Guidelines for the management of blood pressure (BP) in primary care generally suggest that decisions be made on the basis of specific threshold values (e.g. BP 140/90 mmHg); but this fails to adequately accommodate a common cause of variation – the play of chance.To determine the impact of chance variability in BP readings on the clinical decision making of general practitioners (GPs) regarding anti-hypertensive treatment and cardiovascular risk management.We used an internet based study design, where 109 GPs were assigned to manage one of eight case vignettes (guidelines would recommend treatment for only one of the eight) and presented with blood pressure readings that were randomly selected from an underlying population.Seventeen  GPs consulted the vignette for whom treatment was recommended, but only 7/17 (41.2%) GPs prescribed treatment, whereas 14/92 (15.2%) GPs prescribed medication to the other vignettes. When deciding to follow-up a vignette GPs were influenced by threshold values for systolic and diastolic BP, but not by the overall cardiovascular risk. If the first reading was a low BP  GPs were highly likely to discharge the vignette and follow-up a high BP reading (diastolic >90 or systolic BP≥140). Similar factors predicted the decision to prescribe a drug, although the vignette’s cardiovascular risk (>20%) was now statistically significant (p = 0.03).GP decision making, whilst generally consistent with guidelines, appears to be compromised by chance variation leading to under and over treatment. Interventions to adequately accommodate chance variability into clinical decision making are required. Like many other countries, guidelines in the United Kingdom (UK) determine whether a patient is recommended antihypertensive treatment based on their measured blood pressure and their ten-year cardiovascular risk Although guidelines are clear, the decision to start a patient on treatment is not straightforward because within the same individual, measured blood pressure varies. Measured blood pressure exhibits variation of two types: systematic and random (or chance). Systematic variation is caused by a range of patient factors such as flu, the white-coat effect or pain The intrinsic biological variability of blood pressure gives rise to “chance like” variation in blood pressure from beat to beat, minute to minute and day to day. To try and take account of blood pressure variability, clinical diagnosis is based on the average of a number of blood pressure measurements: in an attempt to estimate the true, but unknown, mean blood pressure.Since blood pressure measurement is used as a diagnostic test, variability of blood pressure can lead to false positives – where normotensives misclassified as hypertensive - and false negatives - hypertensives misclassified as normotensive. The Positive Predictive Value Investigating the effect of blood pressure variation on clinical decision-making in clinical practice is challenging because patients with different blood pressures also differ in characteristics such as age, gender or other cardiovascular risk factors and the act of measurement, treatment and management interfere/confound the estimation of the true underlying blood pressure. However simulation, using case vignettes, is one approach to investigating the impact of chance variation on the clinical decision making process because it avoids some of these challenging problems TM sought ethical approval from the South Birmingham Research Ethics Committee and was advised that the project did not require ethical review. All data were handled anonymously.A website was developed that presented one of eight typical case vignettes of a patient consulting for a routine check up in primary care. The eight case vignettes described a 40, 50, 60 and 70 year old white man and a 40, 50, 60 and 70 year old white woman. In each case the vignette was a non smoking, non diabetic, who drank alcohol in moderation and exercised twice a week, with a body mass index of 25 kg/m2. The total cholesterol level and HDL cholesterol level were the mean for a person of this age and sex based on the Health Survey for England of 1998 Participants who accessed the website were presented with information on the patient’s characteristics, with summaries of current UK guidelines and a hyperlink to an on-line cardiovascular risk calculator On accessing the website, participants were randomly allocated to one of the eight case vignettes and presented with a first consultation blood pressure reading. Participants were asked to indicate which of three possible decisions they would take. These decisions were coded as 1 to 3 . If the At subsequent consultations participants were provided with further randomly selected blood pressure drawn from a population of two hundred. If the patient had been started on treatment the subsequent blood pressure was drawn from a population of two hundred blood pressures with a slightly lower mean, reflecting the effects of treatment. Treatment effects were determined from the average effects reported in meta-analysis We aimed to recruit UK based GPs, so participants were recruited by emailing members of a number of the Primary Care Cardiovascular Society, the Royal College of General Practitioners and MidRec (the Midlands Research network). Advertisements were placed in Pulse (a UK magazine read by GPs) to draw attention to the study. All participants could opt to be included in a draw with a prize of £100. Participants were asked to provide basic details: their age, gender and clinical specialty.Party Package ROur exploratory analysis involved the use of Classification and Regression Trees (CART) which are a statistical data mining based technique for constructing decision trees by recursively splitting or partitioning patients into homogenous groups lme4RWe used the insights from the tree models to construct two random effects models logistic regression models using the The first model focused on the decision to follow-up (decision 2) the patient and the second model focused on the decision to prescribe medication (decision 3). These two decisions were considered the most important in our study . The covariates in the random effects models were the patients age (years), patients gender , GPs age (years), GPs gender , patient’s first visit (yes/no), patients systolic BP≥120 (yes/no), patients diastolic BP≥90 (yes/no) and CVD Risk ≥20% (yes/no). We investigated two way interactions between the blood pressure variables and first visit and retained only those interactions which were statistically significant at the 5% level. GPs were included as random effects as were our eight vignettes  Our use and choice of threshold values for systolic BP, diastolic BP and CVD Risk was based on values commonly indicated in clinical guidelines (BP 140/90) which were also seen in our exploratory tree models.Between September 2007 and July 2008, 109 UK GPs, 65 male and 44 female with an average age of 40.28 years (SD: 9.44), accessed the website and answered questions relating to the case vignettes.Specifically if the patient  had a low BP  then the GPs discharged the patient . If the patient  had a low diastolic BP (≤89) but a high systolic BP (>138) then GPs were more likely to invite the patient for a follow-up  and less likely to discharge the patient (5/18 27.7% were discharged). Finally, a high diastolic reading  was most likely to result in a follow-up visit , with 8/67 (11.9%) being discharged and 1/67 (1.5%) being prescribed a drug. The decision to prescribe was related to a one-off high BP reading (systolic 175 and diastolic 107) in vignette eight, which equates to ten year CVD risk calculated with this blood pressure reading would be 20.9% (compared to true underlying CVD risk of 13.9%). At the end of the first consultation, 34% (37/109) of GPs had discharged the patient, whereas 65% (71/109) invited the patient for a follow-up consultation and 1 GP had prescribed medication.If the patient,  was on treatment and presented with a high diastolic BP (>94) then on these occasions, GPs were most likely to add another medication , although in 20/56 such occasions GPs invited the patients for follow-up, on 4/56 such occasions the medication was changed and on one such occasion (1/56) a drug was stopped.If the patient  was on treatment and presented with a low diastolic BP (≤94) then on these occasions, GPs were most likely to invite the patient for a follow-up (74/102), although in 13/102 occasions GPs prescribed an additional BP drug, on 11/102 occasions GPs discharged the patient, on 3/102 occasions GPs stopped one medication and one occasion (1/102) the GP changed the medication.If the patient  was not on treatment and presented with a high diastolic BP (>91) then on these occasions GPs were most likely to invite the patient for a follow-up (44/49), although on 4/49 occasions GPs discharged the patient and on one occasion (1/49) the GP started the patient on medication.If the patient  was not on treatment and presented with a low diastolic BP (≤91) and a low systolic BP (≤137) then in 39/63 such occasions GPs discharged the patient, whilst in 24/63 occasions GPs invited the patient for follow-up.If the patient  was not on treatment and presented with a low diastolic BP (≤91) and a high systolic BP (>137) then in 27/31 such occasions GPs invited the patient for follow-up, whilst on four (4/31) occasions GPs discharged the patient.We further investigated the decision to follow-up the patient using a random effects logistic regression model  and founWe further investigated the decision to prescribe medication using a random effects logistic regression model  and founTo our knowledge this is the first study to explicitly explore how clinician decision making is influenced by chance variation in blood pressure. We found that when deciding to follow-up the patient, GPs appeared not to consider if the vignette’s overall cardiovascular risk was ≥20%, but were influenced by threshold values for systolic and diastolic BP. Furthermore, whilst GPs decisions to prescribe a drug were also affected by similar thresholds, the patient’s cardiovascular risk (>20%) was now seen to be statistically significant. Since cardiovascular risk is only valid in untreated populations we would expect it to influence only the decision to intiate treatment. Finally, we saw evidence of under treatment and over treatment. Under half of practitioners prescribed for the only patient who would be recommended treatment under UK guidelines and about 15% of GPs prescribed medication to non-recommended patients.Treatment seemed to be determined more by blood pressure than by estimated risk of cardiovascular disease. This observation has also been made in UK clinical practice A weakness of this website based study design is that theoretical decisions are made about a theoretical patient, although we found no evidence of nonsensical decision making, indeed the decisions appeared rational. However as observed earlier, this is the only practical design that can investigate the effects of blood pressure variation on standardised patients. Our participating GPs are self-selected volunteers with an average age of 40.3 years and 59.6%  are male. In the UK 52.6% of GPs are male and in England mean age is about 46 A key question about the study design is whether the variability of blood pressure in the vignettes adequately represents what is seen in clinical practice. The coefficient of variation of blood pressure used in the case vignettes (9.9%) is consistent with published reports which range from 7.2% to 10% In the UK it has recently been recommended that 24-hour ambulatory blood pressure measurement be used for diagnosis of hypertension when blood pressures are close to a treatment threshold Whilst further studies to determine the impact of chance variation on clinical decision making would be useful, it is also worth noting that there is little in the clinical guidelines to mitigate against chance variation and interventions that can help clinicians and patients understand and appropriately react to chance variability are also needed, especially because our findings suggest that addressing chance variability could be a promising strategy for reducing under/over treatment and their associated costs.


An increasing body of evidence now implicates acetaldehyde as a major underlying factor for the carcinogenicity of alcoholic beverages and especially for oesophageal and oral cancer. Acetaldehyde associated with alcohol consumption is regarded as 'carcinogenic to humans' (IARC Group 1), with sufficient evidence available for the oesophagus, head and neck as sites of carcinogenicity. At present, research into the mechanistic aspects of acetaldehyde-related oral cancer has been focused on salivary acetaldehyde that is formed either from ethanol metabolism in the epithelia or from microbial oxidation of ethanol by the oral microflora. This study was conducted to evaluate the role of the acetaldehyde that is found as a component of alcoholic beverages as an additional factor in the aetiology of oral cancer.Salivary acetaldehyde levels were determined in the context of sensory analysis of different alcoholic beverages , without swallowing, to exclude systemic ethanol metabolism.The rinsing of the mouth for 30 seconds with an alcoholic beverage is able to increase salivary acetaldehyde above levels previously judged to be carcinogenic in vitro, with levels up to 1000 μM in cases of beverages with extreme acetaldehyde content. In general, the highest salivary acetaldehyde concentration was found in all cases in the saliva 30 sec after using the beverages (average 353 μM). The average concentration then decreased at the 2-min (156 μM), 5-min (76 μM) and 10-min (40 μM) sampling points. The salivary acetaldehyde concentration depends primarily on the direct ingestion of acetaldehyde contained in the beverages at the 30-sec sampling, while the influence of the metabolic formation from ethanol becomes the major factor at the 2-min sampling point.This study offers a plausible mechanism to explain the increased risk for oral cancer associated with high acetaldehyde concentrations in certain beverages. EvidencIn vitro evidence shows that the acetaldehyde DNA-adduct α-methyl-γ-hydroxy-1,N2-propano-2'-deoxyguanosine (Cr-PdG) can be formed in response to acetaldehyde concentrations as low as 100 μM [s 100 μM . Two seps 100 μM , or SV40s 100 μM . Both DNs 100 μM . Acetalds 100 μM .in vitro evidence, the link between acetaldehyde and oral cancer is further substantiated by mechanistic evidence in humans deficient in aldehyde dehydrogenase (ALDH) [Apart from the e (ALDH) ,7. Strone (ALDH) ,13. ALDHe (ALDH) -16, and e (ALDH) .In addition to acetaldehyde metabolism in the gastrointestinal tract and in the liver, the oral and colonic bacterial flora may also contribute considerably to acetaldehyde accumulation ,15,18-25A quantitative risk assessment using the margin of exposure (MOE) approach has estimated the average exposure to acetaldehyde that is a direct component of alcoholic beverages as being 0.112 mg/kg body weight/day. The MOE was calculated at 498, which is considered a public health concern, and the lifetime cancer risk would be 7.6 in 10 000. Higher risk may exist for people exposed to higher acetaldehyde contamination, as we have found in certain alcoholic beverages, and exposure scenarios indicate risks in the range of 1 in 1000 .Theoretical calculations that assume an equal distribution between the beverage and saliva showed that the residual acetaldehyde concentrations in the saliva after swallowing could be, on average, 195 μM for beer, 734 μM for wine, 1387 μM for spirits, or 2417 μM for fortified wine, which are above levels previously regarded as potentially carcinogenic [The present study was conducted to evaluate acetaldehyde found as a direct component of alcoholic beverages as an additional cancer risk factor to acetaldehyde formed from ethanol. Our aim was to provide experimental data to substantiate the theoretical calculations mentioned above. In addition, we focused on differences between sub-groups of alcoholic beverages, as there are some epidemiological findings pointing to an increased risk of oesophageal cancer due to consumption of specific alcoholic beverages .The experiments were conducted within the framework of our function as governmental food and alcohol control institution, which includes a chemical-toxicological as well as an organoleptical evaluation of products by a trained panel of assessors. The experiments included only products legally sold on the market of the European Union (EU). Furthermore, the study only included products that had to be organoleptically tested anyway for other reasons, e.g. to check compliance with EU and national regulations (such as regulation (EC) 110/2008 ). The CVThe assessors were asked to be abstinent for at least one day prior to the experiment. All experiments were conducted more than 1 hour after the last meal or drink to ensure there is no contamination of saliva with interfering substances. The assessors were also asked to uphold their standard dental hygiene (twice daily toothbrush use), but not to use alcohol-containing mouthwashes, and not to ingest alcohol-containing foodstuffs during the trial period. Compliance to these criteria including the study selection criteria was obtained in writing by all participants.® . The system consists of cotton swabs that are gently chewed by the assessors. Afterwards, the swab is replaced in the suspended insert of the salivette®, which is firmly closed using a stopper. The saliva is recovered by centrifugation of the salivette® at 1,000 g for 2 min. The clear saliva supernatant was used for acetaldehyde analysis.The alcoholic beverages were rinsed by the assessors in their mouths for 30 sec and then spit out similar to a wine tasting . Saliva was sampled prior to rinsing, as well as 30 sec, 2 min, 5 min and 10 min after spitting-out. Sampling was conducted using the saliva collection system salivetteThe determination of acetaldehyde in saliva samples was conducted using either enzymatic analysis or gas chromatography. The enzymatic analysis was conducted with aldehyde dehydrogenase according to the method of Lundquist ,38, whicThe test-kit instructions of the manufacturer were followed without modification. 0.2 ml of saliva supernatant were used as sample solution. The enzymatic measurement was conducted immediately (within 1 hour) after saliva sampling to exclude losses of acetaldehyde due to evaporation or oxidation. The spectrophotometric measurements were performed on a Perkin Elmer Lambda 12 dual beam spectrometer equipped with automatic cell changer, which allows the software-controlled measurement of a sample series (n = 13) without manual intervention.The procedure for the gas chromatographic (GC) analysis was previously described in detail for the determination of acetaldehyde in saliva after alcohol-containing mouthwash use . Both thAll data were evaluated using Unscrambler X version 10.0.1  and Origin V.7.5 . Data are summarized as means and standard deviations between assessors for each data point. Statistical dependence between alcoholic strengths and the acetaldehyde contents of the beverages and the salivary acetaldehyde were evaluated using multiple linear regression (MLR) and Analysis of Variance (ANOVA) for all time data points . The regression analysis was also conducted with the area under the curve (AUC) for the complete time period under investigation (0-10 min). Statistical significance was assumed at below the 0.05 probability level.Table Figure The results of ANOVA for the MLR calculations are summarized in Table in vitro and in vivo experiments. These high variations were judged to be predominantly caused by the differences in acetaldehyde production capacity among the oral bacteria [in vitro results of Ernstgård [® saliva collection method, which may stimulate salivary secretion and thus dilute acetaldehyde and ethanol concentrations. Our study therefore could underestimate rather than overestimate the risk.Our results confirm the observation of high inter-individual variations in the acetaldehyde levels in saliva following ethanol exposure previously noted during bacteria ,40,41. WIn our previous experiments on acetaldehyde in saliva after use of alcohol-containing mouthwashes , we did The influence of the directly contained acetaldehyde, however, is short-term and only prevails during the first 2 minutes after rinsing of the mouth with an alcoholic beverage for 30 seconds. Subsequently, the concentration depends on the amount of ethanol available for metabolic oxidation. Further research should be conducted to clarify the influences in the time period between 30 sec and 5 min in more detail, as our approach does not allow to interpolate the exact time at which the change between the two factors occurs.in vivo results confirm our previous theoretical calculations of potentially high short-term acetaldehyde concentrations, as mentioned in the introduction, which were deduced from typical levels found in beverages [Similar findings to our study were generally made by Yokoyama et al. , with a everages .in vitro, as an average of 0.3 μM acetaldehyde occurred in 36 saliva samples without ethanol exposure [in vitro  in a study of Obe and Ristow was suggested as threshold for toxicity evaluation [This now leaves the question regarding how to interpret the health effects of this short-term high exposure to acetaldehyde. Whether a threshold for the carcinogenicity of acetaldehyde exists is still debatable and its potential magnitude is unclear . The natexposure . The lowaluation . This isaluation . After ialuation . Salaspualuation . Linderbaluation  indicateBecause alcohol use significantly increases salivary acetaldehyde above endogenous levels , we ascertain that a "biological threshold" is clearly exceeded during alcohol consumption. The observations of the present study and the suggested molecular mechanisms could conceivably explain the increased oral cancer risk associated with alcohol use seen in epidemiological studies . SalivarCurrently, the acetaldehyde content of most alcoholic beverage types is not regulated. The recent IARC evaluation of acetaldehyde associated with alcohol consumption as a "group 1" carcinogen has not yet been implemented in international risk assessments . Until such assessments become available, we would currently recommend the implementation of the ALARA principle ("as low as reasonably achievable") . In the The authors declare that they have no competing interests.DWL conceived of the study, coordinated the work, and drafted the manuscript. YBM conducted the statistical calculations, and composed the tables and figures. All authors read and approved the final manuscript.


A serendipitous pharmacogenetic finding links the vulnerability to developing levodopa-induced dyskinesia to the age of onset of Huntington's disease. Huntington's disease is caused by a polyglutamate expansion of the protein huntingtin. Aberrant huntingtin is less capable of binding to a member of membrane-associated guanylate kinase family (MAGUKs): postsynaptic density- (PSD-) 95. This leaves more PSD-95 available to stabilize NR2B subunit carrying NMDA receptors in the synaptic membrane. This results in increased excitotoxicity for which particularly striatal medium spiny neurons from the indirect extrapyramidal pathway are sensitive. In Parkinson's disease the sensitivity for excitotoxicity is related to increased oxidative stress due to genetically determined abnormal metabolism of dopamine or related products. This probably also increases the sensitivity of medium spiny neurons for exogenous levodopa. Particularly the combination of increased oxidative stress due to aberrant dopamine metabolism, increased vulnerability to NMDA induced excitotoxicity, and the particular sensitivity of indirect pathway medium spiny neurons for this excitotoxicity may explain the observed increased prevalence of levodopa-induced dyskinesia. Over 45 years after discovery of its beneficial effects , 2, levo GRIN2A variants, rs7192557 and rs8057394, were most frequently associated with dyskinesia in our control group of patients with neurological disorders . It is important to notice that rs7192557 is a tagging SNP for rs1969060, which was previously associated with the age of onset in Huntington's disease, and that SNP rs8057394 was the other variant identified in this study of Arning et al. [Recently, our group has published our serendipitous finding in a small group of patients we used as a control group in a study on tardive dyskinesia (TD) , 12. Ourg et al. . In our g et al. .Huntington's disease (HD) is an inherited neurodegenerative disorder, characterized by progressively worsening chorea, cognitive and psychiatric disturbances involving the basal ganglia and cerebral cortex . HD is c GRIN2A variants and the progression of both diseases. We start with a description of the structure and function of the NMDA receptor and its disfunctioning in HD.In the current paper we try to examine a possible similarity between the pathological mechanisms of LID and HD based on the found association of two GRIN2A gene encodes for a protein which is part of the ionotropic glutamatergic N-methyl-D-aspartate (NMDA) receptor [Thereceptor . The amireceptor .2+ permeability and Mg2+ sensitivity [NR2A and NR2B are relatively large proteins with molecular masses of about 170–180 kDa. They have a large extracellular N-terminal region, which carries a glutamate binding site, three relatively small transmembrane domains , and a small reentry loop (M2), which is critical for Casitivity . Bindingsitivity . Bindingsitivity , 21. Chasitivity . Fan et sitivity . This resitivity . This efsitivity .N = 250) and showed that two additional SNPs (rs8057394 and rs2650427) showed an even stronger association than the originally identified GRIN2A variant [In spite of this selectivity of huntingtin protein for affecting neurotoxicity of NR2B carrying NMDA receptors, also NR2A subunits have been associated with modulation of this neurotoxicity. However, the results are not entirely consistent. Arning et al. described that in a cohort of 167 HD patients a significant part of the variance of age of onset of illness could be attributed to a GRIN2B (rs1806201) and to a lesser extent to a GRIN2A (rs1969060) variant . These a variant . Andrese variant . However variant . GRIN2A gene on chromosome 16 [This type of differences is not very surprising. Variations of the GRIN2A genotype probably only determine the vulnerability to developing HD symptoms to a minor degree and there may exist several factors which contribute to a similar degree. The heterozygosity may vary greatly between various patient populations. As a matter of fact, the C allele frequency of the GRIN2A C/T SNP rs2650427) is 17% in Europe and 52–56% in Asia and Africa [ is 17% iosome 16 . TherefoGlutamate is the most ubiquitous excitatory neurotransmitter in the nervous system, but in HD indirect pathway medium spiny neurons are primarily affected . This caThe mechanism of levodopa-induced dyskinesia is usually studied in animal models in which nigrostriatal dopaminergic neurons have been lesioned with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 6-hydroxydopamine (6-OHDA) , 34. In We want to hypothesize that the pathological basis of LID might be degeneration of indirect pathway medium spiny neurons. This could best be studied by comparing the striatal neuronal composition of patients with PD with and without dyskinesia and that of normal controls. Unfortunately, we did not find such comparisons described in the literature. Calon et al. found increased binding to NR1/NR2B and AMPA glutamate receptors in the lateral putamen associated with the clinical observation of LD-induced motor complications in advanced PD, but this tells relatively little about their subcellular localization and the mechanism behind it .When our hypothesis is accepted, why the clinical presentation of LID is so variable becomes better understandable. The pattern of dyskinesia varies with respect to the time of onset in relation to levodopa intake . This mip = 0.0062) for 101 patients diagnosed with Parkinson's disease and 2.71  for the whole group of 143 patients with neurological disorders. For rs8057394, these figures were 3.59  and 2.86 , respectively. In the same study we found no significant association at all between these two SNPs and tardive dyskinesia in 431 patients with schizophrenia  or schizotypical disorders according to ICD-10 criteria. This might be related to the specific genetic makeup of patients suffering from PD. Schizophrenic patients do not have a genetically determined vulnerability of dopaminergic neurotoxicity which is needed to become specifically vulnerable to this NMDA effect.Why would a change within the functioning of NMDA receptors carrying NR2A subunits result in increased vulnerability to develop LID, as we have found in our Siberian PD patients ? This waWe want to hypothesize that NMDA receptor related excitotoxicity plays an important role in both HD and LID. In HD, excitotoxicity is caused by disinhibition of NR2B carrying NMDA receptors which are more extensively stabilized within the synaptic membrane and therefore are regularly too active (become excitotoxic). In LID, we suggest that the vulnerability to normal excitotoxic insults is potentiated due to the contribution of increased intracellular oxidative stress. This last phenomenon is expected to be caused by the same genetic composition which is causing degeneration of dopaminergic nigrostriatal neurons in these patients. As medium spiny neurons of the indirect pathway are more vulnerable to excitotoxicity than those of the direct pathway, a mismatch between their activities is caused, which results in instability and therefore an increased chance of adverse reactions to stimulation of dopamine receptors.


There is limited effect of tyrosine kinase inhibitors or “naked” antibodies binding EGFR or HER2 for therapy of metastasized urinary bladder cancer and these methods are therefore not routinely used. Targeting radio-nuclides to the extracellular domain of the receptors is potentially a better possibility.EGFR- and HER2-expression was analyzed for primary tumors and corresponding metastases from 72 patients using immunohistochemistry and the internationally recommended HercepTest. Intracellular mutations were not analyzed since only the receptors were considered as targets and intracellular abnormalities should have minor effect on radiation dose.EGFR was positive in 71% of the primary tumors and 69% of corresponding metastases. Local and distant metastases were EGFR-positive in 75% and 66% of the cases, respectively. The expression frequency of HER2 in related lesions was slightly higher (data from previous study). The EGFR-positive tumors expressed EGFR in metastases in 86% of the cases. The co-expression of EGFR and HER2 was 57% for tumors and 53% for metastases. Only 3% and 10% of the lesions were negative for both receptors in tumors and metastases, respectively. Thus, targeting these receptors with radionuclides might be applied for most patients.At least one of the EGFR- or HER2-receptors was present in most cases and co-expressed in more than half the cases. It is therefore interesting to deliver radionuclides for whole-body receptor-analysis, dosimetry and therapy. This can hopefully compensate for resistance to other therapies and more patients can hopefully be treated with curative instead of palliative intention. Evidence for therapy efficacy of such agents in urinary bladder cancer is lacking and it has been claimed that there might, in several cases, be resistance.Biological resistance to both EGFR- and HER2-targeted therapies, due to mutations in for example PI3K/AKT, Ras/Raf/Mek/Erk or other intracellular signal pathways has been observed for many types of cancer.e.g. antibodies, antibody fragments and smaller proteins such as affibody molecules and also with peptides.Therapy with radionuclides is of interest since induced resistance to effects of radiation is not a major problem in cancer therapy. The radionuclides can be delivered to cancer cells with various types of molecules, i.e. HER3 and HER4, has so far not been introduced for clinical applications so we focus only on EGFR and HER2 in this study.EGFR and HER2 belong to the type 1 tyrosine kinase receptor family consisting of four related receptors, forming dimers with each other, and are important for growth of various cancers.e.g. receptor targeted radionuclide therapy is of interest to exploit. We analyzed and discussed in this article whether EGFR and HER2 are expressed with such high frequencies that targeted radionuclide therapy might be a possibility and an alternative or complement to other modalities in the treatment of metastatic urinary bladder cancers.The worldwide incidence of urinary bladder cancer is high with 350–400.000 new cases per year and the incidence is high also in Europe.The study included 72 patients with metastatic urinary bladder carcinoma, where tissue samples from both primary tumors and metastases were available. The study was approved by the institutional review board. In the previous publication 90 patients were analysed232O2 in PBS for 20 min. Then, antigen retrieval was done in 0.05% protease K  in PBS for 10 min at room temperature. The tissue sections were preincubated in PBS for 10 min and then the primary mouse monoclonal antibody  directed against the EGF receptor, diluted 1:100, was added and the sections incubated overnight at 4°C. The secondary biotinylated antibody  and the peroxidase-labeled streptavidin-biotin complex , diluted 1:200, were then added and the samples incubated for 30 min at room temperature. All sections were developed in 0.05% diamino benzidine  for 5 min and counterstained in Harris haematoxylin . Finally, the sections were dehydrated through graded alcohol to xylene and mounted in organic mounting medium .EGFR was assessed by immunohistochemistry, IHC, using a streptavidin-biotin complex technique as previously described.The IHC staining was made as previously described.Evaluation of the IHC staining was performed by four of the authors. Authors JC and MT evaluated the expression in this study. TG, MT and KW evaluated the HER2 expression in the previously published study.22et al.The cellular membrane expression pattern of EGFR is similar to that of HER2. EGFR expression was therefore evaluated using the same scoring criterion as for HER2. Briefly, the EGFR- and HER2-expressions were scored using the HercepTest criterion based on a scale where 0 corresponded to tumor cells that were completely negative, 1+ corresponded to faint perceptible staining of the tumor cell membranes, 2+ corresponded to moderate staining of the entire tumor cell membranes and 3+ was strong circumferential staining of the entire tumor cell membranes creating a fishnet pattern. The Canadian and the DAKO HercepTest guidelinesCytoplasmic staining was considered non-specific and was not included in the scoring. As positive controls we used positive tissue sections; epithelial samples from skin for EGFR-positivity and HER2-positive breast cancer samples complemented with HER2-positive sections from DAKO. All positive controls were stained with the same protocol as applied for the urinary bladder cancer sections. As negative controls we used normal tissues which are expected not to express the receptors such as connective tissue, blood cells and lymphocytes seen in the same sections as the tumor cells. In the metastases sections we used lymphocytes, connective tissue and the surrounding capsule of the lymph nodes as negative internal controls. Thus, all tissues used as negative controls were stained with the same protocol as the urinary bladder cancers since they were in the same sections.Scores 2+ and 3+ were considered receptor positive and scores 0 and 1+ receptor negative. This a “crude” rule but gives an indication of which patients that are candidates for nuclear medicine based receptor analysis and thereafter considered suitable for radionuclide therapy.The EGFR determinations are shown in i.e. 6 upregulations and 7 downregulations, in the metastases in relation to the primary tumors and 11 of these changes were in the distant metastases , dosimetry and therapy is high. Furthermore, targeting of both receptors in the same patient might, in many cases, be a possibility to deliver increased amounts of radionuclides, Radionuclide therapy is of special interest when there is “biological resistance” to EGFR- and/or HER2-targeted therapies probably because of intracellular signal pathway mutationsIt is of methodological interest that discrepancies between gene amplification and receptor expression has actually been reported from studies of urinary bladder cancer.Furthermore, IHC analyses cannot be a basis for radionuclide dosimetry and such calculations were of course beyond the scope of this study. Reliable information related to the spatial distribution of receptor expression and dosimetry for each patient, and possibly also for prediction of therapy results, require a well functioning whole body, non-invasive nuclear medicine based method as suggested for other types of tumors.It is often claimed that positivity for EGFR and HER2 receptors indicate aggressive tumor cells and bad survival prognosisThe expression frequency of EGFR in metastatic urinary bladder cancer has in several articles been reported to be in the range 40–100% for both primary tumors and metastases50The tendency towards lower HER2-expression frequencies with increasing “distance” from the primary tumor as previously indicatedi.e. in the range 25-100% in primary tumorsThe expression frequency of HER2 has also been reported to often be high and vary a lot, The large differences in reported receptor expression frequencies between different studies are probably due to different patient inclusion criteria and etiological differences but differences in receptor scoring methods and IHC retrieval techniques might also be explanations. The values for HER2 positivity in our 2005 article on urinary bladder cancer25Suitable molecules for delivery of radionuclides are antibodies, antibody fragments, affibody molecules and small molecules such as peptides.67Cu, 90Y, 131I, 177Lu, 186Re, 188Re) and α-emitters . The requirement is of course that relevant labeling methods are available that not significantly disturb receptor binding. For receptor mapping (imaging) and dosimetry, radionuclides such as 99mTc, 111In and 123I for gamma cameras (including SPECT) or 18F, 64Cu, 68Ga, 76Br, 86Y, 89Zr and 124I for PET cameras can be applied when relevant labeling methods are at hand.43Radionuclides suitable for therapy are β-emitters  and radiolabeled peptides for therapy of neuroendocrine tumorse.g. somatostatin analogs such as 177Lu-Octreotate). There is potential for more and improved clinical studies since preclinical research and early clinical studies have given interesting results and there is also an intensive search for new targeting agents with advantageous pharmacokinetics and biodistributions.Targeted radionuclide therapy has been available for many years but few methods are routinely used on a large scale. However, during the past decades promising clinical and preclinical research have been made with therapy using radiolabeled antibodies for therapy of lymphomasThe novelty of our previous article published 2005 was to show the high percentage of HER2 expression in urinary bladder cancers and to indicate a possible value of HER2-targeted therapy (with for example lapatinib and/or trastuzumab).In conclusion, it is of interest to try to target EGFR and/or HER2 for radionuclide based therapy of disseminated urinary bladder cancers to decrease the influence of resistance to other forms of therapy. This might improve therapy effectiveness and hopefully allow that more patients can be treated with curative instead of palliative intention.


Type 2 diabetes is a common metabolic disease with the potential for prevention of complications. The prevention requires a high level of lasting actions from the patients, which may be burdensome. The aim of this trial was to evaluate the effectiveness of a training course for general practice nurses in motivation support at 18 months follow-up in the affiliated type 2 diabetes population.1c) -, total cholesterol levels, and well-being measured by the Problem Areas In Diabetes scale (PAID) and the mental component summary score, SF12 . Intention-to-treat analyses were performed. Predefined subgroups analyses were performed.Forty general practices with nurse-led diabetes consultations from the area of Aarhus, Denmark were randomised 1∶1 to either intervention or usual practice. Intervention practices were offered a 16-hour Self-determination theory - based course including communication training for general practice nurses delivered over 10 months. The affiliated diabetes populations (aged 40–74 years) were identified from registers . Primary outcomes were register-based glycated haemoglobin ; 0.08 mmol/l . Differences in median scores adjusted for clustering were for PAID: 1.25; p = 0.31 and SF12, mcs: 0.99; p = 0.15. Women in intervention practices differed from women in usual practices on mean HbA1c: −0.12%-points  and SF12, mcs: 2.6; p = 0.01.The differences between the intervention- and the control practices’ mean HbA1c and total cholesterol levels and the well-being at 18 months of follow-up in a comprehensive register-based diabetes population. Subgroup analyses suggested a possible effect in women, which deserves further attention.Offering a training course for general practice nurses in applying the Self-determination theory in current type 2 diabetes care had no effect compared with usual practice measured by HbANCT01187069).ClinicalTrials.gov (Identifier  Type 2 diabetes mellitus is a common metabolic disease with profound consequences such as visual impairment, renal failure, neuropathy with risk of amputation, myocardial infarction, stroke and increased mortality 1c)-, total cholesterol levels and well-being in the diabetes population at an 18-month follow-up. We expected that the intervention would enhance autonomous motivation in the patients with type 2 diabetes; which would improve their well-being and maintain their motivation for health behaviour changes; which again would reduce their risk profile regarding complications indicated by HbA1c- and total cholesterol levels. We expected that the patients with type 2 diabetes regardless of education level, gender or age would benefit from the intervention.In Danish healthcare, as in many other countries, type 2 diabetes care is provided by general practice in the framework of one annual 30 minute – and three quarterly 15 minutes consultations. In 2009, a growing share of the diabetes care was delegated from general practitioners to nurses employed in the practices (GP nurses) A cluster-randomised pragmatic trial including 40 general practices with nurse-led type 2 diabetes consultations was conducted in the area of Aarhus, Denmark. The median number of nurses in the included practices was 2 (range 1–5). A detailed description of the intervention, the context and the recruitment process has previously been published The 40 general practices were randomised 1∶1 to either intervention or usual practice by a statistician who was blinded to the identity of the practices.The diabetes population aged 40–74 years in October 2009 and listed with the included practices in January 2011  was identified in the Central Denmark Region’s Chronic Disease Database where patients with diabetes were identified by an algorithm based on health registers. We excluded 61 patients, 59 of whom died and 2 emigrated between October 2009 and May 2011. A total of 338 patients did not wish to be contacted for research reasons during the spring of 2011. This left 3635 patients for the study of self-reported outcomes . AnotherThe intervention practices were offered a 16-hour course with interactive training targeted the nurses employed in the practices. The core course was delivered as two coherent afternoons (month 0). Furthermore; in two following single afternoons (months 2 and 5), and a 30-minute visit to the practice by one of the course teachers (month 10), during which implementation issues were addressed. The course was designed to meet the SDT-based recommendations on health care provider behaviour Usual practices were randomly drawn from among intervention practice applicants and were informed by letter about their status as usual practice.1c and total cholesterol levels, and well-being, which were measured by the Problem Areas In Diabetes (PAID) scale The primary outcomes were HbAPAID is a validated 20-item measure of perceived burden of living with diabetes Secondary motivation outcomes were assessed by validated SDT-based questionnaires Cronbach’s α s of the used scales corresponded to previous Danish results 1c and cholesterol measurements were retrieved from the Region’s laboratory database and the self-reported patient data were obtained in a larger survey “Life with Diabetes”; a mailed questionnaire which in addition to being sent to the included diabetes population 16 months after the core course, also was sent to a random sample of 9960 people from the Central Denmark Region’s Chronic Disease Database. Two reminders were sent three weeks apart, and the questionnaires were completed 16–18 months after the core intervention. The register-based blood test data and the questionnaires were pooled by using the unique civil registry number assigned to all Danish citizens. Furthermore, the data were linked to social data  from Statistics Denmark. The following categorisation of data was used; educational level: ≤10 years of education or >10 years of education, ethnicity: Danish background or immigrant, cohabitation: living together with spouse/partner or living alone, and age: <60 years or ≥60 years.Data on HbAEleven months after the core intervention, an evaluation questionnaire was sent to all the nurses who partly or fully completed the course. This questionnaire contained questions on the nurses’ self-reported behavioural changes regarding autonomy support. They were asked to score their perceived autonomy supportive competences Further process data were collected; 1) The patient questionnaire asked questions about whether the patients had participated in a nurse-led diabetes consultation during the follow-up time to ascertain the potential reach of the intervention, and 2) the patients’ perception of autonomy support from the nurses was measured by the HCCQ in the patient questionnaire.1c and total cholesterol baseline values were the average of the values measured within the last 12 months before course start. The follow-up values were the average of the values measured during the “observational window” 15–21 months after the core intervention. Thus, the average follow-up time was 18 months. The effectiveness of the intervention on HbA1c and total cholesterol levels were estimated by the differences between the diabetes populations in the intervention practices and the usual practices with regard to 1) mean HbA1c and total cholesterol, and 2) proportions with HbA1c ≥8%, and proportions with total cholesterol ≥5 mmol/l at follow-up. The differences in mean at follow-up were estimated based on a mixed additive model where adjustments were made for baseline values and random differences between general practices (STATA 12 xtmixed), e.g. analyses were adjusting for clustering within general practices. Furthermore, the differences were adjusted for age (as continuous variable), gender, ethnicity, educational level, cohabitation, and redeemed diabetes medication in the baseline period. The differences in the proportions of patients with a follow-up HbA1c ≥8% and the proportions with a follow-up total cholesterol ≥5 mmol/l were estimated based on mixed additive models that adjusted for baseline status  and random differences between general practices (SAS 9.3 nlmixed).Analyses were performed according to the intention-to-treat principle. HbAThe sum scores for the questionnaire scales were calculated where no item-response was missing. Due to a non-normal distributed data, even after log transformation, the sum scores were presented as medians with quartiles. The hypothesis of no difference between medians was tested by a permutations test using general practices as permutation units, 1,000 simulations and the absolute difference in medians as criteria (STATA 12). Confidence intervals for the difference of two medians were found using two-level bootstrapping by resampling general practices and patients within general practice using 1,000 bootstrap samples (STATA 12). Hence, the differences of the medians were also adjusted for clustering.Subgroup analyses were performed with regard to age, gender and educational level.Results were presented with 95% confidence intervals (CI) and p values. P values of <0.05 were regarded as statistically significant.2-test and by multiple regression analyses.Non-responder analyses were performed for age, gender, educational level, cohabitation and ethnicity by chiRegarding the nurse questionnaire data, for each autonomy supportive competence, the number of nurses was counted for the scores from 1–10 , before and 11 months after the training course. Furthermore, the number of changed autonomy supportive competences per nurse was summarised.A timeframe of the trial is shown by We based the power calculation on the primary outcome HbA1c and a decrease of 0.5% in mean HbA1c https://www.retsinformation.dk/Forms/R0710.aspx?id=29142, and because the intervention addressed the nurses, the informed consent of the diabetes population was not required. According to Danish law, approval by the Ethics committee and written informed consent is not required in questionnaire-based and register-based projects. Additional information is available at The National Committee on Health Research Ethics’ webpage in the “Act on Research Ethics Review of Health Research Projects” §14,2. available at: http://www.cvk.sum.dk/English/actonabiomedicalresearch.aspx.The Danish Research Ethics Committee concluded that the trial was not to be a biomedical intervention cf The Committee Act no. 402 of the 28 May 2003 §7,1 available at: The study was approved by the Danish Data Protection Agency (j.no: 2009-41-3065) and it was registered at ClinicalTrials.gov (Identifier NCT01187069). The data are stored at the Department of Public Health, Aarhus University and at Statistic Denmark.1c at baseline was 6.7% . Among the 1,879 patients where a measurement was performed in the intervention practices, 373 (19.9%) had a baseline HbA1c ≥8% compared with 354 (18.5%) of the 1,910 patients where a measurement was performed in the usual practices. The mean total cholesterol at baseline was 4.6 mmol/l in both groups.The baseline characteristics of the diabetes population are shown in 1c at follow up was 6.8% . No statistically significant difference was found between the groups in the mean follow-up values adjusted for baseline values and clustering  difference was statistically significant. The proportion with a follow-up cholesterol measurement was 6%-points (95% CI: 3 to 9) higher in the intervention practices compared with the usual practices. This tendency was also present in the subgroup analyses regarding the men, the diabetes population <60 years old, and the diabetes population with >10 years education. Adjustment for age, gender, ethnicity, educational level, cohabitation and redeemed diabetes medication in the baseline period did not change the results. The difference in the proportion of the diabetes population with total cholesterol ≥5 mmol/l was not statistically significant between the groups as such , or in tThe overall median score for PAID at follow-up was 12.5 . No statistically significant difference was found between the groups . The oveNo statistically significant differences between the groups were found in the scores for the SDT scales . No otheA total of 34 nurses from 19 of 20 intervention practices had received the core intervention, and 22 nurses from 13 practices had completed the full course . Twenty-One nurse (4%) did not answer the questions, and 7/27 (26%) reported no perceived changes in the autonomy supportive competences. The remaining 19/27 nurses (70%) reported perceived improvement in at least three of the autonomy supportive competences. We do not know whether the nurses who did not complete the evaluation questionnaire have perceived changes in the autonomy supportive competences. The worst case scenario is that 19/34 (56%) perceived improvement in some of the autonomy supportive competences, and the best case scenario is that 27/34 (79%) perceived improvement in some of the autonomy supportive competences. Overall, the nurses reported that they had achieved most from the course in terms of improved communication skills. A total of 24 of the 27 (89%) nurses reported that they had distributed worksheets to patients, but only 1 of 27 (4%) reported that she had distributed at least one patient work sheet to all the diabetes patients attending her consultations.A total of 69% of the diabetes population who completed the questionnaire, reported that they had participated in a nurse-led type 2 diabetes consultation during the follow-up period . The med1c ≥8%. Even when these characteristics were adjusted for each other, they all remained statistically significant.Non-response to the questionnaire was statistically significantly associated with the following characteristics; younger age, being an immigrant, having ≤10 years of education, living alone and baseline HbA1c. Younger age and having a baseline HbA1c ≥8% were found to be independently associated with drop-out in the cholesterol analyses. No social determinants for drop out were found in the blood test analyses.Younger age was statistically significantly associated with not having a follow-up and a baseline HbAThe proportion with no follow-up cholesterol measurement was higher in the usual practices than in the intervention practices, and the total cholesterol baseline values were higher among the patients with no follow-up cholesterol value (mean 4.7 mmol/l) compared to the patients with a follow-up cholesterol value .1c and total cholesterol levels and the well-being at 18 months of follow-up in a comprehensive register-based diabetes population. The difference in mean total cholesterol between the randomisation groups was 0.08 mmol/l (95% CI: 0.01 to 0.15) at follow-up, which favoured usual practice. However, the mean total cholesterol values were below the recommended threshold (4.5 mmol/l) in both groups at follow-up. Furthermore, a higher proportion of the diabetes population had a follow-up total cholesterol measurement in the intervention practices compared with the usual practices, and the patients without a follow-up measurement had higher levels of baseline total cholesterol than those with a follow-up measurement.This trial demonstrated that offering a training course for GP nurses in applying SDT in current type 2 diabetes care had no noticeable effect compared with usual practice measured by HbA1c and self-reported mental health in favour of the intervention among the women. Power calculation was not performed regarding the subgroup analyses. However, the precision of the results are shown by 95% CIs. Regarding the difference in the median scores of mcs, SF12, the 95% CI indicated that the difference could be <0.The discrepancy between the significance testing and the confidence interval for the observed difference can appear when using different methods with different assumptions.The predefined subgroup analyses showed small, but statistically significant differences in mean HbAThe evaluation of the educational process showed that 70% of the nurses who completed the questionnaire perceived that their autonomy supportive skills had improved, especially their communication skills. The process evaluation also showed that implementation of the patient work- sheets was incomplete.1c, total cholesterol and quality of life. Henrich et al. found that their results could partly be explained by a too low training intensity of the nurses and limited time for interaction with patients within the framework of existing diabetes care.The method Motivational interviewing (MI) has been assumed to be largely in accordance with SDT 1c. A high level of usual care including a patient education programme based on Guided Self-Determination may explain the absence of differences between the randomisation groups. The approach in Guided Self-Determination is consistent with SDT and has as previously mentioned, shown an effect in diabetes care Explanatory trials evaluating SDT-based interventions in diabetes care have shown varying results. Recently, a one-year MI programme was evaluated in a Danish diabetes clinic setting including volunteers with type 1 and type 2 diabetes 1cNone of the above-mentioned studies have investigated the gender differential effect of the interventions. West, et al. investigated the effect of adding five individual MI sessions to a group-based behavioural obesity treatment programme for overweight women with type 2 diabetes and reported an effect on weight loss and a decline in HbAWe evaluated the effect of a training course with multiple components as a whole under the conditions in which it would be applied. A quality of the present trial is that the basis of the intervention components and all activities are well-described The near-complete coverage of Danish registers allowed us to evaluate the effectiveness in a comprehensive diabetes population. A limitation of the registers is the inability to distinguish between types of diabetes. However, the number of patients with type 2 diabetes far exceeds the number of patients with type 1 diabetes (85%∶15%), and there should be no reason to assume an unequal distribution between the intervention and the usual practice group. The inclusion of the type 1 diabetes population may however, have contributed to a dilution of a potential effect of the intervention because type 1 diabetes care is primarily delivered by out-patient clinics. For example, the found differences of −0.12%-points in HbA1c and of 2.6 score-points on the 0-100-SF12, mcs scale in our female population could be biased towards the null-hypothesis due to dilution because of the inclusion of the very comprehensive diabetes population.Our recruitment strategy of the general practices was a realistic scenario for future training courses. A previous study showed that the eligible practices for the present trial were associated with a higher quality of diabetes management when compared with practices with no nurse(s) employed and also when compared with practices that did not respond to that survey We used different types of patient outcomes to measure different stages in the expected process from motivation support to improved well-being and changed clinical outcomes. This could speak for multiplicity correction, but because different models and methods were required for the analyses of the outcome measures, a test of no effect based on correlated outcomes was infeasible.Well-being is directly relevant for the patients, but the questionnaire scales’ sensitivity to detect differences caused by interventions is not clear. The ceiling effects in the motivational outcome measures; the SDT scores and the low levels of the PAID-scores showed very little room for improvement.1c - and cholesterol levels are valid surrogates for the development of complications 1c and cholesterol values 1c and total cholesterol measurements as indicators of some occurrence of the guideline recommendations. However, the duration of the follow-up may have been too short to detect a difference in these outcomes. This in particular, because we evaluated the effect under real-life conditions and therefore e.g. made no attempt to ensure the patients’ participation in a diabetes consultation provided by the nurse at certain times during the follow-up time Avoiding complications is highly relevant for the patients and the HbAWhether the current framework of approximately 15 minutes quarterly consultations is adequate for the nurses to sufficiently stimulate the patients’ process of being autonomously motivated could be debatable. A Swedish intervention included besides 1) training of primary care nurses to put more emphasis on the patients’ understanding of their disease in the current framework of diabetes care, also 2) a comprehensive nine months programme with 20 hours of reflective group-discussions on living with diabetes. This explanatory trial showed an intervention effect in terms of statistically significant lower HbA1c-levels in the intervention group compared with the control group after one year (−0.94%-points) and after five years (−1.34%-points) The present trial was not designed to evaluate whether the training course would work under ideal circumstances. As expected under normal, real-life conditions, not all the intervention practices participated in all or even in parts of the course . This ki1c and total cholesterol are narrow and they all show that if effectiveness of the intervention exists, then it is small. However, it could be argued that even small effect sizes found at the diabetes population level in this pragmatic trial design could be worthwhile because of the low intervention costs. The costs of this intervention solely comprised the costs of the education of the nurses because the intervention was delivered to the patients in the framework of existing diabetes care and thereby without additional expenditure. The magnitude of relevant effect sizes seen in relation to intervention costs deserves more attention in the planning of future interventions evaluated in pragmatic trial designs.The underlying assumptions behind sample size calculation of a cluster randomised pragmatic trial like ours can be discussed, e.g. the ICC and the effect size in use 1c and total cholesterol levels and well-being at an 18 month follow-up in a comprehensive register-based diabetes population. Subgroup analyses suggested a possible effect in women on HbA1c and well-being, which deserves further attention.Offering a training course for GP nurses in applying SDT in current type 2 diabetes care had no effect compared with usual practice measured by HbAChecklist S1CONSORT 2010 checklist of information to include when reporting a cluster randomised trial.(DOCX)Click here for additional data file.Checklist S2CONSORT Checklist of Items for Reporting Trials of Nonpharmacologic Treatments.(DOC)Click here for additional data file.Protocol S1Trial protocol detailed description.(PDF)Click here for additional data file.Protocol S2The first draft.(DOC)Click here for additional data file.


Control of airway inflammation is the cornerstone of asthma management. The aim of the present pilot study was to assess the effects of a leukotriene receptor antagonist (LTRA) added to a basic treatment of inhaled corticosteroids (ICS) and long-acting beta-agonist (LABA) on airway hyperresponsiveness, inflammation, and quality of life in well-controlled patients with asthma.Seventeen patients  with well-controlled asthma presenting airway hyperresponsiveness to mannitol and methacholine challenge were given add-on montelukast on a stable ICS + LABA for 4 weeks. Quality of life and selected parameters of airway inflammation were measured at baseline and at study end. ).1 , cumulated higher dose of mannitol  and methacholine  in the provocation test, lower airway reactivity with mannitol and methacholine (response dose ratio (RDR) –50.0%, p = 0.024 and –44.3%, p = 0.006, respectively), and improved airway sensitivity to mannitol and methacholine . Changes in inflammation parameters  were consistent with these findings. Asthma-related quality of life improved significantly in all domains and overall . The main limitation was the absence of a control group.Adding montelukast to ICS + LABA resulted in an increase in mean FEVThe consistency of the changes in airway hyperresponsiveness and inflammation as well as in quality of life observed with an add-on therapy with montelukast in well-controlled asthma patients during 4 weeks suggests that residual inflammation may represent an area for further improvement of asthma control to be explored in adequately powered randomized controlled trials. Thus, control of airway inflammation is considered the cornerstone of asthma management1. Inhaled corticosteroids (ICS) are effective anti-inflammatory agents widely used for improving asthma control1. However, ICS may not suppress all components of airway inflammation3. Oral leukotriene receptor antagonists (LTRAs) have been shown to possess anti-inflammatory properties complementary to ICS5 and may reach small airways not easily accessed by inhaled formulations6. LTRAs have also been shown to attenuate airway hyperresponsiveness to both specific and non-specific stimuli8 and, as an add-on therapy to ICS, to improve asthma control9–12. When added to ICS, LTRAs may primarily have anti-eosinophilic effects14 and/or modulate airway hyperresponsiveness16.Asthma is a chronic inflammatory disorder of the airways in which chronic inflammation is associated with airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing1 from baseline . Airway reactivity is characterized by the response dose ratio (RDR), i.e. the final % fall in FEV1 divided by the total cumulative dose needed to induce that % fall in FEV117. The assessment of airway reactivity by challenge tests is well-established and recommended in the Global Initiative for Asthma (GINA) guidelines for establishing the diagnosis of asthma since 200717.Airway sensitivity to a challenge test is expressed as the cumulated provocative dose (in mg) of either methacholine or mannitol that induces a predefined and substance-specific percentage  of the fall in FEVThe aim of the present pilot study was to evaluate the effects of add-on montelukast to standardized ICS + LABA therapy on airway hyperresponsiveness, selected markers of airway inflammation, and quality of life in a cohort of already clinically well-controlled patients with asthma.All patients were recruited at the outpatient clinic of Allergology and Clinical Immunology of the University Hospital of Bern, Switzerland. Seventeen adult patients older than 18 years and younger than 65 years of age with clinically well-controlled asthma under a fixed combination of budesonide/formoterol  were included after having signed an informed consent form. Clinically well-controlled asthma was defined as the absence of symptoms and exacerbations during the 8 weeks prior to inclusion. In addition, documented airway hyperresponsiveness to mannitol and methacholine as well as current non-smoker status were required for inclusion. Smokers, women planning a pregnancy or pregnant, and patients with any significant illness that could interfere with the feasibility or the results of the provocation tests, such as acute asthma or severe cardiovascular disease, were not included.At baseline, all patients underwent a mannitol provocation test and the fraction of exhaled nitric oxide (FeNO) was measured. If positive, and at least 2 days later, a methacholine provocation test and repeated measurement of FeNO were performed. For standardization all patients were switched to the same dose-equivalent ICS + LABA therapy in a fixed combination with budesonide/formoterol (200/6 µgbid). After a 2-week run-in period on this therapy, 10 mg montelukast was added for a 4 weeks’ treatment duration (interim visit). All patients included attended the final visit in which the provocation tests were repeated.1 was assessed by using a MasterScreen Pneumo spirometer  with filter and according to ATS recommendations18. The best of three values FEV1 repeatable to within 100 ml were recorded and the percentage of predicted values19 were calculated. Mannitol challenge tests were carried out using the protocol described by Anderson et al.20. Mannitol was administered as a dry powder in capsule form, inhaled from a RS01 device . An empty capsule was used as a control at baseline. The test was started with 5 mg and increased doubling the doses up to 160 mg. In case of no response at the 160 mg dose, this dose was repeated up to a maximum cumulative dose of 635 mg. FEV1 was measured 60 seconds after delivery of each dose. The challenge was stopped if FEV1 fell by 15% or more, or when the maximum dose had been administered. The dose causing a 15% fall in FEV1 (PD15 FEV1) was read by interpolation on the plotted dose–response curve. Methacholine testing was carried out according to the protocol of the SAPALDIA Study21 which was identical to the protocol of the European Community Respiratory Health Survey (ECRHS) “method 2 short-protocol”23. The provocation test started with inhalation of saline diluent, and the maximum post-diluent FEV1 recorded 2 minutes later was the control value. All solutions of methacholine were prepared by the pharmacy of University Hospital Bern. Methacholine was delivered using a Mefar MB3 dosimeter  set to deliver the aerosol over a period of 1 second. FEV1 was recorded 2 minutes later and in the absence of a 20% fall in FEV1 from baseline the next dose was given. Methacholine was inhaled with quadrupling doses until a fall in FEV1 greater than 20% from the control value was observed or the maximum cumulative dose of 5.515 mg was reached. The dose causing a 20% fall in FEV1 (PD20 FEV1) was read by interpolation on the plotted dose–response curve. Patients were asked to refrain from short-acting beta-2-agonist for 8 hours, from the combined inhaled therapy (formoterol and budesonide) for 24 hours and from montelukast for 4 days prior to bronchial challenge testing.FEV24 by using an NIOX nitric oxide analyzer with computed biofeedback software by Aerocrine AB, Solna, Sweden.Exhaled nitric oxide was measured with the same equipment throughout the study according to ATS guidelines25. Each item was rated by the patient on a numerical scale ranging from 1 (extreme limitations) to 7 .Asthma-related quality of life (AQLQ) was assessed by applying the self-administered Juniper questionnaire at the baseline, interim and final visit. This validated questionnaire encompasses 32 items clustered in four domains: asthma symptoms, limitations in daily physical activities, emotional function, and exposure to environmental stimuliBlood samples were drawn at the baseline and final visit before any challenge tests, for determination of blood eosinophils and eosinophil cationic protein serum levels .1 in the mannitol test, PD20 FEV1 in the methacholine test, the RDR for mannitol and methacholine, the asthma related quality of life, FeNO, blood eosinophils and eosinophil cationic protein concentrations between baseline and the final visit. Descriptive statistical analysis methods were used. No PD15 or PD20 value was assigned to subjects who did not achieve PD15 or PD20. As all parameters were normally distributed, means, standard deviations and corresponding 95% confidence intervals were calculated. For mean PD15 FEV1, PD20 FEV1, RDR, and FeNO the base 10 antilog (the antilog of the mean of log-transformed data corresponds to the geometric mean) was calculated as previously suggested27. Calculations of statistical significance  and corresponding power for a 5% significance level were performed by Student’s t-test using StatsDirect software version 2.7.7, Altrincham, Cheshire, UK.Predefined parameters of interest were the changes in PD15 FEVThe study was approved by the ethics committee of the University Hospital of Bern (KEK-BE 240/06), Switzerland. The study is registered with ClinicalTrials.gov (NCT01725360).1 (3.35 L) and Tiffeneau index  were consistent with patients with normal lung function, as required by the inclusion criteria. While mean blood eosinophil counts were within normal range, mean serum ECP levels were slightly above. Asthma-related quality of life was high at baseline, scoring 5.3 on a numerical scale up to 7.Seventeen patients , with a mean age of 32 (±11) years were included with baseline characteristics shown in 1 values , significantly higher mean dose of mannitol that could be administered during the provocation test , lower sensitivity to mannitol as suggested by decreasing PD15 FEV1 values , and lower bronchial reactivity as suggested by a significantly lower maximal percent decrease in FEV1  and by a decreasing RDR . Fourteen out of 17 patients had no PD15 to mannitol at the end of the study. As shown in 1, higher cumulated methacholine dose, lower maximal % decrease in FEV1, improved airway sensitivity) and to a significantly decreased airway reactivity . Nine out of 17 patients had no PD20 to methacholine at the end of the study.As given in Changes of blood eosinophil counts, serum ECPs, and FeNO were consistent with the results obtained by the provocation tests. Differences between baseline and final visit did not reach statistical significance; all numerical trends were consistently in favor of a possible modest improvement of airway inflammation .p < 0.001). In addition, 11 patients (65%) experienced a clinically relevant improvement in asthma-related quality of life (>0.5 improvement in score).Asthma-related quality of life assessed by the AQLQ improved significantly in all four domains covered by the Juniper questionnaire . The oveIn this pilot study with 17 well-controlled asthma patients, short-term treatment with montelukast added to a basis therapy with ICS + LABA for 4 weeks improved all parameters of airway hyperresponsiveness and inflammation in a consistent manner, suggesting that, even in clinically well-controlled asthmatics, residual inflammation amenable to therapeutic intervention may persist.28. Indirect challenge tests may be a closer reflection of active airway inflammation than direct challenge tests30. While direct challenge tests (such as the methacholine test) act directly on the airway smooth muscle, indirect challenge tests (such as the mannitol test) act by increasing the osmolarity of the airway surface liquid which leads to the release of mediators from inflammatory cells  and of neuropeptides from sensory nerves. Ultimately, in subjects with bronchial hyperresponsiveness this cascade of events leads to airway narrowing and reduction of FEV1. Thus, as most stimuli that provoke an attack of asthma in daily life do so indirectly, indirect tests may be considered more specific for asthma17. The present study showed improvements in airway hyperresponsiveness when using an indirect challenge test with mannitol. A positive test to mannitol was shown to correlate with asthma severity, asthma worsening during stepwise reduction of ICS, asthma symptoms, and asthma airway inflammation consistent with the presence of inflammatory cells such as eosinophils or their mediators such as prostaglandins, leukotrienes, and histamine31–33. Earlier observations suggested that leukotriene receptor antagonists play a key role in sustaining but not in initiating the airway response to mannitol35, which is consistent with our observations. On the other hand, a negative result may be due to an insufficient number of inflammatory cells (as it may occur in a patient on ICS), insufficient concentration of mediators or lack of airway hyperresponsiveness to mediators (as it may occur in nonasthmatic patients with eosinophilic bronchitis)36. Keeping this limitation in mind, the mannitol test is useful to confirm diagnosis of a currently active asthma in untreated or of insufficiently controlled asthma in treated patients36. In the present study, montelukast added to ICS + LABA over 4 weeks significantly improved bronchial reactivity and lead to a significant increase of the cumulated dose of mannitol administered. On the other hand, montelukast had no effect on airway sensitivity assessed by the mannitol PD15. Thus, the improvements reported in the present study suggest that montelukast might possibly contribute to additionally decrease airway hyperresponsiveness when added to a fixed combination of ICS + LABA even in already well-controlled patients. Bronchial provocation testing with methacholine is generally considered sensitive, i.e. useful to rule out the presence of asthma in a person not taking ICS37. However, 27% of adults reporting physician diagnosed asthma, generally with normal or near normal spirometry, were shown to have a negative methacholine challenge test38. On the other hand, a positive methacholine provocation test does not necessarily rule in the diagnosis of asthma31 so that airway hyperresponsiveness has been described in patients with allergic rhinitis39 and with chronic obstructive pulmonary disease40. Sensitivity to the test is expressed as the PD20, i.e. the cumulated provocative dose of methacholine (in mg) inducing a 20% fall in FEV1. Reactivity is characterized by the response–dose ratio (RDR), i.e. the final % fall in FEV1 divided by the total cumulative dose needed to induce that % fall in FEV141. The latter was first suggested as a simple index of non-specific airway reactivity summarizing the dose–response curve by O’Connor et al.42 and applied to the findings of the Swiss SAPALDIA study to establish reference values for methacholine reactivity43. In the present study, airway reactivity assessed by the methacholine challenge significantly improved after 4 weeks of add-on therapy with montelukast compared to baseline, which is consistent with previous findings in patients with asthma44–46. However, airway sensitivity only numerically improved by up to 48% without reaching statistical significance. These observations support previously reported additional protection against excessive airway narrowing provided by montelukast added to a stable dose ICS in a 12 weeks’ placebo controlled study in patients with controlled asthma. In this study, montelukast significantly improved the FEV1 decline and increased PD20 FEV1 vs. placebo, thus providing additional protection to ICS with regard to excessive airway narrowing47.Airway hyperresponsiveness is a hallmark of asthma that can be assessed by provocation tests using direct and/or indirect stimuli48 and the number of eosinophils in the blood or the sputum, may be of interest to assess the level of inflammation underlying the current asthma status, guide treatment choice, and monitor treatment response49. Eosinophils present in increased numbers in airways release proteins that may damage airway epithelial cells. They also play a role in the release of growth factors and airway remodeling50. Cysteinyl leukotrienes are potent proinflammatory mediators mainly derived from eosinophils and mast cells. They are the only mediator whose inhibition has been associated with an improvement in lung function and asthma symptoms51. Furthermore, there is increasing evidence for the involvement of cysteinyl leukotrienes in the pathophysiology of airway hyperresponsiveness and, subsequently, in airway remodeling52–54. Thus, the trend in numerical decrease of blood eosinophils observed in the present study is consistent with previously published findings showing that montelukast decreases the number of eosinophils and mast cells in airway tissues55 and the blood of asthma patients56.Although their contribution to asthma management remains unclear, non-invasive markers of airway inflammation, such as the levels of exhaled nitric oxide57. Although normal ranges are still to be defined58, NO can be measured in the exhaled air with non-invasive standardized methods59 allowing for longitudinal monitoring. Exhaled NO can be considered a surrogate marker of airway inflammation which increases with loss of asthma control61 and decreases with improving inflammation control62. In our study, exhaled NO levels decreased markedly and early (2 weeks) after initiation of the add-on therapy with montelukast, which is consistent with improved control of airway inflammation, especially when taken together with the effects on the other non-invasive markers of inflammation. It is well-known that ICS dose-tapering in well-controlled patients may lead to loss of asthma control. Airway hyperresponsiveness to mannitol was identified as a predictor of asthma exacerbations after ICS dose reduction32 so that responsiveness to mannitol can be used to predict risk of exacerbation during back titration of steroids63. On the other hand, airway hyperresponsiveness correlates with noninvasive markers of airway inflammation such as exhaled nitric oxide and sputum eosinophils in steroid-naive asthmatics only33. Thus the incremental response of montelukast added to ICS + LABA observed in the present study may suggest that montelukast contributes to improved asthma control via improved control of airway inflammation which is consistent with earlier reports indicating that leukotriene receptor antagonists oppose systemic mediators of inflammation which are not controlled by or not accessible to inhaled corticosteroids64.Nitric oxide (NO) is produced by many cells, including eosinophils. NO can act as a dilator of bronchial and vascular smooth muscle, a neurotransmitter and an immune response mediator65–67. In the present study, the majorities of patients included were women, had no symptoms, and were non-smokers. A potential confounder was the presence or absence of allergic rhinitis which was not assessed at inclusion. A future study should aim at also characterizing such determinants of quality of life in asthmatics not covered by the Juniper questionnaire, e.g. for a stratified analysis. Keeping in mind that the present study had no control group, but given the fact that patients were well-controlled at inclusion, this suggests that montelukast may contribute to improve quality of life in asthmatics by still unknown mechanisms beyond inflammation control and lung function improvement.Quality of life improvements observed in the present study were statistically significant in all domains and clinically relevant in two-thirds of the patients. The determinants of quality of life in asthma are not fully understood. Female sex, the presence of respiratory symptoms, being a current or former smoker, and the presence of an associated allergic rhinitis were independently associated with poorer quality of life in asthmaticsThis pilot study has several limitations. First, this study had no control group such that reported statistical significances relate to differences versus baseline. A future study should ideally include a placebo control group. Furthermore, only patients with well-controlled asthma on ICS and LABA were included. A future study may want to explore the effects of montelukast added to ICS alone and use patients on ICS alone or on ICS plus LABA as a control group. The number of patients included in this pilot study was too small for assessing airway sensitivity but deemed sufficient for assessing airway reactivity. The present results should allow for adequate power calculations, depending on the primary endpoint chosen. Finally, all comparisons were made versus baseline, that is before the 2-week run-in on a standardized ICS + LABA regimen. In a future study, it would be advisable to re-assess the primary outcomes following run-in.In this small cohort of 17 patients consistent trends across all tested dimensions of airway hyperresponsiveness and inflammation suggest that in an adequately powered study, the add-on effects of montelukast added to a basis therapy with ICS + LABA may contribute to improved asthma control. The present results may contribute to facilitate power and thus sample size calculations in future clinical trials aimed at exploring more in depth the effects of montelukast on airway inflammation and hyperresponsiveness.Montelukast added to a fixed combination of ICS + LABA in well-controlled asthma patients was shown to numerically improve airway hyperresponsiveness and selected parameters of airway inflammation. While statistical significance was not reached in this pilot study, the consistency of the reported numerical trends across all parameters of interest deserves further research in adequately designed and powered randomized controlled trials.