with CVD. Besides major improvements in pharmacological treatment with for instance statins, beta-blockers, angiotensin-converting-enzyme inhibitors and antiplatelet agents, interventional treatment modalities such as percutaneous coronary intervention (PCI) have markedly improved the outcome of patients with coronary artery disease. An important complication of percutaneous coronary intervention (PCI) that is still present is coronary restenosis.[7] Coronary restenosis - the renarrowing of the treated obstruction - results in high morbidity [8] and is even reported to be associated with an increased risk of mortality.[9, 10] Data on long-term follow-up of patients with restenosis are, however, scarce. To explore the relation of restenosis development with long-term mortality, we analysed the 10-year survival of the patients included in the GENetic DEterminants of Restenosis (GENDER) study.[11] The second objective of this report was to investigate whether the treatment strategies after PCI, applied in daily clinical practice over the past 10 years, resulted in a shift in mortality rates and causes of death of these confirmed coronary artery disease (CAD) patients compared with the general population.

The techniques and materials used during percutaneous coronary intervention have advanced considerably over the past 3 decades, yet restenosis remains one of the major drawbacks of this procedure. Many innovative technologies, including drug-eluting stents, with or without specific polymers, and fully biodegradable stents have been and continue to be developed in the search for a safe and effective antirestenosis therapy. Remarkable advances in stent design and nanoparticle delivery systems ('nanovehicles') have already fueled revolutionary changes in the prevention and treatment of in-stent restenosis. In this Review we provide an overview of the latest innovations for optimizing outcomes of coronary stenting, and up-to-date information about prevention and treatment of in-stent restenosis.

Introduction With the advent of molecular diagnostics and availability of targeted therapies, treatment of NSCLC has been improved, especially for tumours driven by mutant EGFR or ALK fusions. However, other frequently mutated genes, including KRAS, TP53 and STK11/LKB1, are still not targetable. Murine models of lung cancer that mimic human disease genomics and pathology can help refine diagnostic strategies, allowing discovery of new therapeutic options, identification of biomarkers and validation of the responses in vivo. Material and methods Primary cultures were derived from murine adenosquamous carcinoma (ASC) or adenocarcinoma (AC) tumours driven by mutant Kras and loss of Lkb1 (KL) or Tp53 (KP), and murine lung-derived healthy epithelia. Ex vivo drug sensitivity profiling, biomarker analysis and in vivo response validations were combined to identify NSCLC subtype-specific drug vulnerabilities. To identify biomarkers predictive of response, spatial analyses of murine and human tumours were conducted. Results and discussions By performing ex vivo functional screens, we identified histotype- and genotype-selective drug vulnerabilities. Inhibition of MEK, a downstream KRAS effector, caused a selective transient cytostasis in AC cells. However, long-term MEK inhibition was found to be ineffective in all cultures tested due to adaptive reactivation of MAPK and PI3K/AKT signalling. Resistance to MEK inhibitors was associated with activation of subtype-specific receptor tyrosine kinases: ERBB3 activation in KL;ASCs, adaptive ERBB2 and FGFR1 activation in KL;ACs, or adaptive FGFR1 activation in KP;ACs. Furthermore, while combined inhibition of MEK and ERBB kinases induced cell death in KL;ASC and KL;AC but not in KP;AC ex vivo culture, in vivo this combination showed cytotoxic response only in the squamous cell carcinoma (SCC) regions of KL;ASCs. This was in agreement with baseline ERBB activation occurring selectively in SCC tissue. Interestingly, activation of ERBB family receptors was detected in a significant proportion of human NSCLC tumours. Analysis of spatial signalling activities may therefore identify NSCLC patients that could benefit from combinatorial MEK and ERBB inhibition. Conclusion Our study validates the utility of NSCLC cultures to discover drug resistance mechanisms that can be overcome with subtype-specific drug combinations. Importantly, the combination of ex vivo drug response profiling and in situ spatial signalling shows that Kras-driven lung cancers stratify as functional subgroups.

Despite its unequivocal superiority compared with balloon angioplasty, coronary stenting did not abolish the restenosis problem and even brought along a completely new type of pathology. Bare-metal stents still associate with an approximate 20–30% in-stent restenosis rate and the need for repeat revascularization. Drug-eluting stents (which unfortunately did not completely prevent restenosis either) sometimes determine late-acquired stent malapposition in a significant number of patients. This is occasionally followed by a very serious event – stent thrombosis. Patient comorbidities, stent design, procedural characteristics and antiplatelet therapy influence the risk of poststenting complications. Research in the recent years has also revealed that individual genetic profile plays an important role in adverse outcome after stent implantation. This article reviews the evidence of genetic variations associated with stent restenosis, late-acquired stent malapposition and stent thrombosis.

Objectives:\ud This study sought to investigate the role of secretory phospholipase A2 (sPLA2)-IIA in cardiovascular disease.\ud \ud Background:\ud Higher circulating levels of sPLA2-IIA mass or sPLA2 enzyme activity have been associated with increased risk of cardiovascular events. However, it is not clear if this association is causal. A recent phase III clinical trial of an sPLA2 inhibitor (varespladib) was stopped prematurely for lack of efficacy.\ud \ud Methods:\ud We conducted a Mendelian randomization meta-analysis of 19 general population studies (8,021 incident, 7,513 prevalent major vascular events [MVE] in 74,683 individuals) and 10 acute coronary syndrome (ACS) cohorts (2,520 recurrent MVE in 18,355 individuals) using rs11573156, a variant in PLA2G2A encoding the sPLA2-IIA isoenzyme, as an instrumental variable.\ud \ud Results:\ud PLA2G2A rs11573156 C allele associated with lower circulating sPLA2-IIA mass (38% to 44%) and sPLA2 enzyme activity (3% to 23%) per C allele. The odds ratio (OR) for MVE per rs11573156 C allele was 1.02 (95% confidence interval [CI]: 0.98 to 1.06) in general populations and 0.96 (95% CI: 0.90 to 1.03) in ACS cohorts. In the general population studies, the OR derived from the genetic instrumental variable analysis for MVE for a 1-log unit lower sPLA2-IIA mass was 1.04 (95% CI: 0.96 to 1.13), and differed from the non-genetic observational estimate (OR: 0.69; 95% CI: 0.61 to 0.79). In the ACS cohorts, both the genetic instrumental variable and observational ORs showed a null association with MVE. Instrumental variable analysis failed to show associations between sPLA2 enzyme activity and MVE.\ud \ud Conclusions:\ud Reducing sPLA2-IIA mass is unlikely to be a useful therapeutic goal for preventing cardiovascular events.

Summary Background and objectives Arteriovenous fistula (AVF) failure remains an important cause of morbidity in hemodialysis patients. The exact underlying mechanisms responsible for AVF failure are unknown but processes like proliferation, inflammation, vascular remodeling, and thrombosis are thought to be involved. The current objective was to investigate the association between AVF failure and single nucleotide polymorphisms (SNPs) in genes related to these pathophysiologic processes in a large population of incident hemodialysis patients. Design, setting, participants, & measurements A total of 479 incident hemodialysis patients were included between January 1997 and April 2004. Follow-up lasted 2 years or until AVF failure, defined as surgery, percutaneous endovascular intervention, or abandonment of the vascular access. Forty-three SNPs in 26 genes, related to proliferation, inflammation, endothelial function, vascular remodeling, coagulation, and calcium/phosphate metabolism, were genotyped. Relations were analyzed using Cox regression analysis. Results In total, 207 (43.2%) patients developed AVF failure. After adjustment, two SNPs were significantly associated with an increased risk of AVF failure. The hazard ratio (95% confidence interval) of LRP1 rs1466535 was 1.75 (1.15 to 2.66) and patients with factor V Leiden had a hazard ratio of 2.54 (1.41 to 4.56) to develop AVF failure. The other SNPs were not associated with AVF failure. Conclusions In this large cohort of hemodialysis patients, only 2 of the 43 candidate SNPs were associated with an increased risk of AVF failure. Whether other factors, like local hemodynamic circumstances, are more important or other SNPs play a role in AVF failure remains to be elucidated.

Pharmacogenetics has many possible applications in the drug treatment of cardiovascular diseases. First, individual patients may be screened for genetic polymorphisms to choose agents with the greatest potential for efficacy and least potential for toxicity. Second, pharmacogenetics may be useful for dose adaptations for specific drugs in patients with aberrant drug metabolism. Based on the annual report of the American Heart Association, in 2010, an estimated 785 000 Americans will have a new coronary attack and 610 000 people will experience a new stroke. Despite the advances in the development of more potent platelet inhibitors and more precise revascularisation techniques, 470 000 recurrent coronary attacks and 185 000 recurrent strokes are expected, indicating that optimisation of treatment strategies is warranted.w1 The large number of pharmacogenetic studies undertaken in recent years may provide enough evidence to aid clinicians in prescribing the most optimal drug in the proper concentration in the individual patient. This article discusses the current knowledge of pharmacogenetics of antiplatelet drugs. Human blood platelets are essential and hazardous at the same time. They are, on the one hand, indispensable for appropriate haemostasis upon vascular injury, but, on the other hand, play a major role in the pathophysiology of thrombosis. Since thrombosis of atherosclerotic plaques is the most frequent cause of myocardial ischaemia and embolic stroke, antiplatelet therapy is the cornerstone of modern cardiovascular treatment, as it has been implemented in all major guidelines. It is well known that not all patients derive the same degree of benefit from antiplatelet treatment. Individual differences in the intrinsic rate of platelet reactivity and a wide variability in response to antiplatelet therapy causes a considerable number of patients (up to 25%)1 to be non-responsive and continue to experience new thrombotic events despite guideline treatment. In the literature regarding the pharmacogenetics of platelet inhibitors, the term …

Platelet inhibition during clopidogrel treatment is known to be influenced by genetic variation, especially by reduced function alleles of cytochrome P450 (CYP) 2C19. However, recent data suggests that paraoxonase-1 (PON1) Q192R, and not CYP2C19, is the major contributor to the variability in

Background: DNA repair deficiencies have been postulated to play a role in the development and progression of cardiovascular disease (CVD). The hypothesis is that DNA damage induces cell death that promotes formation of unstable plaques. Using a DNA repair pathway approach we systematically examined whether common genetic variation in DNA excision repair genes influenced the risk of cardiovascular events. Methods: The DNA biobank from 5,244 subjects of the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER) was studied. Cases were defined as patients with fatal/non-fatal myocardial infarction or stroke (N=1,714) and were compared to controls without events (N=3,530). DNA excision repair genes were selected using a systematic pathway approach. A total of 38 single nucleotide polymorphisms (SNPs) within 14 genes were selected. Genotyping was performed using Illumina Human 660-Quad Beadchips. Associations were tested with logistic regression and multi testing correction was applied. Results: A polymorphism in the SMARCA2 gene (rs9298820) was associated with an increased risk of events, OR 1.14 (95% CI 1.05-1.25), p=0.002. Inspection of the genomic region of SMARCA2 revealed 6 independent loci associated with the endpoint, of which the strongest association was found with rs3793516, OR 1.18 (1.08-1.30), p=0.0002. A risk score of the 6 SNPs demonstrated a solid association with cardiovascular events, OR 1.13 (1.07-1.19), p= 9.0E-6. Conclusions: In this explorative study we found an association of cardiovascular events and genetic variation in the SMARCA2 gene, a gene involved in chromatin remodelling. The fact that several loci within the gene were associated and the risk score showed a strong association, increases the likelihood of causal involvement of this gene. These hypotheses-generating findings require extension by replication and functional analysis.

Restenosis is a complex disease for which the pathophysiological mechanisms have not yet been fully elucidated, but are thought to include inflammation, proliferation, and matrix remodeling. Over the years, many predictive clinical, biological, (epi)genetic, lesion-related, and procedural risk factors for restenosis have been identified. These factors are not only useful in risk stratification of patients, they also contribute to our understanding of this condition. Furthermore, these factors provide evidence on which to base treatment tailored to the individual and aid in the development of novel therapeutic modalities. In this Review, we will evaluate the available evidence on the pathophysiological mechanisms of restenosis and provide an overview of the various risk factors, together with the possible clinical application of this knowledge.

Pharmacogenetics is the search for heritable genetic polymorphisms that influence responses to drug therapy. The most important application of pharmacogenetics is to guide choosing agents with the greatest potential of efficacy and smallest risk of adverse drug reactions. Many studies focusing on drug-gene interactions have been published in recent years, some of which led to adaptation of FDA recommendations, indicating that we are on the verge of the clinical application of genetic information in drug therapy. This systematic review provides a comprehensive overview of the current knowledge on pharmacogenetics of all major drug classes currently used in the treatment of cardiovascular diseases.

Introduction. The FSAP-Marburg I polymorphism (1704G > A), which reduces FSAP activity, is associated with late complications of carotid stenosis in humans. Therefore, this study examines the influence of the Marburg I polymorphism and the closely linked Marburg II polymorphism (1280G > C) on various cardiovascular outcomes in two large independent study populations.Methods. The two Marburg polymorphisms in the HABP2 gene encoding FSAP were genotyped in a large population of elderly patients at risk for vascular disease (the PROSPER-study,n=5804) and in a study population treated with a percutaneous coronary intervention (the GENDER-study,n=3104).Results. In the PROSPER study, the Marburg I polymorphism was associated with an increased risk of clinical stroke (HR: 1.60, 95% CI: 1.13–2.28) and all-cause mortality (HR: 1.33, 95% CI: 1.04–1.71). In the GENDER study carriers of this variant seemed at lower risk of developing restenosis (HR: 0.59, 95% CI: 0.34–1.01). The Marburg II polymorphism showed similar but weaker results.Conclusion. The increase in stroke risk in Marburg I carriers could be due to differential effects on smooth muscle cells and on matrix metalloproteinases, thereby influencing plaque stability. The possible protective effect on restenosis could be the result of reduced activation of zymogens, which are involved in hemostasis and matrix remodeling.

Background. Mannose-binding lectin (MBL) is a recognition molecule of the lectin pathway of complement activation, and its serum levels are largely determined by frequently occurring polymorphisms of the MBL gene. We questioned whether MBL deficiency influences infectious complications in patients after simultaneous pancreas-kidney transplantation (SPKT). Methods. Infectious complications in the first year after transplantation were scored retrospectively in 152 consecutive SPKT patients who received their transplant at our center between 1990 and 2005. Pretransplant serum MBL levels were determined with enzyme-linked immunosorbent assay. Results. Every 500 ng/mL increase in baseline MBL was associated with an odds ratio of 0.83 (P= 0.045) for urinary tract infections and an odds ratio of 0.68 (P= 0.029) for urosepsis. Urosepsis was significantly more common in patients with low baseline MBL (

Despite of our increased understanding of cancer biology, the majority of anti-cancer therapies fail at late-stage clinical trials. Thus, there is an urgent need to develop and validate novel preclinical models that could predict drug efficacy in humans. For this purpose, as a part of IMI-PREDECT public-private research consortium, this study describes methodology and infrastructure for characterization and comparison of preclinical models for drug target validation applying a systems pathology approach. Formalin-fixed paraffin-embedded (FFPE) samples from 1050 different in vitro and in vivo models of breast, prostate and lung cancers, as well as 364 clinical tumors from the same origin, were collected from 26 PREDECT collaborators across the EU. We established standard operating procedures for central processing of FFPE samples, including tissue microarray (TMA) construction and immunohistochemistry (IHC) with 15 different antibodies (CK8, Ki67, p-histone H3, ER, AR, p-AKT, p-ERK, p-p38, γH2AX, cleaved caspase 3, p-MET, HIF1α, p63, vimentin, E-cadherin). We constructed 50 TMA blocks, from which sections were cut and stained as well as digitized using whole slide imaging. Images were hosted on a WebMicroscope digital pathology platform and sample metadata on a PREDECT Metadata database (MBase). We developed image analysis methods for the detection and quantification of IHC biomarkers in the 48,800 stained TMA spots. As a proof-of-concept, we compared MCF-7 on several preclinical platforms including cell cultures, xenografts and xenograft tissue slices. Our results of the integrated biomarker phenotype suggest that of the various MCF-7 in vivo and ex vivo complex cell culture models, the xenograft tissue slice model was the most similar model platform to human clinical samples. In summary, we established a systems pathology approach to analyse and compare novel preclinical cancer models with IHC and digital imaging. The intention is that this large database will be made publicly available on the web as images and summary data that could be broadly useful to the community of cancer researchers and drug developers in comparing cancer model systems. The established infrastructure and workflow integrating molecular and digital pathology in a large-scale consortium setting could be applied to quantitative characterisation of consortium data in collaborations similar to PREDECT. Citation Format: Sami Blom, Yinhai Wang, Tauno Metsalu, Tiina Vesterinen, Teijo Pellinen, Anne Grote, Nina Linder, Jenni Säilä, Katja Välimäki, Ruusu-Maria Kovanen, Outi Monni, Panu Kovanen, Emma Davies, Kristin Stock, Marta Estrada, Georgios Sflomos, Sylvia Grünewald, Catarina Brito, Julia Schüler, Ronald de Hoogt, Cathrin Brisken, Heiko van der Kuip, Wytske van Weerden, Simon Barry, Wolgang Sommergruber, Elizabeth Anderson, Matthias Nees, Juha Klefström, Jaak Vilo, Emmy Verschuren, Ralph Graeser, John Hickman, Johan Lundin, Olli Kallioniemi. Systems pathology for characterization of cancer model systems in a multicenter IMI-PREDECT project. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1698. doi:10.1158/1538-7445.AM2015-1698

BackgroundCoronary restenosis after percutaneous coronary intervention (PCI) still remains a significant limitation of the procedure. The causative mechanisms of restenosis have not yet been fully identified. The goal of the current study was to perform gene-set analysis of biological pathways related to inflammation, proliferation, vascular function and transcriptional regulation on coronary restenosis to identify novel genes and pathways related to this condition.MethodsThe GENetic DEterminants of Restenosis (GENDER) databank contains genotypic data of 556,099SNPs of 295 cases with restenosis and 571 matched controls. Fifty-four pathways, related to known restenosis-related processes, were selected. Gene-set analysis was performed using PLINK, GRASS and ALIGATOR software. Pathways with a pResultsSix pathways (cell-extracellular matrix (ECM) interactions pathway, IL2 signaling pathway, IL6 signaling pathway, platelet derived growth factor pathway, vitamin D receptor pathway and the mitochondria pathway) were significantly associated in one or two of the software packages. Two SNPs in the cell-ECM interactions pathway were replicated in an independent restenosis cohort. No replication was obtained for the other pathways.ConclusionWith these results we demonstrate a potential role of the cell-ECM interactions pathway in the development of coronary restenosis. These findings contribute to the increasing knowledge of the genetic etiology of restenosis formation and could serve as a hypothesis-generating effort for further functional studies.

Background Coronary restenosis after percutaneous coronary intervention still remains a significant problem, despite all medical advances. Unraveling the mechanisms leading to restenosis development remains challenging. Many studies have identified genetic markers associated with restenosis, but consistent replication of the reported markers is scarce. The aim of the current study was to analyze the joined effect of previously in literature reported candidate genes for restenosis in the GENetic DEterminants of Restenosis (GENDER) databank. Methodology/Principal Findings Candidate genes were selected using a MEDLINE search including the terms ‘genetic polymorphism’ and ‘coronary restenosis’. The final set included 36 genes. Subsequently, all single nucleotide polymorphisms (SNPs) in the genomic region of these genes were analyzed in GENDER using set-based analysis in PLINK. The GENDER databank contains genotypic data of 2,571,586 SNPs of 295 cases with restenosis and 571 matched controls. The set, including all 36 literature reported genes, was, indeed, significantly associated with restenosis, p = 0.024 in the GENDER study. Subsequent analyses of the individual genes demonstrated that the observed association of the complete set was determined by 6 of the 36 genes. Conclusion Despite overt inconsistencies in literature, with regard to individual candidate gene studies, this is the first study demonstrating that the joint effect of all these genes together, indeed, is associated with restenosis.

Background Dialysis patients have high cardiovascular mortality risk. This study aimed to investigate the association between SNPs of genes involved in vascular processes and mortality in dialysis patients. Methods Forty two SNPs in 25 genes involved in endothelial function, vascular remodeling, cell proliferation, inflammation, coagulation and calcium/phosphate metabolism were genotyped in 1330 incident dialysis patients. The effect of SNPs on 5-years cardiovascular and non-cardiovascular mortality was investigated. Results The mortality rate was 114/1000 person-years and 49.4% of total mortality was cardiovascular. After correction for multiple testing, VEGF rs699947 was associated with all-cause mortality (HR1.48, 95% CI 1.14–1.92). The other SNPs were not associated with mortality. Conclusions This study provides further evidence that a SNP in the VEGF gene may contribute to the comorbid conditions of dialysis patients. Future studies should unravel the underlying mechanisms responsible for the increase in mortality in these patients.