Although gait-related dual-task interference in aging is well established, the effect of gait and cognitive task difficulty on dual-task interference is poorly understood. The purpose of this study was to examine the effect of gait and cognitive task difficulty on cognitive-motor interference in aging. Fifteen older adults  and 20 young adults  performed three walking tasks of varying difficulty  under single- and dual-task conditions. The cognitive tasks were the auditory Stroop task and the clock task. There was a significant Group × Gait Task × Cognitive Task interaction for the dual-task effect on gait speed. After adjusting for education, there were no significant effects of gait or cognitive task difficulty on the dual-task effects on cognitive task performance. The results of this study provide evidence that gait task difficulty influences dual-task effects on gait speed, especially in older adults. Moreover, the effects of gait task difficulty on dual-task interference appear to be influenced by the difficulty of the cognitive task. Education is an important factor influencing cognitive-motor interference effects on cognition, but not gait. Functional community ambulation requires an ability to perform cognitive tasks while walking and an ability to adapt to extrinsic environmental factors that increase the complexity of mobility, such as obstacle avoidance  and time-critical tasks  . A reducA limitation of the existing research on dual-task interference in aging is that it has focused predominantly on dual-task performance during unobstructed walking at preferred gait speed. Therefore, the effect of dual-task interference on gait and cognitive performance during more attention-demanding gait tasks remains largely unknown. Furthermore, because slowing down during unobstructed walking in the gait laboratory is inconsequential for successful completion of the motor task, individuals may place more importance on performing the cognitive task . Indeed,Bock et al.  examinedThe purpose of this study was to determine the effect of gait and cognitive task difficulty on dual-task interference in healthy young adults and older adults. We specifically compare obstructed and unobstructed gait because of the relevance to community ambulation. We hypothesized that as the difficulty of the gait task increased, attention to gait would increase, resulting in smaller dual-task effects relative to simple walking (unobstructed at self-selected speed). We focus on gait speed as the measure of gait performance in dual-task conditions, since meta-analysis results show that cognitive-motor interference effects across a range of cognitive tasks are prominent in gait speed . FurtherTwenty young adults  and 15 older adults  were recruited through advertisements at Northeastern University and local senior centers. To be included, participants had to be 18–30 years or older than 65 years, be able to walk independently in the community for at least 50 meters, have intact cognition according to the Mini-Mental State Exam (score > 23), and have normal or corrected-to-normal hearing and vision. Participants were excluded if they had a history of neurological disorders, any orthopedic conditions affecting gait, reported more than 2 falls in the past year, had an acute hospital stay within the last 3 months, or had a lower extremity amputation. Participants who met the selection criteria signed an informed consent form. The study was approved by the Institutional Review Board at Northeastern University. Demographic information was collected for each participant, including age, gender, and education. Cognitive abilities of the participants were characterized using the Mini-Mental State Exam , Digit SEach participant performed three gait tasks and two different cognitive tasks in isolation (single-task conditions) and each gait task in combination with each of the cognitive tasks . The cognitive tasks were the auditory Stroop  and the The three gait tasks were walking at self-selected speed (SS), walking at fastest comfortable speed (FC), and walking at fastest comfortable speed while stepping over an obstacle (OB). The three gait tasks were chosen to provide differing levels of task difficulty. A critical assumption was that walking at fastest comfortable speed was more attention-demanding than walking at preferred (self-selected) speed, and that walking fast and stepping over an obstacle further increased the demands of the task. All gait tasks involved participants walking across a 6.1-meter Platinum GAITRite walkway, which contains pressure activated sensors. The associated software computes spatio-temporal parameters of gait. The participants started and finished each pass 2 meters beyond the end of the walkway so that only steady state gait data were captured. Participants completed 6 passes of the walkway for each condition and the average of the 6 passes was used for analysis. In the OB condition, a 15 cm high hurdle was placed at the 4.5 m mark of the GAITRite walkway. The order of the three gait tasks was randomized, but was performed in the same order for the single-task and the two dual-task  blocks. Block order  was also randomized across participants. To analyze the effect of task difficulty on dual-task interference, we calculated the dual-task effect (DTE) on both gait speed and cognitive task performance (reaction time and accuracy). Dual-task effects on gait speed (DTEg) and clock and Stroop-task accuracy (DTEacc) were calculated as follows :(1)DTE=Whereas a decrease in gait speed and accuracy represent performance decrement, an increase in reaction time  represents performance decline, therefore DTE on reaction time (DTErt) was calculated as follows :(2)DTE=Thus, for each variable, negative values for DTE indicate that performance deteriorated under dual-task conditions , and positive values represent an improvement in the dual-task condition relative to single-task performance . t-tests. To verify whether the clock task required greater cognitive processing than the Stroop task, we conducted paired t-tests on mean reaction times for the two tasks in the single-task (sitting) condition for each group. To determine whether participants increased their SS gait speed as instructed in the FC and OB conditions, we analyzed changes in gait speed across conditions with a 3 Gait Task  × 3 Cognitive Task  ANOVA for each group. Tukey's post hoc tests were used as needed. The young and older adults were compared on profile measures using independent ηp2) is presented as a measure of effect size for each repeated measures ANCOVA. By convention, 0.01 indicates a small effect size, 0.06 is moderate, and 0.14 represents a large effect [To analyze the effects of gait and cognitive task difficulty on dual-task interference, we applied a 3 Gait Task  × 2 Cognitive Task  × 2 Group  repeated measures ANCOVA with education (years) as covariate to each dependent variable . Significant three-way interactions were followed up with two-way analyses and post hoc tests as needed. The partial eta squared  was significantly longer than that for the Stroop task  for both young adults, t(19) = −10.5, P < .001, d = 2.9, and older adults, t(14) = −9.5, P < .001, d = 2.7. Thus, consistent with our assumption, the clock task required greater cognitive processing than the Stroop task. In both tasks, mean reaction time of older adults was slower than young adults. The mean reaction time while sitting (single-task) for the clock task  = 3.17, P = .050, ηp2 = .10. Although the statistical significance was marginal, the effect size was large. Follow up two-way ANCOVAs revealed that the three-way interaction occurred because the Cognitive Task × Group interaction was significant for the FC gait task , but not for SS  or OB   . Specifi2 = .03) . In SS aP < .001), with lower mean accuracy in the clock task  than the Stroop task  (P = .045). Mean values for DTErt and DTEacc for each gait task and cognitive task are presented in Young adults had mean accuracy of 99% (SD 0.01) in the Stroop task and 97.2% (SD 0.1) in the clock task. Older adults, on average, were significantly less accurate than young adults ; lower levels of education were weakly associated with larger dual-task costs in accuracy on the clock task during obstacle crossing. Before adjusting for education, the three-way interaction on DTEacc was significant due to a Gait Task × Cognitive Task interaction for older adults but not young adults: dual-task cost on accuracy was significantly greater in the clock task during OB than in any other task among older adults; the effect was not significant after adjusting for education. Education was significantly related to DTEacc in the OB condition  affect dual-task interference. Furthermore, we assumed a hierarchical increase in gait task difficulty between walking at self-selected speed, walking at fastest comfortable speed, and walking fast while stepping over an obstacle. However, we did not ask the participants of their perceptions of the tasks. Finally, the findings from this study may be limited in their generalizability due to the small sample size and the predominance of women in the sample. Thus, the findings should be viewed as preliminary; investigations involving larger, more representative samples are needed to examine the interactions between age group, gait task difficulty, and cognitive task difficulty on cognitive-motor interference. In the future, manipulating the timing of the onset of the stimulus in the obstacle negotiation path may provide more insight into the interactions between attentional processing associated with obstacle avoidance and the attention processing of an additional cognitive task.In conclusion, the results of this study suggest that obstacle negotiation at fast walking speed, such as when stepping up a curb to avoid traffic, is highly attention-demanding for older adults and significantly compromises the ability to maintain walking speed. This study provides evidence that gait task difficulty influences dual-task effects on gait speed, especially in older adults. Moreover, the effects of gait task difficulty on dual-task interference appear to be influenced by the difficulty of the cognitive task. Education and/or cognitive reserve may be an important factor influencing cognitive-motor interference, especially in terms of performance of the cognitive task.


Postnatal depression (PND) is under-diagnosed and most women do not access effective help. We aimed to evaluate comparative management of (PND) following screening with the Edinburgh Postnatal Depression Scale, using three best-practice care pathways by comparing management by general practitioners (GPs) alone compared to adjunctive counselling, based on cognitive behavioural therapy (CBT), delivered by postnatal nurses or psychologists.This was a parallel, three-group randomised controlled trial conducted in a primary care setting  and a psychology clinic. A total of 3,531 postnatal women were screened for symptoms of depression; 333 scored above cut-off on the screening tool and 169 were referred to the study. Sixty-eight of these women were randomised between the three treatment groups.Mean scores on the Beck Depression Inventory (BDI-II) at entry were in the moderate-to-severe range. There was significant variation in the post-study frequency of scores exceeding the threshold indicative of mild-to-severe depressive symptoms, such that more women receiving only GP management remained above the cut-off score after treatment (p = .028). However, all three treatment conditions were accompanied by significant reductions in depressive symptoms and mean post-study BDI-II scores were similar between groups. Compliance was high in all three groups. Women rated the treatments as highly effective. Rates of both referral to the study (51%), and subsequent treatment uptake (40%) were low.Data from this small study suggest that GP management of PND when augmented by a CBT-counselling package may be successful in reducing depressive symptoms in more patients compared to GP management alone. The relatively low rates of referral and treatment uptake, suggest that help-seeking remains an issue for many women with PND, consistent with previous research.NCT01002027.The study is registered at ClinicalTrials.gov, Trial Registration Number  Postnatal depression (PND), defined as an episode of major or minor depression occurring in the first 12 months postpartum, has a point prevalence of 13% at 3 months postpartum . Whilst General Practitioners (GPs) and postnatal nurses are key primary care professionals engaged with mothers during the postnatal period. It is therefore important to determine whether best-practice management of PND in primary care can offer an effective pathway resulting in alleviation of depression for the majority of women.Further as many women are reluctant to take antidepressants during lactation, due to potential side effects on the newborn  readily Whilst CBT is generally delivered by mental health specialists such as psychologists, some evidence for the ability of nurses to deliver psychological interventions for PND in primary care has been published. However, studies conducted to date have not explicitly compared such interventions to management by GPs. To our knowledge, in the postnatal period, five controlled trials have evaluated psychologically-informed interventions delivered by primary care practitioners  -14. OnlyEffective and manualised psychological interventions can be successfully translated to widespread delivery by a range of primary care professionals and could be a valuable resource for health systems around the world. For example, in Australia, the advent of the National Perinatal Depression Initiative (NPDI ) will seThe present study similarly sought to examine the effectiveness of counselling informed by the principles of CBT and delivered by primary care practitioners to women with PND. In addition, this study sought to address currently unanswered questions: Is the same treatment delivered by different professionals similarly effective (e.g. trained nurses versus psychologists)? In this RCT we compare three model care pathways: management by trained GPs alone and management by trained GPs augmented with a counselling-CBT intervention delivered either by a trained nurse or a psychologist.The study  took place in three municipalities in Melbourne, Australia with approval from Austin Health Human Ethics Research Committee. Postnatal women with infants < 12 months of age were screened by nurses working in primary care at maternal child health centres during regular routine visits. The Edinburgh Postnatal Depression Scale (EPDS) , is a sibeyondblue; available at http://www.beyondblue.org.au/index.aspx?link_id=7.102) was also provided. GPs were free to prescribe antidepressant medication in all three groups , supported by detailed printed materials, to enhance their ability to manage PND. This involved systematically working through a 25-page training manual covering screening, diagnosis with standard psychiatric criteria (DSM-IV), risk assessment and management, engagement, a biopsychosocial model of PND, medication during lactation, common patient concerns, onward referral and principles of treatment . Telephone consultation with a psychiatrist was available to provide additional advice on medication for PND. A GP-specific, one-page PND Management Guide (developed by settings ). A totaTwenty two nurses completed a half-day training workshop in the counselling-CBT intervention . The traWomen allocated to this group were managed as usual by their own GP (trained in PND management).Women allocated to this group received six sessions (one per week over six weeks) of the manualised Overcoming Postnatal Depression Program. This counselling-CBT program was delivered by a trained nurse at maternal and child health centres and was an adjunct to GP management.Women allocated to this group received six sessions (one per week over six weeks) of the same Overcoming Postnatal Depression Program as group B. This counselling-CBT was delivered by an experienced psychologist at a hospital Psychology department. Again this was delivered as an adjunct to GP management.The main outcomes were levels of depressive symptoms and the proportion of participants with symptoms below the cut-off score indicative of mild to severe depressive symptoms. Two validated measures of depressive symptoms were used and were administered at baseline, again after 3 weeks, and immediately post-study. The Beck Depression Inventory II (BDI-II ) was theIn addition, women completed questionnaires rating the perceived effectiveness of treatment on binary (Yes/No) and Likert-type (1 to 10) scales. Information on medication use was collected post-study. As all outcome measures were self-report, it was not possible to obtain blinded measures of symptomatology.2 (8.4/7.1)2 = 22.0, at 80% power with p = 0.05. We therefore continued recruitment until at least n = 22 had been achieved in all 3 groups.Based on the average baseline BDI-II score in a previous study  a post-treatment improvement of 30% (7.1 points) would take average scores to the midpoint of the "mild" range of depressive symptoms (BDI-II = 14-19). Applying these numbers we calculated: n = 2(0.84 + 1.96)per se, and also whether there was a difference between counselling-CBT by psychologists compared to nurses. This required two, a priori, orthogonal contrasts as follows: Contrast i) Group A vs [Group B + Group C]/2. Contrast ii) Group B vs Group C.The BDI-II score classifications given by Beck et al  were useThe primary analysis was by intention-to-treat  using maFigure 2 = 1.59, df = 2, p = .45).Seventy one percent of GP appointments were kept . Similarly, attendance at the 6 counselling-CBT sessions averaged 4.6 and 4 sessions for groups B and C respectively. Of the 68 participants, 50 returned post-study questionnaires. This attrition was demonstrably random with respect to group . Neither planned Contrast i) GP management versus counselling-CBT, nor planned Contrast ii) Adjunctive counselling-CBT from nurse versus psychologist, were significant (p = 0.347 and p = .247 respectively).Graphical inspection of Figure depression, anxiety and stress (p > 0.05).Figure 2, df = 2, p = .028). The same information is re-expressed in terms of Relative Risk at the bottom of Table An observed-case frequency analysis of remittance rates based on categorising BDI-II scores as above or below threshold Table , found tThere was a poor return rate from women regarding other services accessed and medication use with only one third of the sample returning these questionnaires. Based on the available data there was no difference in post-study outcome between women known to be taking antidepressants  and all other women .Forty six women responded to the questions on treatment efficacy. A majority in all groups indicated that treatment was sufficient . On a scale of 1 to 10, respondents rated perceived effectiveness of their treatment highly in all groups , and significantly more highly in group B .per se. Possibly, the GP training component made any additional effect of adjunctive counselling-CBT more difficult to detect. Nonetheless, we also found that women in GP management continued to exhibit a higher frequency of above-threshold depressive symptoms post-study. These data may suggest that adjunctive counselling-CBT involving either psychologists or nurses could be a promising model of collaborative PND management in primary care.This study compared three pathways of care for managing PND, all treatments requiring training the key primary care health professionals involved. An important question in the management of perinatal mood disorders is whether different "real world" care pathways actually result in amelioration of depressive symptoms, and whether they differ consistently in efficacy . On averA number of other positive outcomes were found. Firstly, anxiety, (which is often a co-morbid problem with PND) was also effectively reduced by treatment. Secondly, compliance rates were good and women in all groups showed significant reductions in post-study symptoms of depression. Interestingly, there is some suggestion that adjunctive counselling-CBT was most effective when delivered by nurses. This is consistent with some previous findings on the effectiveness of PND treatment programs delivered by both specialist and trained non-specialist practitioners ,12,27,14The study has a number of limitations. First, the sample size was relatively small, and attrition reduced this further at follow-up, limiting our ability to generalise from the results. Second, the "control" group itself involved an enhancement of current care, by training GPs. For ethical reasons it was inappropriate to include a wait-listed control group in this study. However the observed improvements in mood (a drop of 17.3 BDI-II points on average) are of a magnitude at least as large as post-treatment effect sizes observed in studies involving psychological interventions versus routine care for PND . FurtherEarly intervention for PND is essential due to the negative consequences for women and for their close family members in terms of mental health and child socio-emotional development ,29. The Even among those who agreed to referral to this study, most did not ultimately take up treatment, although some cited improved mood or had already accessed other treatment options. Low referral rates to, and participation rates in a particular research study such as this may also reflect the reluctance of women to take part in research.However, given the current evidence, it seems clear that specific research on how to increase women's engagement with treatment would be valuable. Whilst systematic screening for PND offers one possibility for increasing detection (the first step to accessing treatment) data on the ultimate usefulness of screening programs for PND in terms of increased treatment uptake are still relatively scarce. As has been pointed out elsewhere, the introduction of screening in isolation will have little impact ,33. In tIn summary, for the majority of those who received treatment, all three possible models of care appeared effective. It therefore appears that for the management of moderate-to-severe PND, best practice primary care management routes are effective for the majority of women. GP management coupled with adjunctive counselling-CBT yielded promising results. In practice these models of PND management are deliverable by existing primary care professionals. However, rates of both referral to treatment (51%), and subsequent treatment uptake (40%) were low, suggesting help-seeking remains an issue in clinical practice that needs to be addressed by comprehensive research on methods to overcome this obstacle. Training key primary care professionals and strengthening their collaboration is likely to remain centrally important for improving current treatment pathways for PND following screening, under Australia's National Perinatal Depression Initiative, and for similar universal programs in other countries.The authors declare that they have no competing interests.JM, JE, AG and AB conceived the study. JM, JE, and BL contributed to the design of the GP training. CS and BL delivered the training. CH and BL oversaw data collection and monitored the adherence to study protocols. AG and CH designed and executed data analyses. AG (50%) BL (25%) and CH (25%) wrote a first draft of the manuscript. JM, JE, AG, CS, AB, CH and BL all edited subsequent drafts for important intellectual content and all authors agreed on the submitted version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/11/95/prepub


One of the most consistent biological findings in autism is the elevated blood serotonin levels. Immune abnormalities, including autoimmunity with production of brain specific auto-antibodies, are also commonly observed in this disorder. Hyperserotonemia may be one of the contributing factors to autoimmunity in some patients with autism through the reduction of T-helper (Th) 1-type cytokines. We are the first to investigate the possible role of hyperserotonemia in the induction of autoimmunity, as indicated by serum anti-myelin-basic protein (anti-MBP) auto-antibodies, in autism.Serum levels of serotonin and anti-MBP auto-antibodies were measured, by ELISA, in 50 autistic patients, aged between 5 and 12 years, and 30 healthy-matched children.Autistic children had significantly higher serum levels of serotonin and anti-MBP auto-antibodies than healthy children . Increased serum levels of serotonin and anti-MBP auto-antibodies were found in 92% and 80%, respectively of autistic patients. Patients with severe autism had significantly higher serum serotonin levels than children with mild to moderate autism (P < 0.001). Serum serotonin levels had no significant correlations with serum levels of anti-MBP auto-antibodies in autistic patients (P = 0.39).Hyperserotonemia may not be one of the contributing factors to the increased frequency of serum anti-MBP auto-antibodies in some autistic children. These data should be treated with caution until further investigations are performed. However, inclusion of serum serotonin levels as a correlate may be useful in other future immune studies in autism to help unravel the long-standing mystery of hyperserotonemia and its possible role in the pathophysiology of this disorder. Autoimmunity to CNS may have a pathogenic role in autism . This maSerotonin is formed by hydroxylation and decarboxylation of tryptophan. Serotonin is known to play a role in brain development prior to the time it assumes its role as a neurotransmitter. Disruption of serotonergic development can leave permanent alterations in brain function and behavior. This may be the case in autism ,17. It wSerotonin, being well known for its role in depression, has been shown to modulate immune responses. Serotonin may contribute to asthma pathogenesis through reduction of Th1-type cytokines . In addiWith this background, this study was conducted to investigate the relationship between serum levels of serotonin and anti-myelin-basic protein (anti-MBP) auto-antibodies, which are possible indicators of autoimmunity to CNS, in a group of autistic children.This case-control study was conduced on 50 children who had classic-onset autism. The patients were fulfilling the criteria for the diagnosis of autism according to the 4th edition of the Diagnostic and Statistical Manual of Mental Disorders .The autistic group comprised 41 males and 9 females. They were recruited from the Autism Research and Treatment Center, Faculty of Medicine, King Saud University, Riyadh, Saudi Arabia. Their ages ranged between 5 and 12 years (mean ± SD = 8.22 ± 2.28 years).1-Patients who had no associated neurological diseases .2-Patients who had no associated metabolic disorders (eg. Phenylketonuria) because these associated comorbidities with autism may influence the results of serum serotonin and anti-MBP levels.3-Patients who were not receiving any medications.The control group comprised 30 age-and sex-matched apparently healthy children. They included 25 males and 5 females. They were the healthy older siblings of the healthy children who attend the Well Baby Clinic, King Khalid University Hospital, Faculty of Medicine, King Saud University, Riyadh, Saudi Arabia for routine follow up of their growth parameters. The control children were not related to the children with autism, and demonstrated no clinical findings suggestive of immunological or neuropsychiatric disorders. Their ages ranged between 5 and 12 years (mean ± SD = 8.23 ± 2.36 years).The local Ethical Committee of the Faculty of Medicine, King Saud University, Riyadh, Saudi Arabia, approved this study. In addition, an informed written consent of participation in the study was signed by the parents or the legal guardians of the studied subjects.This was based on the clinical history taking from the caregivers, clinical examination and neuropsychiatric assessment. In addition, the degree of the severity of autism was assessed by using the Childhood Autism Rating Scale (CARS)  which raSerum serotonin levels were assayed by using serotonin EIA kit (Biosource Europe S.A. rue de l'industrie 8 1400 Nivelles Belgium). Principally, the competitive serotonin EIA kit uses the microtiter plate format. Serotonin is bound to the solid phase of the microtiter plate. Acylated serotonin and solid phase bound serotonin compete for a fixed number of antiserum binding sites. When the system is in equilibrium, free antigen and free antigen-antiserum complexes are removed by washing. The antibody bound to the solid phase serotonin is detected by anti-rabbit peroxidase. The substrate TMB/peroxidase reaction is read at 450 nm with a filter wave length 620 nm. The corresponding serotonin concentrations are determined from the standard curve by matching their mean absorbance readings with the corresponding serotonin concentrations in ng/ml. To increase accuracy, all samples were analyzed twice in two independent experiments to assess inter-assay variations and to ensure reproducibility of the observed results (P > 0.05).This was done by using ELISA kit that allows for the specific measurement of human anti-MBP . This assay recognizes recombinant and natural human MBP antibodies. Principally, the microtiter plate provided in this kit has been pre-coated with MBP protein. Samples are added to the appropriate microtiter plate wells and incubated. Horseradish Peroxidase (HRP) conjugated to anti-antibody is added to each microplate well and incubated. Finally a TMB  substrate solution is added to each well. Only those wells that contain anti-MBP and enzyme-conjugated anti-antibody exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wave length of 450 nm ± 2 nm. To increase accuracy, all samples were analyzed twice in two independent experiments to assess inter-assay variations and to ensure reproducibility of the observed results (P > 0.05). No significant cross-reactivity or interference was observed.th and 75th percentiles. Mann-Whitney test was used for comparison between these data. Chi-square test was used for comparison between qualitative variables of the studied groups. Spearman's rho correlation coefficient "r" was used to determine the relationship between different variables. For all tests, a probability (P) of less than 0.05 was considered significant. Patients were considered to have elevated serum serotonin or anti-MBP if their levels were above the highest cut-off values  which were the 95th percentiles of serum serotonin and anti-MBP levels of healthy controls as the distribution of the data was non-parametric.The results were analyzed by commercially available software package . The data were non-parametric, thus they were presented as median and interquartile range (IQR), which are between the 25Autistic children had significantly higher serum serotonin levels [median (IQR) = 243.5 119) ng/ml] than healthy controls [median (IQR) = 41 (31) ng/ml], P < 0.001  = 268 (46) ng/ml] than children with mild to moderate autism [median (IQR) = 160 (84) ng/ml], P < 0.001  = 251 (115) ng/ml and 220 (92) ng/ml, respectively], P = 0.32. In addition, serum serotonin levels had no significant correlations with the age of the children with autism (P = 0.81).Autistic children had significantly higher serum levels of anti-MBP auto-antibodies than healthy controls, P < 0.001  = 476 (346) ng/ml and 365 (361) ng/ml, respectively], P = 0.92. In addition, serum anti-MBP auto-antibodies had no significant correlations with the age of the children with autism (P = 0.75).There was a non-significant difference in serum levels of anti-MBP auto-antibodies between autistic patients with hyperserotonemia and patients with normal serum serotonin levels, P = 0.74 table . In addiAutism without a discernible cause may be a genetic disorder of serotonin metabolism . Immune Elevated serotonin level in platelets, whole blood and serum is the most consistent biochemical abnormality found in autism . In our In the present work, all autistic patients were not receiving any medications, including the selective serotonin reuptake inhibitors. This may explain the increased frequency of autistic patients with hyperserotonemia in this study. In addition, it was reported that serum serotonin levels are affected by age as postpubertal subjects had lower serum serotonin levels than prepubertal subjects . Thus, tPlatelet hyperserotonemia has been detected in 25-60% of autistic children. The significant increase of the level of serotonin mRNA in the platelets of some autistic patients could suggest serotonin system dysregulation in some patients with autism . ResearcAmong the potential environmental factors, hyperserotonemia during pregnancy and its effect on brain development could be playing a role in autism. Hyperserotonemia during fetal development may result in a dysfunction of the hypothalamo-pituitary axis, affecting the amygdala as well as pro-social hormone oxytocin regulation. Dysfunction of the amygdala and abnormal oxytocin levels may underlie many clinical features of autism . HyperseIn the present work, patients with severe autism had significantly higher serum serotonin levels than patients with mild to moderate autism (P < 0.001). In addition, the frequency of hyperserotonemia was significantly higher in children with severe autism (100%) than patients with mild to moderate autism (76.5%), P = 0.01. This may indicate that the extent of the elevation of serum serotonin levels was closely linked to the degree of the severity of autism. Previous research also reported a close association between serum serotonin levels and the severity of autism . It is nSome studies reported that autistic patients with elevated blood serotonin levels had elevated serotonin transport into platelets . HoweverAutoimmunity to CNS may have a pathogenic role in autism . This maMBP is a protein believed to be important in the process of myelination of nerves in the central nervous system. Interest in MBP has centered on its role in demyelinating diseases, particularly multiple sclerosis (MS). Several studies have shown a role for antibodies against MBP in the pathogenesis of MS ,45.The reason behind the formation of some brain auto-antibodies in some patients with autism is not fully understood. It is speculated that autoimmune reaction to neurons might be trigged by cross-reacting antigens in the environment resulting in the release of neuronal antigens. These neuronal antigens may result in induction of autoimmune reactions through the activation of inflammatory cells in genetically susceptible individuals. The environmental antigens may include food allergies to certain peptides such as gliadin, cow's milk protein and soy  infectioIn our series, there was a non-significant difference in serum levels of anti-MBP auto-antibodies between children with severe autism and patients with mild to moderate autism, P = 0.15. In addition, there was a non-significant difference in the frequency of seropositivity of anti-MBP auto-antibodies between patients with severe autism (81.8%) and children with mild to moderate autism (76.5%), P = 0.46. These findings may indicate that the induction of the production of brain auto-antibodies in some patients with autism may occur regardless the degree of the disease severity.Previous research had also found an increased frequency of autoimmunity in families of autistic children -15. ThisTo date, a definitive relationship between autism and autoimmunity has not been fully established. On the basis of the preliminary results reported in this study, however, there seems to be a suggestive evidence in support of the induction of autoimmune reaction against brain in some patients with autism. Additional investigation designed to expand on these data is warranted. Therapy in patients who are seropositive for serum auto-antibodies is directed at reducing the antibody concentration, blocking the effector mechanisms and depleting the monoclonal B cells. The recent availability of a monoclonal antibody suppressing B-cell clones, which is not myelosuppressive and does not cause secondary malignancies, allows for early targeted intervention . PrelimiSerotonin has been shown to modulate some immune responses and hyperserotonemia may explain some of the abnormal cellular immune responses seen in autism. Serotonin has an important role in initiation of delayed-type hypersensitivity responses , which aIt is possible that a common factor could account for both hyperserotonemia and immune abnormalities seen in autism. Such common factor may be the serotonin transporter (5-HTT) which transports serotonin into platelets. 5-HTT is also found on immune cells where it can influence immune function . Some stIn this work, we have tried to find a possible relation between the elevated serum levels of serotonin and anti-MBP auto-antibodies. There was a non-significant difference in serum levels of anti-MBP auto-antibodies between autistic patients with hyperserotonemia and autistic patients with normal serum serotonin levels, P = 0.74. In addition, there was a non-significant difference in the frequency of seropositivity of anti-MBP auto-antibodies between autistic patients with hyperserotonemia (80.4%) and autistic children with normal serum serotonin levels (75%), P = 0.6. Furthermore, there was a non-significant correlation between serum serotonin and anti-MBP levels in autistic patients (P = 0.39). We could not trace data in the literature concerning the relationship between hypersrotonemia and brain auto-antibodies in autistic patients to compare our results. We are the first to study such a relationship. This warrants further research to determine the possible link between hyperserotonemia and the elevated serum levels of brain auto-antibodies which are detected in a subgroup of autistic patients.The results of this study may indicate that hyperserotonemia is not one of the contributing factors to the increased frequency of anti-MBP auto-antibodies in some autistic children. However, this initial report can not rule out the possible association between hyperserotonemia and immune abnormalities that had been reported in autism, but rather raises additional questions. So, these data should be treated with caution until further investigations are performed. Therefore, studies should be conducted to investigate the possible association between hyperserotonemia and immune abnormalities in autism.Hyperserotonemia may not be one of the contributing factors to the increased frequency of serum anti-MBP auto-antibodies in some autistic children. These data should be treated with caution until further investigations are performed. However, inclusion of serum serotonin levels as a correlate may be useful in other future immune studies in autism to help unravel the long-standing mystery of hyperserotonemia and its possible role in the pathophysiology of this disorder. In addition, the possible role of blood serotonin lowering drugs in amelioration of immune abnormalities in hyperserotonemic autistic children should also be studied.(BBB): blood brain barrier; (CARS): Childhood Autism Rating Scale; (CNS): central nervous system; (IQR): interquartile range; (MBP): myelin basic protein; (SERT): serotonin transporter; (Th): T helper cells; (5-HTT): serotonin transporter.The authors declare that they have no competing interests.Both authors designed, performed and wrote the research. In addition, both authors have read and approved the final manuscript.


More than half of veterans who use Veterans Health Administration (VA) care are also eligible for Medicare via disability or age, but no prior studies have examined variation in use of outpatient services by Medicare-eligible veterans across health system, type of care or time.To examine differences in use of VA and Medicare outpatient services by disability-eligible or age-eligible veterans among veterans who used VA primary care services and were also eligible for Medicare.A retrospective cohort study of 4,704 disability- and 10,816 age-eligible veterans who used VA primary care services in fiscal year (FY) 2000. We tracked their outpatient utilization from FY2001 to FY2004 using VA administrative and Medicare claims data. We examined utilization differences for primary care, specialty care, and mental health outpatient visits using generalized estimating equations.p < 0.001) and more VA specialty care visits (p < 0.001) than age-eligible veterans. They were more likely to have mental health visits in VA (p < 0.01) and Medicare-reimbursed visits (p < 0.01). Disability-eligible veterans also had more total (VA+Medicare) visits for primary care (p < 0.01) and specialty care (p < 0.01), controlling for patient characteristics.Among Medicare-eligible veterans who used VA primary care, disability-eligible veterans had more VA primary care visits (Greater use of primary care and specialty care visits by disability-eligible veterans is most likely related to greater health needs not captured by the patient characteristics we employed and eligibility for VA care at no cost. Outpatient care patterns of disability-eligible veterans may foreshadow care patterns of veterans returning from Afghanistan and Iraq wars, who are entering the system in growing numbers. This study provides an important baseline for future research assessing utilizations among returning veterans who use both VA and Medicare systems. Establishing effective care coordination protocols between VA and Medicare providers can help ensure efficient use of taxpayer resources and high quality care for disabled veterans. The Medicare program and the health care system of Department of Veterans Affairs (VA) are the two major public health care programs funded by the federal government of the United States (US).The Medicare program, the largest public health program in the US, provides health care for 47 million elderly and disabled Americans, with an annual budget of $510 billion in 2010. The majority of Medicare beneficiaries are in the fee-for-service Medicare plans. Individuals under 65 can become disability-eligible for Medicare if they have sufficient work history for their age and receive Social Security Disability Insurance (SSDI) for 24 months, develop end-stage renal disease (ESRD) with a 3-month waiting period, or have amyotrophic lateral sclerosis/Lou Gehrig's disease and receive SSDI (no waiting period). About 14% of Medicare enrollees were disability-eligible beneficiaries in 2004 [The VA health care system, the largest integrated health care system in the US, provides comprehensive medical care to veterans with a budget of $48 billion in 2010. Eligibility for VA health care benefits is determined by a system of eight Priority Groups based on the factors including the level of service-connected injuries and conditions and incomes. The VA system had 153 hospitals and more than 900 outpatient clinics, served 5.5 million users, and provided 60 million outpatient visits in 2008. More than half of veterans who use VA are also eligible for Medicare [Compared to the general non-elderly U.S. population, non-elderly people with disabilities are less likely to be employed, more likely to have poor or fair self-reported health status, and have lower incomes ,7,8. PeoWhile some prior research has examined use of Medicare and Medicaid services by disability-eligible beneficiaries ,11-14, mCompared to age-eligible Medicare beneficiaries, disability-eligible beneficiaries are more likely to be in poorer health and to have cognitive or mental health impairments ,13,18 whResearch on Medicare-eligible veterans has focused almost exclusively on age-eligible veterans. Age-eligible veterans obtain a significant proportion of their inpatient care outside the VA -29. A stPetersen and colleagues  found thThis paper examined differences in outpatient utilization in VA and Medicare between disability-eligible and age-eligible veterans who used VA primary care services. Based on prior work in non-veteran samples -10,15, wThe results of this analysis can inform VA and Medicare policy by identifying the outpatient services in each public insurance system that may be in great demand by current and future disability-eligible veterans and by identifying Medicare-eligible veterans who will require improved coordination of care to minimize duplication and contain federal health expenditures . As soldThis was a retrospective cohort study conducted using administrative data covering Fiscal Year (FY) 2000-2005 (10/1/1999-9/30/2005). We identified the patient cohort, who were VA primary care users and also eligible for Medicare in the baseline year of FY2000 and tracked their outpatient utilization obtained from both VA and Medicare sources for the four subsequent years (FY2001-FY2004).The sample was drawn from a prior study  of 66,36Additional inclusion and exclusion criteria were applied for this analysis. First, we excluded veterans who died before the end of FY2001, the first year of the follow-up period for the study n = 4,033). We identified deaths using dates of death from VA and Medicare data. Second, we excluded those who did not enrolled in Medicare or were Medicare-enrolled but without both Medicare Parts A (inpatient coverage) and B (outpatient coverage) during the entire study period  as indicated by the Medicare entitlement/buying indicator of the Medicare Denominator files. The requirement of both Part A and Part B data was because this analysis focused on outpatient services. Third, we excluded veterans who developed end stage renal disease (ESRD) and became eligible for the Medicare ESRD program prior to or during the study period, 2000-2004 (n = 422), because their benefits under the Medicare ESRD program and their health needs were likely to be significantly different from non-ESRD patients. ESRD status was determined by the ESRD code in the Medicare status indicator, current reason for entitlement, or original reason for entitlement variable in the Medicare denominator files. Fourth, we excluded all Medicare managed care enrollees using the Health Maintenance Organization (HMO) enrollment indicator in the Medicare Denominator files , because Medicare databases do not collect service utilization data on these patients. Finally, we excluded Medicare-eligible veterans who did not use primary care in VA in FY2000  as defined below. After applying these exclusion criteria, the final sample included 15,520 Medicare-enrolled veterans located in 49 of 50 US states who used VA primary care in 2000 with 6,556 hospital-based primary care users and 8,964 community-based primary care users. The criteria for the selection of community clinics and VA hospitals in the prior study  led to a,033. We We defined disability- versus age-eligible based on the original reason for Medicare eligibility. This definition of disability included veterans, who were 65 years or older in FY2000, who became Medicare-eligible because of disability before 65 years old.Data sources included 1999-2004 Medicare claims data, FY2000-FY2004 VA administrative data and 2000 Census data. We identified primary care, specialty care and mental health care utilization outcomes from FY2001-FY2004 VA Outpatient Care Files and Medicare Carrier Standard Analytical Files. Medicare Denominator files were used to identify Medicare and Medicaid eligibility and managed care enrollment. We obtained patient characteristics at baseline (2000) from Medicare Denominator files and VA Outpatient Care files. Finally, we used 2000 US Census data for the ZIP level characteristics where the patient lived.The outcomes of interest in this study were the number of visits for three types of outpatient care: primary care, specialty care, and mental health in VA and Medicare in each year from FY2001-FY2004. To compare outpatient use between VA and Medicare, we developed a classification algorithm for different types of outpatient care by using provider specialty and CPT procedure codes that are available in both systems. This approach allows us to construct comparable visits by type of outpatient care from two data systems, which have different data generating processes. The classification algorithm is described in detail in the paper by Burgess and colleagues . The outThe independent variable of interest is whether a veteran was disability- or age-eligible for Medicare based on the original reason for Medicare eligibility.We controlled for other patient level covariates at the baseline year (FY2000) based on the theoretical framework described by the Behavioral Health model for vulnerable populations  that heaEnabling factors included both the patient level and community level measures at the baseline year (FY2000). Patient characteristics included Medicaid coverage, veteran's VA copayment status, type of VA clinic where a patient received primary care , and the distance from patient's residence to the closest VA facility . We classified VA copayment status for each veteran into three groups: 1) receiving free care in VA due to a military service-related disability at 50% or higher, 2) receiving free care in VA due to low income, and 3) not eligible for free care in VA and required to pay copayments . Three community level measures were used as potential measures of resource availability of the community where the patient resides, including the median income at the ZIP code level, the proportion of high school graduates at the ZIP code level, and the population density at the county level.The need factors were the military service related disability and the Diagnostic Cost Group (DCG) risk adjustment measure that accounts for multiple comorbid conditions. The DCG system is a widely used diagnosis-based case-mix adjustment system based on both inpatient and outpatient diagnoses obtained from administrative files or claims databases . The DCGDescriptive and bivariate statistics, including t-tests and chi-squared tests, assessed differences in patient characteristics between disability-eligible and age-eligible veterans in the cohort identification year (FY2000). The unit of analysis for utilization comparisons was the person year. Due to the skewness of utilization data, we used Wilcoxon nonparametric rank-sum tests to compare unadjusted differences in utilization. For multivariate analysis, we estimated the annual number of primary care and specialty care visits in FY2001-2004 using generalized estimating equations (GEEs) with a negative binomial distribution and a log link function.We used longitudinal two-part models for mental health visits because a significant proportion of veterans did not use mental health care. The first part of the model uses GEE logistic regressions to estimate the odds of having any mental health visit. The second part uses the GEE negative binomial regression, described above, to estimate the number of mental health visits among veterans who had one or more mental health visits. We employed the independent-correlation and the robust option.The multivariate analysis adjusted for patient characteristics, zip code level income and education, county-level population density and year fixed effects. Incidence rate ratios (IRRs) and differences in the predicted number of outpatient visits were generated to examine whether VA, Medicare and total (VA+Medicare) outpatient visits differed between disability-eligible and age-eligible veterans. Significance was lowered from 0.05 to 0.01 to adjust for multiple comparisons.All analyses were weighted for the sampling strategy used by the original community clinic evaluation study . Analysep = 0.001), more likely to be enrolled in Medicaid , more likely to receive free care in VA  due to disability  or low income , and had a higher level of service-related disability  (Table p < 0.0001), white , or receive primary care in community clinics . For community characteristics, compared to age-eligible veterans, disability-eligible veterans were more likely to live in areas with lower per capita incomes (p = 0.001) or a lower percentage of high school graduates (p = 0.001).Compared to age-eligible veterans, disability-eligible veterans were younger . The proportion of veterans using primary care in both VA and Medicare remained stable over time , and disability-eligible veterans were less likely to use both VA and Medicare primary care in all years. The proportions of veterans obtaining primary care only in Medicare increased over time, but in all years disability-eligible veterans were less likely than age-eligible veterans to receive primary care only in Medicare .For both disability-eligible veterans and age-eligible veterans, the largest proportion of these veterans used primary care only in VA, but this proportion declined over time  , but fewer primary care visits financed by Medicare . Use of VA primary care decreased in both groups over time, while use of Medicare primary care services increased over time , but the proportions of both disability- and age-eligible veterans obtaining specialty care only in VA decreased over time. About half of veterans used specialty care in both VA and Medicare and this proportion remained stable over time for both disability- and age-eligible veterans. The proportion of veterans obtaining specialty care only in Medicare increased over time, but disability-eligible veterans were less likely to obtain specialty care only in Medicare in all years .The patterns of specialty care use from the overall sample were also observed in both disability- and age-eligible veterans, respectively  in FY2001-4, fewer Medicare specialty care visits  and fewer total specialty care visits   in FY2001, with the majority of visits occurring in VA for both groups   with the majority of visits occurred in VA (0.9-1.0 visits)  than age-eligible veterans and 7% (0.6 visits) more total (VA+Medicare) visits , but similar numbers of primary care visits in Medicare (p = 0.84). Disability-eligible veterans had 19% (2.5 visits) more specialty care visits in VA  and 7% (0.6 visits) more total specialty care visits  than age-eligible veterans, but similar numbers of specialty care visits in Medicare (p = 0.82).After adjusting for patient characteristics, zip code-level characteristics and year fixed effects Table , disabilp < 0.001), any Medicare mental health visits , and any mental health care visits in either system  (Table p = 0.45), Medicare mental health visits (p = 0.23), and total mental health care visits (p = 0.96).Disability-eligible veterans were more likely than age-eligible veterans to have any VA mental health visits (Odds Ratio (OR) = 1.74, This is one of the first studies to compare use of outpatient services in VA and Medicare between disability-eligible and age-eligible veterans among VA primary care users. We found that disability-eligible veterans were more reliant on VA for primary care, specialty care and mental health care than age-eligible veterans following a fixed cohort from FY2001-FY2004. Both groups sought primary care most commonly in VA, which differs from results of a study examining 1997-1999 outpatient care patterns for male, elderly Medicare-eligible veterans from three states in New England . Our finDual use of specialty care in VA and Medicare was common (47-50%) and similar in the two groups, while dual use of primary care was somewhat less common and less likely among disability-eligible veterans. We also found that, as expected, disability-eligible veterans had more total (VA+Medicare) primary care and specialty care visits than age-eligible veterans, which was largely driven by more VA visits.Dual use of specialty care in VA and Medicare may indicate strategic choices about the source of particular types of specialty care by Medicare-eligible veterans, such as having a Medicare cardiologist but a VA pulmonologist. This pattern may be less duplicative than obtaining primary care from two different sources, but still fragments both care and the medical record, which may impact chronic disease management, continuity of care, and quality measurement at provider and system levels. Future research should explore whether disability-eligible and age-eligible veterans differ in the types of specialty care they obtain in VA or Medicare and assess quality outcome effects of fragmentation.We also found that disability-eligible veterans were more likely to have one or more mental health visits in VA or Medicare than age-eligible veterans, and obtained outpatient mental health care almost exclusively in VA. The greater likelihood of mental health services may reflect a greater burden of mental health impairment among disability-eligible veterans ,13,18 anThis study has several limitations. This is not a random sample of Medicare eligible veterans in the VA system, though it was determined by trying to balance urban and rural locations. In focusing on Medicare and VA, the study does not include utilization data provided under other insurance coverage, including Medicaid and private insurance, though the emphasis on the disabled population for outpatient care lessens the likely impact of this focus. This study did not include Medicare-eligible veterans who solely relied on Medicare. Further, these results show the experience of Medicare-eligible veterans who used VA primary care in FY2001-FY2004 before the implementation of Medicare Part D, so these results may not generalize to more current experience of veterans facing different choices regarding access to pharmaceuticals. However, VA pharmaceutical prices and policies have remained attractive to veterans thus far. The current medication copayment is $9 for a 30-day supply among veterans who are not eligible for free VA care. The study cohort also precedes the influx of returning veterans from the Afghanistan and Iraq wars, which has resulted in large increases in VA budgets and provision of more specialty care and mental health care aimed at these younger veterans. Further research should assess utilization among these young veterans. Our cohort was somewhat similar to a cohort of Afghanistan and Iraq veterans who used VA in 2001-2005 that were examined in a recent study . Our cohThis study shows greater outpatient care needs among disability-eligible veterans than age-eligible veterans, especially for VA care. This has important implications for care of younger veterans returning from recent wars, who are entering the system in growing numbers  and whosThe authors declare that they have no competing interests.CFL conceived of the study, obtained grant funding, designed the analyses, interpreted the results, and drafted and revised the manuscript. CLB, JFB, and MLM conceived of the study, obtained grant funding, designed the analyses, interpreted the results, and participated in manuscript preparation. MP cleaned and linked all of the data files, conducted all of the statistical analyses, and participated in manuscript preparation. NS participated in coordination of the study, interpretation of results, and manuscript preparation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/12/51/prepub


Prenatal exposure to persistent organic pollutants, e.g. polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) has been suggested to negatively affect birth weight although epidemiological evidence is still inconclusive. We investigated if prenatal exposure to PCBs and PBDEs is related to birth weight in a Swedish population with background exposure.ortho PCBs  and tetra- to hexa- brominated PBDEs . Simple and multiple linear regression models were used to investigate associations between lipid-adjusted, ln-transformed PCB and PBDE concentrations, and birth weight. Covariates included in the multivariate regression model were PCB and PBDE exposure, maternal age, pre-pregnancy BMI, weight gain during pregnancy, education, smoking, gender of the infant and gestational length. The effect of including fish consumption was also investigated.Breast milk was sampled during the third week after delivery from first-time mothers in Uppsala county, Sweden 1996–2010 (POPUP cohort) (N = 413). Samples were analysed for di-ortho PCBs was significantly associated with increased birth weight . The result did not change when gestational length was added to the model. An inverse association between PBDE(4)  and birth weight was observed in the multivariate model including gestational length . Maternal pre-pregnancy BMI and weight gain during pregnancy were important confounders of the association between di-ortho PCBs and birth weight. The associations were not alleviated after adjustment for fish consumption, a major source of PCB and PBDE exposure. The observed associations were stronger for boys than for girls.In the multivariate model, prenatal exposure to di-ortho PCBs and PBDE(4) may influence birth weight in different directions, i.e. PCB exposure was associated with higher birth weight and PBDE exposure with lower birth weight. Maternal pre-pregnancy BMI and weight gain during pregnancy were important confounders that may hide positive association between di-ortho PCB exposure and birth weight if they are not included in the statistical model. We speculate that even small PCB- and PBDE-induced shifts in the distribution of birth weight may influence future public health in populations with background exposure.Our results indicate that prenatal exposure to di- Among other potential effects, PCBs and PCDD/Fs are suggested to cause reduced birth weight [Toxic persistent organic pollutants (POPs), such as polychlorinated biphenyls (PCBs), polychlorinated dibenzo-h weight -4 and thh weight ,5,6. Amoh weight ,8. Both h weight -11 and ch weight ,13. Possortho PCBs and tetra- to hexa brominated PBDEs are related to birth weight in a Swedish population of first-time mothers with background exposure. The level of di-ortho PCBs is a good marker for dioxin-like PCBs and PCDD/Fs in Sweden [The aim of this study was to investigate if prenatal exposures to di-n Sweden , and then Sweden . Fish con Sweden , and at n Sweden -19. Furtn Sweden -22.ortho PCBs and tetra- to hexa brominated PBDE and birth weight in a cohort of Swedish first-time mothers. Prenatal exposure was estimated by levels in breast milk sampled three weeks after delivery.In this article we report crude and adjusted associations between prenatal exposure to di-The POPUP study  started in 1996, and up to 2010 we recruited 526 first-time mothers born in Nordic countries and living in Uppsala County. In 1996 to 1999, participants were randomly recruited in late pregnancy and donated a blood sample in gestational week 32–34 (N = 316) . 203 of Data on maternal and child characteristics were obtained from questionnaires or from the Swedish Medical Birth Register at the National Board of Health and Welfare ,23. 118 ortho substituted PCB congeners PCB 138, PCB 153 and PCB 180 (N = 413), and a randomly selected number of samples (N = 364) for four PBDE-congeners . The analyses were performed at the Swedish National Food Agency (NFA), and have been described earlier [ortho PCBs were analysed by gas chromatography coupled to high-resolution mass spectrometry [All breast milk samples were analysed for the di- earlier . After etrometry . To comptrometry . PBDEs wtrometry . The totortho PCBs and PBDEs were used to estimate prenatal exposure since these levels give a good estimate of the maternal body burden during pregnancy, and consequently the exposure of the fetus during pregnancy [Lipid-adjusted breast milk levels of di-regnancy ,27. For Correlation analysis (Pearson) and one-way analysis of variance (ANOVA) were used to investigate univariate associations between birth weight and individual characteristics of the mothers.ortho PCBs  and PBDE(4)  and birth weight. Logarithmically transformed exposure data were used since the distribution of data closely followed a log-normal distribution. Birth weight was normally distributed in the study population.We used simple and multiple linear regression models  to analyse associations between prenatal exposure to di-ortho PCBs or PBDE(4) and birth weight were investigated in separate models. In the adjusted model, di-ortho PCBs and PBDE(4) were included simultaneously. In addition, covariates included in the adjusted model were maternal age, pre-pregnancy BMI, weight gain during pregnancy, education, smoking during pregnancy and sex of the child. These variables have all been associated with birth weight [In the crude analyses, the associations between di-h weight -33. Covah weight  to the sCharacteristics of the participating women and children are presented in Table ortho PCBs and PBDEs among the women recruited in 1996–2010 are presented in Table ortho PCB data was available for all study participants (N = 413) and PBDEs in samples from 364 randomly selected participants from 1996 to 2010. PCB 153 was the di-ortho PCB congener with the highest mean concentration and the highest mean level among the PBDEs was shown for BDE-47. The levels of di-ortho PCBs were about thirty times higher than the levels of PBDE(4).Data on breast milk levels of di-ortho PCB exposure and birth weight (Table ortho PCB concentration. This corresponds to a mean difference in birth weight between the 25th (64 ng/g lipid) and 75th (132 ng/g lipid) percentile of di-ortho PCB exposure of approximately 100 g. When the analysis was restricted to individuals with data on gestational length, the p-value increased to 0.08, but the β-coefficient was essentially unchanged  and birth weight became stronger when fish consumption was included in the multivariate model, β decreased with 29% from −106 to −137 (p = 0.01). When female and male infants were studied separately, the multivariate models showed significant inverse associations between PBDE(4) and birth weight in male infants, but not in females  and birth weight was inverse in the unadjusted as well as in the multivariate model Table . When th and birtortho PCBs and birth weight. A shift towards higher birth weights at higher PCB exposures may be non-beneficial since positive associations between birth weight and risk of obesity later in life have been demonstrated [ortho PCBs on birth weight and a negative effect at higher exposure levels. For endocrine disrupting chemicals, different effects at low and high doses are currently discussed [Our study showed a weak but significant positive association between background prenatal exposure to di-nstrated ,35. In cnstrated ,36. Amonnstrated ,37-41, wnstrated -48. One nstrated  is unceriscussed .ortho PCB exposure and birth weight in the multivariate model was observed only after adjustment for maternal pre-pregnancy BMI, weight gain during pregnancy and concurrent exposure to PBDE(4). Increased pre-pregnancy BMI and weight gain are both associated with increased birth weight as well as with decreased levels of PCBs in breast milk and maternal blood [In our study, the positive association between di-al blood ,23, and Many studies like ours adjust their results for maternal pre-pregnancy BMI, but among studies showing inverse associations or no associations between birth weight and PCB exposure, Lopez-Espinosa et al.  is the oortho PCB exposure and birth weight. It could be speculated that PCBs and PBDEs influences birth weight via biochemical pathways in such way that exposure to PBDE(4) may to some extent mask the association between di-ortho PCBs and birth weight and vice versa. When the multivariate regression analysis was restricted to individuals with data on gestational length, the association did not change, although it became non-significant most probably because of a decreased statistical power  was an important covariate in the analysis of associations between di-er Table . Adding er Table , indicatortho PCBs, if causal. Maternal estrogen levels during pregnancy have been linked to increased birth weight [in vitro and in vivo bioassays [ortho PCBs alone are not responsible for the positive association with birth weight. In addition to di-ortho PCBs and PBDEs, a selected number of breast milk samples from the POPUP cohort have previously been analysed for non-ortho PCB congeners, 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) and for p,p’-DDE (N = 242-383) [ortho PCB levels and the levels of non-ortho PCB TEQs , PCDD/F TEQs and p,p’-DDE were strong (Pearson correlation coefficients 0.7-0.9), showing that di-ortho PCB is a good marker for exposure to the other compounds. Consequently, it is difficult to separate the influence of different chlorinated compounds on birth weight from each other, and it is possible that di-ortho PCB exposure is a proxy for exposure to the total mixture (‘cocktail’).We can only speculate about a possible mechanism responsible for the observed positive association between birth weight and di-h weight ,52, and ioassays -56. It i-DDE N = 2-383 [24ortho PCB exposure and gestational length as covariates. A β-coefficient of −106 corresponds to a mean difference in birth weight between the 25th (1.6 ng/g lipid) and 75th (3.5 ng/g lipid) percentile of PBDE(4) exposure of approximately −80 g. The association was similarly significant in the multivariate model , when the analysis was restricted to individuals with data on gestational length  and birth weight. The association was significant in the multivariate regression model including di- and birtThere are few other studies on associations between PBDEs and birth weight, and these studies report no ,58 or inortho PCBs. Consequently, our results strongly suggest that it is important to control for concurrent exposure to different contaminants, such as PCBs and PBDEs, in studies of POPs and birth weight. Including several pollutants in the same model may however introduce over-adjustment bias if the pollutants are strongly correlated. In our study, the correlation between levels of di-ortho PCBs and PBDE(4) was weak (Pearson correlation coefficient 0.19) and collinearity did not bias the associations. The weak correlation between di-ortho PCBs and PBDE(4) is probably due to differences in exposure pathways. Food is the main source of human exposure to di-ortho PCBs, while both diet and indoor dust may be important sources of PBDEs [Despite considerably lower exposure levels in the POPUP cohort than in the studies by Foster et al.  and Harlof PBDEs .Possible mechanisms responsible for the observed negative association between PBDE(4) exposure and birth weight are unknown but could involve effects on endocrine systems. For example, animal as well as epidemiological studies have shown that PBDEs can alter thyroid hormone levels -66, whicortho PCBs as well as PBDEs may be a proxy for fish consumption since fish is the major contributor to the dietary intake of these substances in the Swedish population [ortho PCBs exposure and birth weight was not appreciably changed when fish consumption during pregnancy was added to the regression model, thus indicating that omega-3 fatty acids in fish are not responsible for the observed association for di-ortho PCB. However, the inverse association between PBDE(4) and birth weight became stronger when fish consumption was added to the regression model. Fish consumption may consequently confound this association because of its positive association with both PBDE exposure and birth weight.Exposure to di-pulation . Maternapulation . Long-chpulation ,18,67. IWhen female and male infants were studied separately, the observed associations were only significant in males Table . The obsortho PCBs and PBDEs. This made it possible to adjust associations for concurrent exposure. The study is also relatively large in comparison to previously published studies, although statistical power is a problem in the analyses of e.g. sex differences. The long recruitment period could have influenced the results since the breast milk levels of PCBs and PBDEs have decreased in the POPUP cohort over the years [ortho PCBs became stronger . Available data on several life-style factors, including dietary habits, enabled us to evaluate several possible confounders. Some of the variables included in the final regression model were self-reported which adds uncertainty to the results, although a strong correlation between register and self-reported data was found for some variables. Data on gestational length was only available for 71% of the participants, and it is therefore difficult to conclude whether the observed effects on birth weight are related to gestational length  or fetal growth .An important strength of our study is the exposure assessment including chemical analysis of both di-he years . Howeverortho PCBs and PBDE(4) indicates that these substances may influence birth weight in different directions, especially in boys. Exposure to di-ortho PCBs tended to be associated with higher birth weight while the association between PBDE(4) and birth weight was inverse. Importantly, we were able to adjust for maternal weight gain during pregnancy, a factor that should be consider in future studies since failure to control for this may result in false negative associations between di-ortho PCB exposure and birth weight. The observed associations may be causal, but it is also possible that some unmeasured or unknown confounding factors have influenced the results. The differences in birth weight associated with an increase in exposure from the 25th to the 75th percentile of exposure were relatively small (+100 and −80 g for di-ortho PCBs and PBDE(4) respectively). This can be compared with a difference in birth weight between non-smoking and smoking mothers of about 200 g [Our relatively large study of background prenatal exposure to di-ut 200 g ,70. Bothut 200 g . We specp-dioxin; PCDF: Polychlorinated dibenzofuran; POP: Persistent organic pollutant; POPUP: Persistent Organic Pollutants in Uppsala Primiparas; SD: Standard deviation; SE: Standard error; PBDE(4): sum of BDE-47, BDE-99, BDE-100, BDE-153; TEQ: Toxic equivalent; VIF: Variance inflation factor.ANOVA: Analysis of variance; BMI: Body mass index; LOD: Limit of detection; LOQ: Limit of quantification; NFA: Swedish National Food Agency; PBDE: Polybrominated diphenyl ether; PCB: Polychlorinated biphenyl; PCDD: Polychlorinated dibenzo-The authors declare that they have no competing interests.All authors have read the final version of the manuscript and are in agreement that the work is ready for submission to a journal. SL took part in the planning of the study and the acquisition of data, conducted the statistical analyses and did most of the writing. AG planned and designed the study, helped interpreting the results and took part in the preparation and reviewing of the manuscript. MA was responsible for the chemical analyses, for the quality control of analytical results and for the reviewing of the parts of the manuscript involving chemical analyses. POD planned the study and helped interpreting results and critically revise the manuscript. AH helped interpreting the results in a toxicology context and revised the manuscript. SCL helped planning the statistical analyses, interpreting the results and revised the statistical parts of the manuscript.


As aCGH is a rather complex method, we translated the BRCA1aCGH profile to a Multiplex Ligation-dependent Probe Amplification (MLPA) assay, to identify both BRCA1-mutated breast cancers and sporadic cases with a BRCA1-likeaCGH profile.Our group has previously employed array Comparative Genomic Hybridization (aCGH) to assess the genomic patterns of BRCA1-mutated breast cancers. We have shown that the so-called BRCA1-likeaCGH breast cancers and 45 non-BRCA1-likeaCGH breast cancers, which had previously been analyzed by aCGH. The BRCA1-likeaCGH group consisted of germline BRCA1-mutated cases and sporadic tumours with low BRCA1 gene expression and/or BRCA1 promoter methylation. We trained a shrunken centroids classifier on the training set and validation was performed on an independent test set of 40 BRCA1-likeaCGH breast cancers and 32 non-BRCA1-likeaCGH breast cancer tumours. In addition, we validated the set prospectively on 69 new triple-negative tumours.The most important genomic regions of the original aCGH based classifier  were mapped to a set of 34 MLPA probes. The training set consisted of 39 BRCA1-likeBRCAness in the training set of 84 tumours could accurately be predicted by prediction analysis of microarrays (PAM) (accuracy 94%). Application of this classifier on the independent validation set correctly predicted BRCA-like status of 62 out of 72 breast tumours (86%). Sensitivity and specificity were 85% and 87%, respectively. When the MLPA-test was subsequently applied to 46 breast tumour samples from a randomized clinical trial, the same survival benefit for BRCA1-like tumours associated with intensified alkylating chemotherapy was shown as was previously reported using the aCGH assay.Since the MLPA assay can identify BRCA1-deficient breast cancer patients, this method could be applied both for clinical genetic testing and as a predictor of treatment benefit. BRCA1-like tumours are highly sensitive to chemotherapy with DNA damaging agents, and most likely to poly ADP ribose polymerase (PARP)-inhibitors. The MLPA assay is rapid and robust, can easily be multiplexed, and works well with DNA derived from paraffin-embedded tissues. Breast cancers are genomically highly instable, and array Comparative Genomic Hybridization (aCGH) analysis shows numerous gains and losses of whole chromosomes or parts of chromosomes. Previous studies have shown that hereditary breast cancer tends to develop specific genomic alterations, based on which they can be distinguished from sporadic tumours -5. Our gBRCA1 mutated cancers are more sensitive to DNA damage inducing chemotherapy than their sporadic counterparts . This caaCGH profile was strongly predictive for benefit from intensive carboplatin-based alkylating chemotherapy . Th. Th10]. In this study, we describe the design, testing and validation of a Multiplex Ligation-Dependent Probe Amplification (MLPA) kit to determine BRCA1-like genomic profiles. Our findings suggest that this classifier can be used both in clinical genetic testing and in treatment response prediction. In the clinical genetic setting, the classifier could be used in addition to conventional BRCA1 mutation testing: as a tool to classify BRCA1 variants of unknown significance or to identify potential BRCA1 mutations other than the mutations that are currently screened for. MLPA is a rapid, cost-efficient method, suitable for FFPE tissue derived DNA, and, therefore, potentially useful for routine clinical application.In previous work, we developed an aCGH-based classifier recognizing the genomic pattern of BRCA1-mutated breast tumours. We showed that this classifier could identify hereditary breast tumours, for which no mutation was identified by routine clinical screening . In addiThe clinical application of a DNA test is associated with more requirements for practicability than an assay used for research purposes only. It must be rapid as the patient and the physician are in need of a result within days; it should be feasible using standard equipment; and it must be cost effective. In addition, it is an advantage if the test can be performed on FFPE tissue, as this is the material routinely available in clinical pathology laboratories. MLPA fulfils all these criteria. Therefore, we believe that this assay makes BRCAness testing a viable option for triple-negative tumours. The kit is now ready for prospective clinical validation and it can already be used along with aCGH in the clinical genetics testing mentioned previously.aCGH. When we compared misclassifications between the BRCA1 aCGH and MLPA classifiers we found that five BRCA1-mutated tumours were not classified as BRCA1-like with MLPA and five were missed by aCGH (not completely overlapping). Based on these numbers we can conclude that MLPA and aCGH perform approximately equally well in identifying BRCA1 mutation carriers. Interestingly, one of the misclassified BRCA1 mutated cases was positive for ER staining. About 5% of BRCA1-mutated tumours are ER+ [Currently, there is no gold standard for BRCAness. In this study we used MLPA as a surrogate, in the previous, aCGH. Others have used gene expression , methyla are ER+ . We obse are ER+ .Based on aCGH data, both a BRCA1- and a BRCA2-like genomic profile can be obtained ,8. A limMLPA pattern, in the clinical diagnostics setting, we can continue with extra analysis sorting out the defect . In the therapy selection setting, the patient can be eligible for protocol treatment in an intensified alkylating chemotherapy study . This sIn conclusion, we have shown that the BRCA1-like MLPA assay can be both applied in clinical genetic testing and in treatment benefit prediction. As the MLPA assay is rapid and robust, can easily be multiplexed, and works well with DNA derived from paraffin-embedded tissues, it is a suitable method for a clinical application. However, both its use in the clinical genetics as in the treatment prediction setting should be validated in large prospective series.aCGH: array Comparative Genomic Hybridisation; DSB: double strand break; FFPE: formalin-fixed, paraffin-embedded; MLPA: Multiplex Ligation-dependent Probe Amplification; PAM: prediction analysis of microarrays; PARP: poly ADP ribose polymerase; TN: triple-negative.Nadja Laddach and Suvi Savola are employees of MRC-Holland. The other authors do not have competing interests.EHL, PMN and SR conceived and designed the study. EHL, NL, SPS, MAV AMMO, ALTI and JW acquired the data. EHL and LFAW analysed and interpreted the data. EHL, JW, PMN and SR prepared and revised the manuscript. All authors read and approved the final version of the manuscript.


Cancers behave as organoids, composed not only by genetically altered tumor cells, but also by a variety of non-tumoral cells, such as fibroblasts, macrophages and endothelial cells among others. Cellular interactions within this organoid dictate the rate of tumor progression, and therefore the accumulation of heterogeneous genotypes which characterize the tumor mass at the moment of diagnosis. Evidence for both homotypic and heterotypic cooperation between cells within the tumor microenvironment exist. We will show evidence for fluctuations in the expression of a specific tumor marker, namely galectin-3.Galectin-3 is an animal lectin that is found in diverse cellular compartments, depending on the cellular context and functional state of cells. Normal cells tend to accumulate galectin-3 in the nucleus. Upon malignant transformation, galectin-3 may concentrate in the cytoplasm and can be secreted to the extracellular matrix, where it acts as a matricellular protein, interfering with integrin function among other cell surface glycoproteins. We had shown a focal accumulation of galectin-3 in glioblastomas. Galectin-3 expression was associated to a very specific tumor microenvironment, the pseudopalisades- areas of viable tumor cells surrounding necrotic areas that are commonly found in advanced stages of glioblastomas. Galectin-3 expression in hypoxic and nutrient deprived cells is associated the activation of HIF-1a and it was inhibitable by NF-kB antagonists. In glioblastoma cells, galectin-3 expression was associated with its translocation to mitochondria, suggesting its involvement in mitochondrial homeostasis. Inhibition of galectin-3 expression led to increased cell death, indicating that galectin-3 acts as a prosurvival factor in very specific tumor microenvironments associated with glioblastoma progression. The protumoral role of galectin-3 was also observed in murine melanomas, engrafted in both wild type and galectin-3-deficient mice. Absence of galectin-3 from both tumoral and stromal compartments was associated with attenuated tumor growth, as compared to an experimental condition where both tumor and stromal cells expressed galectin-3. Intriguingly, de novo expression of galectin-3 was frequently observed in tumors derived from cells that did not express galectin-3 when these cells were engrafted in galectin-3 deficient mice. This observation prompted us to investigate whether galectin-3 expressing cells would favor tumor growth when admixed with galectin-3 negative cells. Indeed, when cells are admixed, tumors grew much more efficiently than those engraftments of either cell alone, indicating cooperation between galectin-3 expressing cells and galectin-3 negative cells. Further experiments indicated that galectin-3 expression is related with the induction of a proangiogenic tumor environment. Thus, targeting galectin-3 expression in selected tumors may prove to be an effective strategy to control tumor growth.Angiogenesis and vasculature function are critically altered in cancers, which are usually ill-perfused. Evidence for activation of both angiotensin and bradykinin-dependent pathways within the tumor microenvironment of melanomas will be discussed. Blockage of vasoactive peptide-dependent pathways may control tumor angiogenesis and therefore may control tumor growth. Indeed, epidemiological studies are now indicating that individuals receiving specific blockers of angiotensin receptors may be relatively protected from the development of tumors. In a clinical setting, however, antiangiogenic drugs would be preferably used in combination with chemotherapeutic drugs. Vasculature function, however is also critical for the proper delivery of chemotherapeutic drugs to tumors, specific therapeutic windows for the application of antiangiogenic drugs need to be identified. This notion will be further discussed. Altogether, the examples above call for a necessary understanding of tumor physiology, which seems to be under appreciated in the design of experimental/clinical trials.There are no competing interests in this presentation.


EGFR inhibition has emerged to be an important strategy in the treatment of non-small cell lung cancer (NSCLC). Small molecule tyrosine kinase inhibitors (TKIs) and mono-clonal antibodies (mAbs) to the EGFR have been tested in multiple large randomized phase III studies alone or combined with chemotherapy, as well as small phase I-II studies which investigated their efficacy as radiosensitizers when combined with radiotherapy. In this review, we described the current clinical outcome after treatment with EGFR TKIs and mAbs alone or combined with chemotherapy in advanced stage NSCLC, as well as the early findings in feasibility/phase I or II studies regarding to whether EGFR TKI or mAb can be safely and effectively combined with radiotherapy in the treatment of locally advanced NSCLC. Furthermore, we explore the potential predictive biomarkers for response to EGFR TKIs or mAbs in NSCLC patients based on the findings in the current clinical trials; the mechanisms of resistance to EGFR inhibition; and the strategies of augmenting the antitumor activity of the EGFR inhibitors alone or when combined with chemotherapy or radiotherapy. The epidermal growth factor receptor (EGFR), a 170 kD transmembrane protein consisted of a N-terminus extracellular ligand-binding site, a hydrophobic transmembrane domain, and a C-terminus intracellular region with tyrosine kinase activity, is the first of the ErbB family of receptor tyrosine kinases (RTKs) . The othAs the leading cause of cancer mortality worldwide, 50-80% of non-small cell lung cancer (NSCLC) is associated with EGFR overexpression; with 65% of them also found to have an increased EGFR gene copy number -11. GiveNumerous phase III studies on TKI alone or combined with chemotherapy as first line treatment for stage IIIB-IV NSCLC have been conducted since Gefitinib has been approved by the Food and Drug Administration (FDA) in 2003, and Erlotinib has been approved by the FDA in 2004 for chemotherapy-resistant stage IIIB-IV NSCLC -23. In 2Despite the disappointment with the INTACT and TRIBUT studies, the importance of selecting patients who will respond to TKIs has been recognized, which is suggested by the prolonged survival observed in never smokers ,17. Thisvs. 6.7%, p <0.001). Better ORR associated with Gefitinib was also reported . Among 437 patients whose EGFR mutation data can be evaluated, 261 had EGFR mutations identified. Out of these 261 patients, 53.6% had exon 19 deletions, and 42.5% had a missense mutation at exon 21. A predictor of TKI resistance [vs. 47.3%, p <0.001). The opposite was found in patients without EGFR mutations . No significant difference in toxicity profile between the two treatment arms was found. This study established that EGFR mutation, such as those on exon 19 and 21, can be used as a predictive marker for response to EGFR TKIs, which may be more effective than chemotherapy in patients with these mutations. Its findings were further validated in several other phase 3 randomized studies [To further classify the patients who will respond to EGFR TKIs, a phase III randomized study (IPASS) comparing upfront Geftinib and Carboplatin/Paclitaxel in stage IIIB-IV patients with the following characteristics has been conducted in East Asia: never or former light smokers with a diagnosis of adenocarcinoma . A totalsistance , a mutat studies . All thend line chemotherapy in randomized phase 3 studies [While effective only in EGFR mutation positive patients as first line treatment, both Gefitinib and Erlotinib have been shown to achieve similar ORR, PFS, and OS when compared with 2 studies ,29. EGFR studies . In addivs. 29%, p = 0.010) and median survival . Only 10% of the patients experienced an acne-like rash on the Cetuximab arm, and no >grade 3 acne-like rash was observed in this study. The superior response associated with the addition of Cetuximab to first line chemotherapy was confirmed in another phase III randomized study (BMS 099) randomizing 676 patients to Carboplatin, plus a taxane or Caboplatin-based chemotherapy combined with Cetuximab, which is given till disease progression. However, no survival benefit was demonstrated in that study  was observed with the addition of cetuximab [The EGFR has been known to play a key role in the activation of DNA repair and anti-apoptotic proteins, as well as tumor cell repopulation after irradiation ,46. Uponetuximab . As an Eetuximab . Despiteet al., a concurrent TKI and radiotherapy to 70 Gy in stage III-IV patients achieved a median survival of 21.8 months, which is at least comparable to what has been reported after concurrent chemoradiation [In pre-clinical studies of NSCLC, radiosensitizing effects of both EGFR TKIs and mAbs have been reported ,50-54. Tadiation ,64,65. Hadiation ,59. Furtadiation . Disappoadiation . This imadiation . Due to adiation ,62. AgaiA limited number of studies investigating the radiosensitizing effects of a mAb have been reported as well Table . Among tet al., T790M appears to be more prevalent in patients who has not had any TKI treatment (25.2% - 31.5%), and it becomes even more prevalent after TKI treatment (83.3%) [et al., NSCLC patients who harbor mutations in exons 19, 20, and 21 can still benefit from TKI, with superior PFS to that in patients without any of these mutations [The findings from large phase III randomized studies demonstrated that patient selection is essential in the application of EGFR TKI in the treatment of stage IIIB-IV NSCLC. Patients who harbor a activating EGFR mutation usually responds very well to treatments including EGFR TKI or mAb alone or combined with chemotherapy and/or radiotherapy -42,45,55 (83.3%) . This fiutations . Therefoutations . This agutations .et al., WZ4002, a mutant EGFR-TKI, and E7050, a mutant selective dual inhibitor of Met and VEGFR-2, were able to inhibit tumor growth in Erlotinib resistant NSCLC cells in vitro and in vivo when given together with successful inhibition of the EGFR, Met, and their downstream PI3K-AKT pathway [nd generation TKIs and MET inhibitors with radiotherapy and/or chemotherapy still need to be further investigated in future studies. In addition to MET signaling stimulated PI3K signaling, BRAF mutation in the RAS-RAF-MEK-MAPK pathway, and loss of PTEN expression may also lead to acquired resistance to an EGFR TKI [Increased signaling through the PI3K pathway through MET-trigger ErbB3 signaling in TKI-resistant NSCLC cells overexpressing MET oncogene has been shown to be a classic example of acquired resistance through bypassing the EGFR, which can be overcome through dual EGFR and MET inhibition . This st pathway . The comEGFR TKI ,81.As a another potential predictive biomarker for a lack of response to EGFR TKIs in NSCLC, KRAS mutation was only shown to be associated with treatment outcome in selected studies ,41. It iin vitro and in vivo [Due to differences of the EGFR inhibition mechanism by the mAb, which can generate antibody dependent cellular cytotoxicity; and TKIs ability to inhibit multiple types of ErbB receptors, the role of dual blockade should be explored further in the clinical as they have been shown to increase the potency of EGFR inhibition  in vivo ,84. Targ in vivo ,86. At lThe authors declare that they have no competing interests.AC, SR and WT have generated the idea of conducting this review and AC has writing the manuscript with SR and WT reviewed, given suggestions on modifications, and approved the final version. All authors read and approved the final manuscript.