Ischemic Stroke Temporally Associated With New-Onset Atrial Fibrillation: A Population-Based Registry-Linkage Study.

BACKGROUND: Limited data exist on the temporal relationship between new-onset atrial fibrillation (AF) and ischemic stroke and its impact on patients' clinical characteristics and mortality. METHODS: A population-based registry-linkage database includes all patients with new-onset AF in Finland from 2007 to 2018. Ischemic stroke temporally associated with AF (ISTAF) was defined as an ischemic stroke occurring within ±30 days from the first AF diagnosis. Clinical factors associated with ISTAF were studied with logistic regression and 90-day survival with Cox proportional hazards analysis. RESULTS: Among 229 565 patients with new-onset AF (mean age, 72.7 years; 50% female), 204 774 (89.2%) experienced no ischemic stroke, 12 209 (5.3%) had past ischemic stroke >30 days before AF, and 12 582 (5.8%) had ISTAF. The annual proportion of ISTAF among patients with AF decreased from 6.0% to 4.8% from 2007 to 2018. Factors associated positively with ISTAF were higher age, lower education level, and alcohol use disorder, whereas vascular disease, heart failure, chronic kidney disease cancer, and psychiatric disorders were less probable with ISTAF. Compared with patients without ischemic stroke and those with past ischemic stroke, ISTAF was associated with ≈3-fold and 1.5-fold risks of death (adjusted hazard ratios, 2.90 [95% CI, 2.76-3.04] and 1.47 [95% CI, 1.39-1.57], respectively). The 90-day survival probability of patients with ISTAF increased from 0.79 (95% CI, 0.76-0.81) in 2007 to 0.89 (95% CI, 0.87-0.91) in 2018. CONCLUSIONS: ISTAF depicts the prominent temporal clustering of ischemic strokes surrounding AF diagnosis. Despite having fewer comorbidities, patients with ISTAF had worse, albeit improving, survival than patients with a history of or no ischemic stroke. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04645537. URL: https://www.encepp.eu; Unique identifier: EUPAS29845.

Exploring a revised interprofessional learning curriculum in undergraduate health education programs at Linköping University.

BACKGROUND: Interprofessional education aiming at providing competencies require evaluation in order to ensure that outcomes match the needs and ambitions. Health professionals today need a broad range of skills and competencies in order to provide high quality care, including interprofessional competence. Linköping University has been a pioneer in interprofessional learning for decades and this study provides one example of how a curriculum revision can be carried out. The aim of this study was to study the intentions and outcomes of a revised interprofessional learning curriculum in health professions education programs. METHODS: This was a qualitative study, including documents (n = 143) and complementary interviews with key individuals (n = 4). Data included syllabuses, study guides, educational program plans, supervisor guides, and interview transcripts. A qualitative document analysis and a content analysis with a directed approach was used, applying a theoretical framework for curriculum development that guided the analysis. RESULTS: The analysis resulted in one overarching theme named "A planned, lived, and attended curriculum" including four main categories inspired by a theoretical framework. The findings demonstrate a variety of aspects relating to the why and how of curriculum revision. The introduction of a programme director in interprofessional learning, with a mandate equal to respective program directors, seemed to contribute to legitimacy. Further, the partnership between the university and the healthcare sector had an impact on the curriculum revision, in that healthcare had a say in the revision regarding what suggestions to implement or not. The expectations of the teachers involved were high, although clear support structures seemed to be lacking. CONCLUSIONS: This study has identified some of the important links between teachers, organizational prerequisites, and healthcare when revising an existing fully integrated curriculum in interprofessional learning for health professions education programs. The aim of this curriculum revision was to legitimize and provide education that is up to date with current healthcare needs and to provide students with competencies to collaborate in teams to ensure patient safety. When redesigning a curriculum there seems to be a fine balance between pedagogical innovation and pragmatism. This study identified that the links provided between organizational support structures and the expectations on teachers were not aligned.

Social Insurance Literacy Among the Sick-listed-A Study of Clients' Comprehension and Self-Rated System Comprehensibility of the Sickness Insurance System.

INTRODUCTION: Sickness insurance systems and their processes have been studied in terms of transparency, comprehensibility and fairness, highlighting the importance of just procedures that make sense to clients. Related research demonstrates differences between groups of clients, pointing towards a social gradient. The concept of social insurance literacy and the Social Insurance Literacy Questionnaire (SILQ) was recently developed and serves as a measure for client's ability to obtain, understand and act on information in a sickness insurance system, relating to the comprehensibility of the information that the system provides. OBJECTIVE: The purpose of this study was to investigate social insurance literacy among clients on sick leave and its associations with perceived justice, being granted sickness benefits and background factors. METHODS: This was a questionnaire study with clients on sick leave in Sweden. In the selection process 3993 clients were invited, of which 1173 recently had their sickness benefits withdrawn. Those who answered the SILQ (n = 1152) also answered a perceived justice measure and accepted sharing register data from the Swedish Social Insurance Agency. Data were analyzed through regression analysis. RESULTS: The findings demonstrate that clients' perceptions of system comprehensibility and the status of their sick leave case was significantly associated with perceived justice, and being granted sickness benefits, while their individual abilities to obtain, understand, and act on information had lesser influence. CONCLUSIONS: The system's ability to provide understandable information seems more important than clients' abilities to comprehend it. From a client perspective, a just system seems to be related to their experiences of the sick leave process (i,e., whether they had an ongoing or closed case) rather than their skills to obtain the correct information.

Intrinsically Disordered Flanking Regions Increase the Affinity of a Transcriptional Coactivator Interaction across Vertebrates.

Interactions between two proteins are often mediated by a disordered region in one protein binding to a groove in a folded interaction domain in the other one. While the main determinants of a certain interaction are typically found within a well-defined binding interface involving the groove, recent studies show that nonspecific contacts by flanking regions may increase the affinity. One example is the coupled binding and folding underlying the interaction between the two transcriptional coactivators NCOA3 (ACTR) and CBP, where the flanking regions of an intrinsically disordered region in human NCOA3 increases the affinity for CBP. However, it is not clear whether this flanking region-mediated effect is a peculiarity of this single protein interaction or if it is of functional relevance in a broader context. To further assess the role of flanking regions in the interaction between NCOA3 and CBP, we analyzed the interaction across orthologs and paralogs (NCOA1, 2, and 3) in human, zebra fish, and ghost shark. We found that flanking regions increased the affinity 2- to 9-fold in the six interactions tested. Conservation of the amino acid sequence is a strong indicator of function. Analogously, the observed conservation of increased affinity provided by flanking regions, accompanied by moderate sequence conservation, suggests that flanking regions may be under selection to promote the affinity between NCOA transcriptional coregulators and CBP.

Hearing and Functioning in Everyday Life Questionnaire: Development and Validation of an ICF-Based Instrument.

OBJECTIVES: Self-assessment instruments are commonly used in audiological rehabilitation. However, several studies highlight the lack of multidimensionality in existing outcome measures, with the consequence that they only partially capture aspects of functioning in everyday life for people living with hearing loss. This study aimed to develop and investigate the content validity of a self-assessment instrument based on the validated Brief International Classification of Functioning, Disability, and Health Core Set for Hearing Loss. DESIGN: The design was a two-part instrument development study. The first part focused on the item-generation process of the instrument, named the Hearing and Functioning in Everyday Life Questionnaire (HFEQ) during an experts' workshop. The second part focused on international content validation of the instrument using group interviews. Strategic sampling was used and 30 adults with hearing loss from India, South Africa, and the United States participated in the group interviews. RESULTS: The expert's workshop resulted in the first version of the HFEQ containing 30 items. The results from group interviews show that the content of the HFEQ was considered to be valid concerning its relevance, comprehensiveness, and comprehensibility. A majority (73%) of the HFEQ items were perceived by the participants as relevant and easy to comprehend. For the remaining 27% of the items, the content was perceived to be relevant in all countries, but some terms and expressions were reported to require rewording or clearer examples. These modifications will be made in the next step of the development process. CONCLUSION: Content validation of the HFEQ demonstrates promising results, with participants perceiving the content as relevant and comprehensible. Further psychometric validation is required to investigate other psychometric properties, such as construct validity and reliability. The HFEQ has the potential to become a valuable new instrument for assessing everyday functioning in people with hearing loss in audiological rehabilitation and in research.

The Social Insurance Literacy Questionnaire (SILQ): Development and Psychometric Evaluation.

PURPOSE: For clients to understand social insurance decisions and processes, information from authorities needs to be comprehensible, and clients need sufficient individual abilities. These dimensions are captured by the concept social insurance literacy, which has been operationalized into a measure, the Social Insurance Literacy Questionnaire (SILQ). The aim of this study was to describe the development of the SILQ and evaluate its psychometric properties using Rasch measurement theory. METHODS: The development of the SILQ included a Delphi study and cognitive interviews. A preliminary version, divided on four scales corresponding to the domains of the concept (obtaining information, understanding information, acting on information, and system comprehensibility) was psychometrically evaluated according to Rasch measurement theory, in a survey to a stratified random sample of people on sick leave (n = 1151) sent out in the fall of 2020. RESULTS: Overall, the items in the final version of the SILQ demonstrated good fit to the Rasch model, and the response scale worked as intended. Unidimensionality was supported for all scales, but minor problems with local dependency was detected for three items. The person separation was 0.80 for the Obtain scale, 0.82 for the Understand scale, 0.68 for the Act scale, and 0.81 for the System scale. Corresponding ordinal alpha values were 0.91, 0.91, 0.86, and 0.91, respectively. CONCLUSION: This study is a first step toward exploring literacy in the social insurance field. The SILQ covers individual abilities and systems' comprehensibility, and the results show that it has acceptable psychometric properties.

Exploring Interactions in the Sickness Insurance System in Terms of Power and Trust.

Purpose Activation policies and efforts to reduce sick leave rates has influenced sickness insurance systems in Western countries, which has led to social security being more connected with work and attempts to expose malingering among the sickness absent. The aim of this study was to explore how power and trust are expressed by clients and stakeholders within the Swedish sickness insurance system. Methods This was a longitudinal qualitative study based on semi structured interviews and case files from 31 clients on sick leave in Sweden. Data was analyzed using a thematic analysis. Results The main theme 'Acts of power and distrust' illustrates how stakeholders' express suspicions towards each other, and how clients need to demonstrate desire and efforts to return to work which other stakeholders verified. Conclusions The clients desire to prove themselves able to contribute to society was prominent in this study and power relations need to be acknowledged, in particular between client and the SIA. Further, to preserve citizens trust in the system, the system needs to demonstrate trust also in the clients.

Functional interplay between protein domains in a supramodular structure involving the postsynaptic density protein PSD-95.

Cell scaffolding and signaling are governed by protein-protein interactions. Although a particular interaction is often defined by two specific domains binding to each other, this interaction often occurs in the context of other domains in multidomain proteins. How such adjacent domains form supertertiary structures and modulate protein-protein interactions has only recently been addressed and is incompletely understood. The postsynaptic density protein PSD-95 contains a three-domain supramodule, denoted PSG, which consists of PDZ, Src homology 3 (SH3), and guanylate kinase-like domains. The PDZ domain binds to the C terminus of its proposed natural ligand, CXXC repeat-containing interactor of PDZ3 domain (CRIPT), and results from previous experiments using only the isolated PDZ domain are consistent with the simplest scenario for a protein-protein interaction; namely, a two-state mechanism. Here we analyzed the binding kinetics of the PSG supramodule with CRIPT. We show that PSG binds CRIPT via a more complex mechanism involving two conformational states interconverting on the second timescale. Both conformational states bound a CRIPT peptide with similar affinities but with different rates, and the distribution of the two conformational states was slightly shifted upon CRIPT binding. Our results are consistent with recent structural findings of conformational changes in PSD-95 and demonstrate how conformational transitions in supertertiary structures can shape the ligand-binding energy landscape and modulate protein-protein interactions.

Mapping the transition state for a binding reaction between ancient intrinsically disordered proteins.

Intrinsically disordered protein domains often have multiple binding partners. It is plausible that the strength of pairing with specific partners evolves from an initial low affinity to a higher affinity. However, little is known about the molecular changes in the binding mechanism that would facilitate such a transition. We previously showed that the interaction between two intrinsically disordered domains, NCBD and CID, likely emerged in an ancestral deuterostome organism as a low-affinity interaction that subsequently evolved into a higher-affinity interaction before the radiation of modern vertebrate groups. Here we map native contacts in the transition states of the low-affinity ancestral and high-affinity human NCBD/CID interactions. We show that the coupled binding and folding mechanism is overall similar but with a higher degree of native hydrophobic contact formation in the transition state of the ancestral complex and more heterogeneous transient interactions, including electrostatic pairings, and an increased disorder for the human complex. Adaptation to new binding partners may be facilitated by this ability to exploit multiple alternative transient interactions while retaining the overall binding and folding pathway.

Validation of the Brief International Classification of Functioning, Disability and Health (ICF) core set for hearing loss: an international multicentre study.

OBJECTIVE: Hearing loss (HL) affects the everyday functioning of millions of people worldwide. The Brief International Classification of Functioning Disability and Health (ICF) core sets for HL was developed to meet the complex health care needs of adults with HL. Because the brief core set for HL has not yet been validated internationally, this study aimed to investigate its validity from an international perspective. DESIGN: A cross-sectional validation study based on data from structured interviews with adults with HL. STUDY SAMPLE: Participants (n = 571) from India, South Africa, Sweden and the US were included. RESULTS: A six-factor solution explained 71% of the variance, focussing on issues related to communication, the social environment, participation in society, health care services, support, relationships and emotions (α = 0.915). Three ICF categories demonstrated low reliability - temperament and personality functions, seeing functions and school education. CONCLUSION: The Brief ICF core set for HL is valid for adults with HL internationally. However, to further increase its international validity, we recommend adding the categories d920 recreation and leisure and replacing d850 school education with the more inclusive block, d810-d839 education.

Emergencies in freestanding ambulatory surgery centre.

PURPOSE OF REVIEW: Ambulatory surgery is increasing, more procedures as well as more complex procedures are transferred to ambulatory surgery. Patients of all ages including elderly and more fragile are nowadays scheduled for ambulatory surgery. Enhanced recovery after surgery (ERAS) protocols are now developed for further facilitating readily recovery, ambulation, and discharge. Thus, to secure safety, a vigilant planning and preparedness for adverse events and emergencies is mandatory. RECENT FINDINGS: Proper preoperative assessment, preparation/optimization and collaboration between anaesthetist and surgeon to plan for the optimal perioperative handling has become basic to facilitate well tolerated perioperative course. Standard operating procedures for rare emergencies must be in place. These SOPs should be trained and retrained on a regular basis to secure safety. Check lists and cognitive aids are tools to help improving safety. Audit and analysis of adverse outcomes and deviations is likewise of importance to continuously analyse and implement corrective activity plans whenever needed. SUMMARY: The present review will provide an oversight of aspects that needs to be acknowledged around planning handling of rare but serious emergencies to secure quality and safety of care in freestanding ambulatory settings.

A structurally heterogeneous transition state underlies coupled binding and folding of disordered proteins.

Many intrinsically disordered proteins (IDPs) attain a well-defined structure in a coupled folding and binding reaction with another protein. Such reactions may involve early to late formation of different native structural regions along the reaction pathway. To obtain insights into the transition state for a coupled binding and folding reaction, we performed restrained molecular dynamics simulations using previously determined experimental binding Φb values of the interaction between two IDP domains: the activation domain from the p160 transcriptional co-activator for thyroid hormone and retinoid receptors (ACTR) and the nuclear co-activator binding domain (NCBD) of CREB-binding protein, each forming three well-defined α-helices upon binding. These simulations revealed that both proteins are largely disordered in the transition state for complex formation, except for two helices, one from each domain, that display a native-like structure. The overall transition state structure was extended and largely dynamic with many weakly populated contacts. To test the transition state model, we combined site-directed mutagenesis with kinetic experiments, yielding results consistent with overall diffuse interactions and formation of native intramolecular interactions in the third NCBD helix during the binding reaction. Our findings support the view that the transition state and, by inference, any encounter complex in coupled binding and folding reactions are structurally heterogeneous and largely independent of specific interactions. Furthermore, experimental Φb values and Brønsted plots suggested that the transition state is globally robust with respect to most mutations but can display more native-like features for some highly destabilizing mutations, possibly because of Hammond behavior or ground-state effects.

Coupled Binding and Helix Formation Monitored by Synchrotron-Radiation Circular Dichroism.

Intrinsically disordered proteins organize interaction networks in the cell in many regulation and signaling processes. These proteins often gain structure upon binding to their target proteins in multistep reactions involving the formation of both secondary and tertiary structure. To understand the interactions of disordered proteins, we need to understand the mechanisms of these coupled folding and binding reactions. We studied helix formation in the binding of the molten globule-like nuclear coactivator binding domain and the disordered interaction domain from activator of thyroid hormone and retinoid receptors. We demonstrate that helix formation in a rapid binding reaction can be followed by stopped-flow synchrotron-radiation circular dichroism (CD) spectroscopy and describe the design of such a beamline. Fluorescence-monitored binding experiments of activator of thyroid hormone and retinoid receptors and nuclear coactivator binding domain display several kinetic phases, including one concentration-independent phase, which is consistent with an intermediate stabilized at high ionic strength. Time-resolved CD experiments show that almost all helicity is formed upon initial association of the proteins or separated from the encounter complex by only a small energy barrier. Through simulation of mechanistic models, we show that the intermediate observed at high ionic strength likely involves a structural rearrangement with minor overall changes in helicity. Our experiments provide a benchmark for simulations of coupled binding reactions and demonstrate the feasibility of using synchrotron-radiation CD for mechanistic studies of protein-protein interactions.

Loss of protein tyrosine phosphatase, non-receptor type 2 is associated with activation of AKT and tamoxifen resistance in breast cancer.

Breast cancer is a heterogeneous disease and new clinical markers are needed to individualise disease management and therapy further. Alterations in the PI3K/AKT pathway, mainly PIK3CA mutations, have been shown frequently especially in the luminal breast cancer subtypes, suggesting a cross-talk between ER and PI3K/AKT. Aberrant PI3K/AKT signalling has been connected to poor response to anti-oestrogen therapies. In vitro studies have shown protein tyrosine phosphatase, non-receptor type 2 (PTPN2) as a previously unknown negative regulator of the PI3K/AKT pathway. Here, we evaluate possible genomic alterations in the PTPN2 gene and its potential as a new prognostic and treatment predictive marker for endocrine therapy benefit in breast cancer. PTPN2 gene copy number was assessed by real-time PCR in 215 tumour samples from a treatment randomised study consisting of postmenopausal patients diagnosed with stage II breast cancer 1976-1990. Corresponding mRNA expression levels of PTPN2 were evaluated in 86 available samples by the same methodology. Gene copy loss of PTPN2 was detected in 16% (34/215) of the tumours and this was significantly correlated with lower levels of PTPN2 mRNA. PTPN2 gene loss and lower mRNA levels were associated with activation of AKT and a poor prognosis. Furthermore, PTPN2 gene loss was a significant predictive marker of poor benefit from tamoxifen treatment. In conclusion, genomic loss of PTPN2 may be a previously unknown mechanism of PI3K/AKT upregulation in breast cancer. PTPN2 status is a potential new clinical marker of endocrine treatment benefit which could guide further individualised therapies in breast cancer.

VAV3 mediates resistance to breast cancer endocrine therapy.

INTRODUCTION: Endocrine therapies targeting cell proliferation and survival mediated by estrogen receptor α (ERα) are among the most effective systemic treatments for ERα-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERα transcriptional regulatory plasticity. Herein we identify VAV3 as a critical component in this process. METHODS: A cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERα was evaluated by molecular docking analyses, an agonist fluoligand assay and short hairpin (sh)RNA-mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot analysis of signaling and proliferation markers, and shRNA-mediated protein depletion in viability and clonogenic assays, were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine its association with therapeutic response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression. RESULTS: The compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase), but instead was likely a result of binding to ERα. VAV3 was selectively reduced upon exposure to YC-1 or ERα depletion, and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with the response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 × 10-4). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy. CONCLUSIONS: This study proposes VAV3 as a biomarker and a rationale for its use as a signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer.

Hearing and Functioning in Everyday Life Questionnaire: Psychometric Evaluation and Revision.

PURPOSE: The aim of the current study was to explore the construct validity and internal consistency reliability of the International Classification of Functioning, Disability and Health (ICF)-based original English version of the Hearing and Functioning in Everyday Life Questionnaire (HFEQ) and to revise the HFEQ based on the results. METHOD: This study used a cross-sectional survey design. The data were collected using an online survey. Adults with self-reported hearing disability (n = 513) from the United States were included. The ICF components of body functions, activity and participation, and environmental factors were tested as the underlying structure of the HFEQ using confirmatory factor analysis and then adjusted by triangulation with previous content validation. RESULTS: The results of the current study confirmed the ICF components of body functions, activity and participation, and environmental factors as underlying constructs of the HFEQ. However, after triangulation with previous content validation, fine adjustments were made. The revised version of the HFEQ includes two removed items and a fine-tuned factor structure. CONCLUSION: The results confirm that the structure of the HFEQ aligns with the ICF, and the overall results indicate that HFEQ has acceptable construct validity and internal consistency.

Temporal Relation Between Myocardial Infarction and New-Onset Atrial Fibrillation: Results from a Nationwide Registry Study.

Myocardial infarction (MI) and atrial fibrillation (AF) are commonly seen in the same patient. In this study, we evaluated the temporal relations and prognosis of MI and AF. This is a substudy of the nationwide registry-based Finnish Anticoagulation in Atrial Fibrillation (FinACAF) study, comprising all Finnish patients with new-onset AF from 2010 to 2017. Patients with MI and AF were divided into groups depending on the temporal relation between the disease onsets: (1) MI before AF (MIAF), and (4) no MI. The 1-year mortality in the groups were studied with the Cox proportional hazards model. Of the 153,207 patients with new-onset AF (mean age 72.7 years, 50.0% women), 16,265 (10.6%) were diagnosed with MI. Altogether, 8,889 (54.7%) of the patients with MI were in the MIAF group. Of all MIs, 42.2% were diagnosed within 1 year from new-onset AF. The MI>AF group had the worst survival, with an adjusted hazard ratio for death of 3.08 (confidence interval [CI] 2.89 to 3.27) compared with patients without MI. For the MI

Folded Alpha Helical Putative New Proteins from Apilactobacillus kunkeei.

The emergence of new proteins is a central question in biology. Most tertiary protein folds known to date appear to have an ancient origin, but it is clear from bioinformatic analyses that new proteins continuously emerge in all organismal groups. However, there is a paucity of experimental data on new proteins regarding their structure and biophysical properties. We performed a detailed phylogenetic analysis and identified 48 putative open reading frames in the honeybee-associated bacterium Apilactobacillus kunkeei for which no or few homologs could be identified in closely-related species, suggesting that they could be relatively new on an evolutionary time scale and represent recently evolved proteins. Using circular dichroism-, fluorescence- and nuclear magnetic resonance (NMR) spectroscopy we investigated six of these proteins and show that they are not intrinsically disordered, but populate alpha-helical dominated folded states with relatively low thermodynamic stability (0-3 kcal/mol). The NMR and biophysical data demonstrate that small new proteins readily adopt simple folded conformations suggesting that more complex tertiary structures can be continuously re-invented during evolution by fusion of such simple secondary structure elements. These findings have implications for the general view on protein evolution, where de novo emergence of folded proteins may be a common event.

The mTOR effectors 4EBP1 and S6K2 are frequently coexpressed, and associated with a poor prognosis and endocrine resistance in breast cancer: a retrospective study including patients from the randomised Stockholm tamoxifen trials.

INTRODUCTION: mTOR and its downstream effectors the 4E-binding protein 1 (4EBP1) and the p70 ribosomal S6 kinases (S6K1 and S6K2) are frequently upregulated in breast cancer, and assumed to be driving forces in tumourigenesis, in close connection with oestrogen receptor (ER) networks. Here, we investigated these factors as clinical markers in five different cohorts of breast cancer patients. METHODS: The prognostic significance of 4EBP1, S6K1 and S6K2 mRNA expression was assessed with real-time PCR in 93 tumours from the treatment randomised Stockholm trials, encompassing postmenopausal patients enrolled between 1976 and 1990. Three publicly available breast cancer cohorts were used to confirm the results. Furthermore, the predictive values of 4EBP1 and p4EBP1_S65 protein expression for both prognosis and endocrine treatment benefit were assessed by immunohistochemical analysis of 912 node-negative breast cancers from the Stockholm trials. RESULTS: S6K2 and 4EBP1 mRNA expression levels showed significant correlation and were associated with a poor outcome in all cohorts investigated. 4EBP1 protein was confirmed as an independent prognostic factor, especially in progesterone receptor (PgR)-expressing cancers. 4EBP1 protein expression was also associated with a poor response to endocrine treatment in the ER/PgR positive group. Cross-talk to genomic as well as non-genomic ER/PgR signalling may be involved and the results further support a combination of ER and mTOR signalling targeted therapies. CONCLUSION: This study suggests S6K2 and 4EBP1 as important factors for breast tumourigenesis, interplaying with hormone receptor signalling. We propose S6K2 and 4EBP1 as new potential clinical markers for prognosis and endocrine therapy response in breast cancer.

Activation of Akt, mTOR, and the estrogen receptor as a signature to predict tamoxifen treatment benefit.

The frequent alterations of the PI3K/Akt/mTOR-growth signaling pathway are proposed mechanisms for resistance to endocrine therapy in breast cancer, partly through regulation of estrogen receptor α (ER) activity. Reliable biomarkers for treatment prediction are required for improved individualized treatment. We performed a retrospective immunohistochemical analysis of primary tumors from 912 postmenopausal patients with node-negative breast cancer, randomized to either tamoxifen or no adjuvant treatment. Phosphorylated (p) Akt-serine (s) 473, p-mTOR-s2448, and ER phosphorylations-s167 and -s305 were evaluated as potential biomarkers of prognosis and tamoxifen treatment efficacy. High expression of p-mTOR indicated a reduced response to tamoxifen, most pronounced in the ER+/progesterone receptor (PgR) + subgroup (tamoxifen vs. no tamoxifen: hazard ratio (HR), 0.86; 95 % confidence interval (CI), 0.31-2.38; P = 0.78), whereas low p-mTOR expression predicted tamoxifen benefit (HR, 0.29; 95 % CI, 0.18-0.49; P = 0.000002). In addition, nuclear p-Akt-s473 as well as p-ER at -s167 and/or -s305 showed interaction with tamoxifen efficacy with borderline statistical significance. A combination score of positive pathway markers including p-Akt, p-mTOR, and p-ER showed significant association with tamoxifen benefit (test for interaction; P = 0.029). Cross-talk between growth signaling pathways and ER-signaling has been proposed to affect tamoxifen response in hormone receptor-positive breast cancer. The results support this hypothesis, as an overactive pathway was significantly associated with reduced response to tamoxifen. A clinical pre-treatment test for cross-talk markers would be a step toward individualized adjuvant endocrine treatment with or without the addition of PI3K/Akt/mTOR pathway inhibitors.