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Background

Testosterone therapy is increasingly promoted. No randomized placebo-controlled trial has been implemented to assess the effect of testosterone therapy on cardiovascular events, although very high levels of androgens are thought to promote cardiovascular disease.

Methods
A systematic review and meta-analysis was conducted of placebo-controlled randomized trials of testosterone therapy among men lasting 12+ weeks reporting cardiovascular-related events. We searched PubMed through the end of 2012 using “(“testosterone” or “androgen”) and trial and (“random*”)” with the selection limited to studies of men in English, supplemented by a bibliograph ic search of the World Health Organization trial registry. Two reviewers independently searched, selected and assessed study quality with differences resolved by consensus. Two statisticians independently abstracted and analyzed data, using random or fixed effects models, as appropriate, with inverse variance weighting.

Results
Of 1,882 studies identified 27 trials were eligible including 2,994, mainly older, men who experienced 180 cardiovascular-related events. Testosterone therapy increased the risk of a cardiovascular-related event (odds ratio (OR) 1.54, 95% confidence interval (CI) 1.09 to 2.18). The effect of testosterone therapy varied with source of funding ( P-value for interaction 0.03), but not with baseline testosterone level (P-value for interaction 0.70). In trials not funded by the pharmaceutical industry the risk of a cardiovascularrelated event on testosterone therapy was greater (OR 2.06, 95% CI 1.34 to 3.17) than in pharmaceutical industry funded trials (OR 0.89, 95% CI 0.50 to 1.60).

Conclusions
The effects of testosterone on cardiovascular-related events varied with source of funding. Nevertheless, overall and particularly in trials not funded by the pharmaceutical industry, exogenous testosterone increased the risk of cardiovascular-related events, with corresponding implications for the use of testosterone therapy.

Keywords:
Testosterone; Cardiovascular; Men; Trial

Background
In observational studies low serum testosterone is associated with cardiovascular disease [1,2]. Testosterone may protect or be a secondary risk marker of other processes [1-3]. On the precautionary principle, expert advice and reviews, largely based on observational evidence, warn that cardiovascular disease may be increased by androgen deprivation therapy [4] and low testosterone [5,6]. Awareness of low testosterone as a treatable condition is being raised [7,8]. Testosterone use is increasing [9-11], possibly as self-medication in response to advertising. In 2004 the Institute of Medicine (IOM) reviewed the evidence on testosterone therapy and concluded, largely based on placebocontrolled trials, that „there is not clear evidence of benefit for any of the health outcomes examined‟ [12]. The IOM recommended small-scale trials to establish the efficacy of testosterone therapy where other treatments were not available [12]. To our knowledge, no trial has been designed to assess the effect of testosterone therapy on cardiovascular morbidity or mortality. Previous metaanalyses of randomized placebo-controlled trials found that testosterone therapy resulted in a non-significantly higher risk of cardiovascular events, based on adverse events, but only included trials through March 2005 [13,14]. A more recent meta-analysis included trials through August 2008 but only reported on three specific cardiovascular outcomes, that is, arrhythmia, coronary bypass surgery and myocardial infarction[15]. Given the widespread use of testosterone, the high prevalence of cardiovascular disease in older men and no comprehensive assessment of the effect of testosterone therapy on cardiovascular events, an up-to-date meta-analysis may help inform clinical practice. We carried out a meta-analysis of adverse events from randomized placebocontrolled trials to examine the overall risk of cardiovascular-related events associated with testosterone therapy.

Methods
This meta-analysis follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist [see Additional file 1] and a published protocol (CRD42011001815)[16]. Two reviewers (LX and CMS) independently searched for and selected trials, resolving any differences by consensus. Two statisticians (GF and BJC) extracted information from the selected trials. Additional file 1. PRISMA 2009 Checklist. Format: DOC Size: 63KB Download file This file can be viewed with: Microsoft Word Viewer

Data sources and searches
We (LX and CMS) systematically searched PubMed until 31st December 2012 using “(“testosterone” or “androgen”) and (random*) and trial” with the selection limited to studies in men in English, because a preliminary search only found studies in Engl ish. We (LX and CMS) searched the World Health Organization (WHO) International Clinical Trials Registry Platform for any trial using testosterone as an intervention. From this search, we (LX and CMS) discarded any studies both agreed were irrelevant based on title or abstract and read the remaining. We did a bibliographic search of the selected trials and relevant reviews.

Study selection
We included randomized placebo-controlled trials giving cardiovascular-related events by study arm, because a report may focus on a particular aspect of the trial [17,18] and not report all events that have occurred [17-19]. We excluded trials that only gave treatment-related events in the testosterone arm because these might not include full reporting of events in the placebo arm. Initially, we intended to exclude trials that only reported withdrawals as potentially less comprehensive than reporting of adverse events [18,19], however this turned out to be a very fine distinction, so we included any trial reporting cardiovascular-related events by study arm. We included any randomized controlled trial (RCT) of testosterone, but not other androgens, compared with placebo, including a comparison against a background of other treatments, because men likely to be taking testosterone may also be in treatment for other conditions. We excluded trials of less than twelve weeks‟ duration to assess long-term rather than acute effects of testosterone therapy. We checked for duplication based on overlapping authorship, study description, number of participants and participant characteristics. When duplication occurred we used the study with the most comprehensive description of adverse events.

Outcome
The primary outcome was composite cardiovascular-related events; because we anticipated too few events for robust assessment by cardiovascular event type; and a system-wide composite outcome may be most suitable for adverse events [20]. Cardiovascularrelated events were defined as anything reported as such by the authors, that is, events reported as cardiac disorders, cardiovascular complaints, cardiovascular events, vascular disorders, cardiac or cardiovascular, or where the event description fell within the International Statistical Classification of Disease (ICD) version 10 chapter IX (I00 to I99). Most trials only reported serious adverse events, but a few also reported a wider range of events, so we also examined the effect of testosterone therapy by seriousness. Seriousness was based on the US Department of Agriculture definition of serious adverse events and the type of cardiovascular event generally considered serious [21]. Serious cardiovascular events were defined as cardiovascular-related events which the authors described as serious adverse events or where the outcome was death, life-threatening, hospitalization, involved permanent damage or required medical/surgical intervention, or was one of the following types of cardiovascular event: myocardial infarction, unstable angina, coronary revascularization, coronary artery disease, arrhythmias, transient ischemic attacks, stroke or congestive heart failure but not deep vein thrombosis.

Data extraction and quality assessment
A statistician (GF) extracted the number of participants randomized and cardiovascular-related events by trial arm. Event classification was checked by a physician (LX). A second statistician (BJC) checked the information extracted. Where trials reported cardiovascular-related events without giving the study arm, we contacted the authors twice by email to ask for cardiovascular-related events by study arm. The reviewers (LX and CMS) independently used an established tool to evaluate the quality of each trial [22], focusing on the quality of reporting of cardiovascular-related adverse events. First, we reported whether cardiovascular-related events were individually listed in a table by study arm, because these are easier to identify unambiguously. Second, we reported whether the type and severity of cardiovascular-related events reported was either pre-specified or identified before the allocation was revealed, because issues have been expressed about the reporting of adverse events [23,24]. Cardiovascular-related events vary in severity making the selection criteria and categorization crucial to an outcome assessed from adverse events.

Sensitivity analysis
We initially planned only to assess whether the effects of testosterone on cardiovascular-related events varied with average baseline testosterone, because we did not expect sufficient trials for sub-group analysis by type of testosterone product or by type of cardiovascular-related event. However, the reporting of adverse events may be open to interpretation [23], and may not be comprehensive [25]. Given potential lack of clarity as to the selection of the cardiovascular-related adverse events reported, we also examined whether the effect of testosterone therapy varied with funding source. Finally, we also considered cardiovascular-related death as an outcome.

Of these 169 papers.733 testosterone and 1. that is. Two trials were stopped early [49. We used meta-analysis regression.Data synthesis and analysis We used the number of participants randomized as the denominator and included all cardiovascular-related events from the start. Initial analysis show ed the pooled odds ratio was similar using a Peto or a Mantel-Haenszel estimate.15].a beneficial effect of testosterone [on mortality] by continuing the study‟ [49].1. we used inverse variance weighting for consistency with the meta-regression.40. using the „rma‟ function of the „metafor‟ package in R 2. but not other cardiovascularrelated events. using fixed effects models where there was low heterogeneity (I 2 <30%). one of the others that gave cardiovascular deaths.42-48. including one where the cardiovascular-related events were surmised from a P-value [65].35-37. Table 1 shows the 27 trials over 25 years of 2.261 placebo) with low testosterone and/or chronic diseases. which does not require ethics committee approval. Six authors never responded [37. Most of the trials were in Western settings. Results The initial search yielded 1. We sought clarification concerning events by study arm for 10 trials as set out in Additional file 2. Thirteen trials were supported by the pharmaceutical industry. with inverse variance weighting.in the foreseeable future . We used I 2 to assess heterogeneity between trials. but not all cardiovascular-related events by study arm [44]. For 17 trials cardiovascular-related events were surmised from withdrawals and/or adverse events as given in Additional file 4.40.994 mostly middle-aged or older men (1. The search found one additional trial [39] potentially relevant to the earlier metaanalyses [13. otherwise using random effects models.[13. Two additional trials [35. to assess whether the effects of testosterone therapy varied with baseline testosterone or funding source.14. 31 concerned different placebo-controlled randomized trials among men of testosterone therapy of 12+ weeks reporting cardiovascular-related events by study arm.36] selected for full text scrutiny had cardiovascular-related events shown in a plot from one previous meta-analysis [13]. We did not find any additional such trials from a bibliographic search of these 32 papers or from eight reviews [27-34]. missing trials.15] or in the relevant publications [35-37]. We included this later trial [48]. Additional file 2.50]. We used funnel plots and „trim and fill‟ to assess publication bias.46.42-45].47] and one provided information [48]. The search did not find one small non-randomized trial [38] included in two previous meta-analyses [13. one because of adverse events among the men allocated to testosterone [50] and one because „it would not be feasible to demonstrate . of which 169 were selected for full text scrutiny. Austr ia). Characteristics of placebo-controlled randomized trials giving the effects of testosterone therapy on cardiovascularrelated events among men Quality assessment The quality assessment (see Additional file 3) shows that two trials provided a table with a comprehensive list of cardiovascularrelated events by study arm and eight trials provided a summary table of cardiovascular-related events by study arm. Trials where authors contacted for additional information and responses. The type and severity of adverse events to be reported was pre-specified in one trial [61].14].41] potentially relevant to the most recent meta-analysis [15] and 11 subsequent trials.14. but not the other meta-analyses [14. Table 1. three responded but did not provide any relevant information [36.100]. two additional trials [40. We obtained the pooled odds ratio. Vienna. by study arm [46] and one that gave vascular events. We found one additional recent trial from the WHO International Clinical Trials Registry Platform [26]. Selection process for the placebo-controlled randomized trials of the effects of testosterone therapy on cardiovascular-related events. This study is an analysis of published data.882 papers. Figure 1 shows the search strategy resulting in 27 placebo-controlled randomized trials.1 (R Development Core Team. using the „metabin‟ function of the „meta‟ package in R 2. Format: DOCX Size: 32KB Download file Figure 1. In the two trials terminated early the adverse events motivated termination and were identified . who experienced 180 cardiovascular-related events.

Sensitivity analysis The cardiovascular-related event rate was lower in trials funded by the pharmaceutical industry (4% (66/1. In a meta-regression model.57 (95% CI 0. Forest plots of placebo-controlled randomized trials examining the pooled effect of testosterone therapy on cardiovascular-related events. In trials funded by the pharmaceutical industry testosterone had no effect on cardiovascular-related events. odds ratio (OR) 1.61 (95% CI 1. Finally.[26].13) by trim and fill.[39].[48]-[68].14) by trim and fill.38).[48]-[67].50].[41]. Quality assessment of the selected placebo-controlled RCTs of the effects of testosterone therapy on cardiovascular-related events (CRE)[19]. the estimate was very similar (OR 1. Figure 3.09 to 2. Funnel plot of placebo-controlled randomized trials examining the effects of testosterone therapy on cardiovascular-related events.before treatment allocation was known [49. 33 cardiovascular-related deaths were identified (22 testosterone arm and 11 placebo arm).[26].01 (95% CI 1.42 (95% CI 0.[39]. whose categorization is shown in Additional file 4. Otherwise it was sometimes unclear whether the definition or classification was made by masked assessors.[101].651)) than in other trials (8% (114/1. for which the odds ratio was similar 1.21 to 2. When the analysis was restricted to serious events. Figure 4.69 (95% CI 1. however. Testosterone increased the risk of a cardiovascular-related event in a fixed effect model.[44]. Format: DOCX Size: 61KB Download file Data synthesis The funnel plot (Figure 2) shows several small studies (on the left hand side) where testosterone reduced cardiovascular-related events.18.[46].70 to 2. but in the other trials testosterone therapy substantially increased the risk of a cardiovascular-related event (Figure 4).[46]. and was revised to 1.[44].8%). The forest plot (Figure 3) shows that the trials were homogeneous (I2 = 7.343)).30 to 3. Forest plots of placebo-controlled randomized trials examining the pooled effects of testosterone therapy on cardiovascular-related events by source of funding: upper panel funded by the pharmaceutical industry and lower panel not funded by the pharmaceutical industry. risk of cardiovascular-related events on testosterone therapy varied with the source of funding (P-value for interaction 0.54.[41]. 95% confidence interval (CI) 1. Description of cardiovascular-related events in the selected placebo-controlled RCTs [19].70). there were no similar small studies where testosterone increased cardiovascular-related events.78 to 3. Additional file 3. Trim and fill revised the OR to 1.03) but not with baseline testosterone ( P-value for interaction 0. Figure 2.56)) and was revised to 2. Format: DOCX Size: 54KB Download file Additional file 4. Discussion .01 to 2.89) to the estimate for all cardiovascular-related events.

2] and with testosterone declining with age when cardiovascular disease increases with age. some accidental unmasking may have occurred. Fourth. with a much larger number of participants than previous metaanalyses [13-15]. as has long been suggested [71] and recently substantiated by the effective use of anti-androgens in prostate cancer at castrate levels of serum testosterone [72. Exogenous estrogens do not protect men against cardiovascular disease [74]. hematocrit and thromboxane [15. consistent with the observational evidence[1. Trials favoring testosterone may be unpublished. by raising or lowering androgens would also bring clarity. Finally. This meta-analysis based on many more trials (27). However.85] and could be due to different reporting of adverse events in industry funded trials. if at all. It is possible. such as androgen glucuronides [94]. Strengths and limitations Despite providing a meta-analysis of all known placebo-controlled randomized trials. Few trials have examined the effects of dihydrotestosterone administration and have usually focused on prostate rather than cardiovascular effects [76-78]. although unusual. However. the funnel plot (Figure 2) and „trim and fill‟ suggested trials favoring the placebo may be missing. did not corroborate protective effects of endogenous testosterone on cardiovascular disease risk factors [69]. using genetic variants as an instrumental variable for endogenous testosterone. The difference between the estimates by funding source is consistent with other observations [84. The funnel plot and analysis by funding source are consistent with that possibility. Industry funded trials reported fewer cardiovascular-related events. which all indicated a non-significantly higher risk of testosterone therapy for a composite cardiovascular outcome of the events considered.80]. such as estrogens or dihydrotestosterone. for the effects of treatment to be „crossed‟ by age [86]. Testosterone compared to placebo could be beneficial for glucose metabolism [65]. such as testosterone. consideration could be given to whether further trials should be of agents that raise or lower testosterone. Natural experiments suggest that lower lifetime endogenous androgens are associated with a relatively lower risk of death from ischemic heart disease. exogenous testosterone lowers HDL-cholesterol [15] and raises hemoglobin. depression[87. not all trials of testosterone therapy reported all cardiovascular-related events by study arm [36. Assuming the increased risk of cardiovascular-related events seen here with testosterone therapy would give a number needed to harm of at most 90 per year of testosterone therapy. From a clinical perspective the issue is ensuring that the benefits of testosterone therapy outweigh the potential risks. we searched broadly and found several potentially eligible trials that had not been included in previous meta-analyses. some men in the testosterone arm stopped treatment because of increased prostate specific antigen or polycythemia.92]. sexual dysfunction [26. but other metabolites of exogenous testosterone.88]. Third. Second. could mediate cardiovascular-related events. Industry funded trials tended to be in younger men.89]. bone density [90] and HIV wasting syndrome [91. The 10-year risk of a cardiovascular event for US men aged 65 to 69 years is about 28% [93]. despite bias to the null by the competing risk of death from prostate cancer. RCTs are not always tagged as such and could be missed [96]. our study cannot include on-going trials. The interplay of testosterone and dihydrotestosterone is complex and challenging to disentangle in RCTs [79]. in a trial of a therapy. such as Mendelian randomization. a recent Mendelian randomization study. all of which might contribute to cardiovascular disease. a meta-analysis of RCTs of androgen deprivation therapy found a non-significantly lower risk of cardiovascular mortality among men allocated to androgen deprivation [83]. A very large market is at stake [9]. First. with endogenous testosterone being protective. Moreover. Second. which may change how men feel or their sex drive. despite discrepancies in some studies [13. with the confidence interval no longer including no effect.46]. and to other study designs less open to biases. which would bias towards the null. Our findings are consistent with the three previous meta-analyses [13-15]. limitations exist. Fifth. that is. However. for example by confirming that the observed negative associations of serum testosterone with specific cardiovascular diseases extends to other androgen biomarkers. As such. Differences by funding source could also be due to differences between trials. many more men (2.This updated meta-analysis of placebo-controlled randomized trials. The risk of testosterone therapy was particularly marked in trials not funded by the pharmaceutical industry. further research might focus on obtaining evidence without interventions. However. Few trials assessed or reported this possibility. Another possibility is that serum testosterone is not a good indicator of androgen activity [70].73]. although whether testosterone is better than established treatments for these conditions has not been clearly established. but more precise estimate. older men. a gene in the steroidogenesis pathway (CYP17A1) is reliably associated with coronary artery disease [95]. Third. such as the Testosterone Supplementation and Exercise in Elderly Men trial (NCT00112151) and The Testosterone Trial . Similarly. First. Establishing if CYP17A1 acts. adds to the previous findings by showing that testosterone therapy increases cardiovascular-related events among men. which reduces power although it should not affect the direction of effect. endogenous testosterone may be beneficial. endogenous and exogenous testosterone may have different effects. higher free testosterone rather than higher estradiol appeared to mediate the cardiovascular events in a recent trial of testosterone therapy [75]. raised by testosterone therapy. based on legally castrated men [81] and men with Klinefelter‟s syndrome[ 82]. Almost a decade after the IOM‟s report [12] the efficacy of testosterone therapy for health outcomes where treatments are not already available remains uncertain.994) and correspondingly more events (180) produced a similar. the reporting of adverse events may be open to conflicts of interest [24]. Thromboxane promotes clotting and blood vessel constriction. Nevertheless. Several possible explanations exist for our findings. Cardiovascular disease is common in typical users of testosterone therapy.14]. The risks of cardiovascular-related events were similar by baseline testosterone.

Seventh. but the severity varied between trials. All authors read and approved the final manuscript. from a public health perspective the issue is identifying side-effects. they also reviewed the manuscript critically. it would be against the general run of evidence to date making interpretation uncertain because of heterogeneity [97]. Authors’ contributions LX carried out the systematic search and drafted the manuscript. caution needs to be exercised to ensure that the associated health benefits of testosterone therapy outweigh the potential increased risk of cardiovascular-related events. IOM: Institute of Medicine. so hospitalization may represent a particularly significant event. Abbreviations CI: confidence interval. these trials are not designed to assess the effect of testosterone on cardiovascular events and will take time to complete. Conclusions Appropriately prescribed testosterone is undoubtedly beneficial. because they may be particularly prone to hospitalization. Arguably. However.50] which reduces power and could affect the estimates. On the other hand.. most trials only reported fairly serious cardiovascular-related events. and the events reported are more or less severe symptoms of cardiovascular disease on the pathway to cardiovascular mortality. However. References Background . However. carried out the systematic search and helped draft the manuscript. two larger trials were terminated early [49. do not apply to frail older men. testosterone therapy increased the risk of cardiovascular-related events overall. Sixth. RCT: randomized controlled trial. WHO: World Health Organization. but was limited by relying on events the authors had chosen to describe in detail by study arm and was revised upwards by trim and fill. including hospitalization.(NCT00799617). where a composite outcome relating to a particular system (here the cardiovascular system) has been recommended [20]. GF and BJC did the data extraction and analysis. Systematic review reference number CRD42011001815. the terminations took place towards the end of the planned trials and did not specifically concern cardiovascular-related events. however. CYP17A1: cytochrome P450 17A1. Such sub-group analysis would undoubtedly be etiologically valuable. Ninth. the abstractors were not blinded. However. although for RCTs the reporting of events should be comparable within trials. and consults for Crucell MV. CMS originated the idea. the standard definitions of event seriousness. All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. BJC has received research funding from MedImmune Inc. Subsequent large RCTs rarely reverse the direction of effect from metaanalysis [99]. Meta-analysis may overstate the benefits of treatment [98] however. given the lack of detailed cardiovascular-event reporting secondary analysis by type of cardiovascular event was not possible. Notably. LX is the guarantor. Nevertheless. Acknowledgements The authors thank Steffie Woolhandler. Competing interests All authors declare: no support from any organization for the submitted work. have been similar. PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses. Finally. OR: odds ratio. although meta-analyses are a mainstay of evidence-based medicine they may be less reliable than large RCTs. However. an estimate based solely on deaths also had a similar point estimate. early terminations may have slightly increased the estimate and widened the confidence intervals. Eighth. the difference observed by source of funding could just be chance variation. no other relationships or activities exist that could appear to have influenced the submitted work. Tenth. although the confidence interval included no effect because of low power. particularly in older men where cardiovascular disease is common. If new trials show testosterone therapy to be strongly protective against cardiovascular disease. ICD: International Statistical Classification of Disease. they are less prone to overstate the harms [98]. David Himmelstein and Heidi Jones for their support. the interpretation would. most likely. An analysis restricted to events which could be identified as serious gave a similar estimate. frail older people may also be most affected by any decrement to their already poor health.

liver disease[21]. A lifetime history of mood and anxiety disorders was identified using the Structured Clinical Interview for DSM-IV-TR Research Version. have been shown to be related to several medical conditions. Anthropometric measurements and socioeconomic status (SES) were determined and information on medication use and lifestyle was obtained via questionnaire. results vary according to the type and severity of the chronic disease and methods of ascertainment [3. Furthermore. The causal pathway between comorbid mental and physical illness is complex and remains unclear. Over time.14]. Rates of depression and anxiety are generally higher among the physically ill than in the general population. cancer [18]. psychodynamic and cognitive factors) [44] and social (for example. migraine and chronic pain. immune and endocrine changes) [43]. as recently reviewed by Culpepper and colleagues.10]. and health risk factors (body mass index.24]. psoriasis [17]. psychological (for example. yet an incomplete understanding prevails[1. behavioural. peptic ulcer. GORD and other gastrointestinal disorders. blackouts and/or epilepsy. whilst anxiety disorders were significantly associated with thyroid.2]. thyroid disorders [35]. chronic headaches [24]. Conclusions Our study provides further population-based evidence supporting the link between mental and physical illness in men. arthritis and pulmonary disease are also associated [2]. pulmonary disease [20]. patients with depression have been found to have a significantly higher number of somatic symptoms [5] and a higher risk of chronic diseases [6-8]. affects medication response and treatment adherence [38]. recurrent headaches. cancer [18]. Medical burden. socioeconomic status. diabetes [13. links to the deteriorating patient‟s health status and functioning [7. Physical illness Background For centuries. an evidence base has grown to support this notion. psychotropic . gastrointestinal problems [34]. In addition. neurological disorders [19]. and psoriasis. and to verify whether these psychiatric disorders are linked to overall medical burden. Understanding these associations is not only necessary for individual management. liver disorders and pulmonary disease in older people. and social disengagement) [45]. stroke and dementia [12]. thyroid disorders [22] and asthma [23. loneliness. Similarly. congestive heart failure [11]. and increases the risk of complications [39] and even death [37]. Logistic regression models were used to test the associations. Increased odds of high medical burden were associated with both mood and anxiety disorders. psoriasis [17] and a higher number of medical symptoms [36].42] including biological (for example. osteoarthritis and rheumatoid arthritis [16].37]. mood disorders were associated with gastro oesophageal reflux disease (GORD). 20 to 97 years old. anaemia [26].The mind-body nexus has been a topic of growing interest. it has been hypothesised and debated that a mind-body interaction exists. metabolic. diabetes. increased proinflammatory cytokines. The co-occurrence of mood or anxiety disorders and physical illness worsens the impact of symptom burden [36]. gastro oesophageal reflux disease (GORD) [15]. as well as non-specific syndromes such as obesity [25]. impaired social support. Non-patient edition (SCID-I/NP). Methods This study examined data collected from 942 men. hypothalamic-pituitary axis deregulation. GORD [15]. The presence of medical conditions (lifetime) was self-reported and confirmed by medical records. irritable bowel syndrome [33]. Further data are however required to understand the specific relationship between mood and anxiety disorders and individual physical health conditions. Within clinical samples. but also to inform the delivery of health promotion messages and health care. medication use or clinical data. anxiety disxorders.4]. the comorbidity between mental and physical illness is relevant in terms of role impairment and work performance [24]. The strength of the association as well as the extent of the evidence varies for each medical condition. such as coronary heart disease [9. Results After adjustment for age. Population-based study. renal dysfunction [27]. autonomic nervous system dysfunction. Moreover. however. chronic fatigue syndrome [28]. such as cardiovascular disease [30]. serotonin depletion. Keywords: Mood disorder. as well as quality of life and health service use and costs [40]. and chronic pain [29]. obesity [31] and other metabolic disorders [32]. Anxiety disorder. impacts disease course. physical activity and smoking). Comorbidity. It has been suggested that the bidirectionality of this relationship may involve several mechanisms [41. participating in the Geelong Osteoporosis Study.

one or more standard deviations below the young normal mean at the femoral neck or spine (L2-L4. minor depression. psoriatic arthritis.540). generalised anxiety disorder. Diagnosis of GORD was based on self-report as were gastrointestinal diseases which include peptic ulcer. treatment for these disorders may influence each other [50]. All interviews were conducted by personnel with qualifications in psychology. we aimed to describe the relationship between comorbid mental and physical illness in a large. and/or an anxiety disorder including panic disorder. resulting in a final sample of 942 eligible men. Thus. kg/m2) was calculated from these measurements. substance-induced anxiety disorder or anxiety disorder not otherwise specified. 139 were not able to be contacted and 225 declined to participate resulting in 978 participants (81% of the eligible baseline sample). 141 had died. Graves‟ disease. a study originally designed to investigate the epidemiology of osteoporosis in Australia. The Barwon Statistical Division is situated in South-Eastern Australia. text revision (DSM-IVTR) Research Version. utilising structured clinical interviews. thyroiditis and other unspecified thyroid conditions. low trauma fracture. dysthymia. Clinical measures Height was measured to the nearest 0. This study was approved by the Barwon Health Human Research Ethics Committee and written informed consent was obtained from all participants. Furthermore. For this analyses. Of those invited to participate in the five-year follow-up study (n = 1. chronic diarrhoea.1 kg using electronic scales. who were trained using live and videotaped interviews under the supervision of a psychiatrist. irritable bowel syndrome. and clinical and self-reported data collected as part of the Geelong Osteoporosis Study (GOS) [51]. Medical conditions The presence of medical conditions (lifetime) was self-reported and confirmed by medical records. representative sample of men residing in Australia. WI. including major depressive disorder. Hypertension was characterised by a combination of self-report and current medication use (antihypertensive. falls and bleeding. those who did not undergo psychiatric assessment (n = 17) or provide medical information (n = 19) were excluded. mood disorder due to general medical condition (GMC) and substance-induced mood disorder.540. medication use or clinical data. defined by self-report or current medication use (oral hypoglycaemic agents and insulin preparations). Musculoskeletal disease was defined by low BMD or self-reported osteoarthritis. muscle disease or weakness. Thyroid disorders included self-reported or medicated (thyroid hormones and anti-thyroid agents) hyperthyroidism. Assessments Psychiatric measures The Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders. Given these data. posterior-anterior projection) [53]. gastric surgery. response 67%. Non-patient edition (SCID-I/NP) [52] was used to identify those who had ever experienced a mood disorder. 4th edition. hypothyroidism. agoraphobia. an age-stratified. Cardiovascular disease was defined by a diagnosis of hypertension (as . beta-adrenergic blocking agents and diuretics) or a systolic blood pressure of >140 mmHg or diastolic blood pressure of >90 mmHg.medication use has been linked to a higher incidence of an array of health outcomes. where possible. population-based sample of adult men (n = 1. Lumbar spine and femoral neck bone mineral density (BMD) was measured by dual energy X-ray absorptiometry (DXA) using GE Lunar Prodigy (Prodigy GE Lunar Madison. Originally. Low bone mass was classified according to the World Health Organisation definition. Further details of the study have been published elsewhere [51]. anxiety disorder due to a medical condition. Enhancing the quality of care for those with mental illness not only can improve mood and anxiety symptoms. chronic gastritis and causes of malabsorption.1 cm using a wall-mounted stadiometer and body weight was measured to the nearest 0. 16 were unable to provide written informed consent. but also physical health in patients with comorbid mental and physical illness [49]. with regard to syndrome reciprocal influences and medication interactions. comprising both rural and urban communities. or self-reported or medicated gout (agents used in gout or hyperuricaemia). USA). osteoporosis. 20 to 97 years old) was randomly-selected from the Australian Electoral Commission (AEC) rolls for the Barwon Statistical Division between 2001 and 2006. rheumatoid arthritis. Metabolic risk factors included a diagnosis of diabetes. ankylosing spondylitis. such as pancreatitis. Hashimoto‟s disease. obsessive compulsive disorder. estimating the prevalence and understanding the association between mood and anxiety disorders and physical illness is important in order to make more accurate diagnoses and to provide integrated and effective treatment. Long term stability of the machine was tested three times weekly by scanning an anthropomorphic phantom (Hologic). including diabetes. and sudden cardiac death [46-48]. Body mass index (BMI. specific phobia. self-reported or medicated hypercholesterolemia (use of cholesterol-lowering agents) and hypertension. inflammatory bowel syndrome and coeliac disease. medical records. 41 had left the study region. bipolar disorder (types I and II). Methods Participants Data were derived from the GOS male cohort. social phobia. obesity defined by a BMI >30 kg/m2[54].

9 (5. 23 (38.096). PA. 5 (8. 10 (6.46. 3 (1. SES.002).75. 95% CI 1. arrhythmia and valve and vascular diseases. which accounts for various socioeconomic parameters including high and low income. P = 0. and some measure of wealth (such as owning a car.30. Statistical analyses were performed using Minitab (version 16.027).41.29 to 4. Reported medication use was crossreferenced with the participants‟ list of medications or containers and was considered current if the participants reported u se at the time of assessment.05). Age was an effect modifier in the association between mood disorders and pulmonary disease. 5 (8. The relative SES of participants was ascertained by categorizing the sample into quintiles based on cutpoints for the study region [57-59]. adrenergic stimulants. fainting and dizzy spells. Mood disorders tended to be associated with musculoskeletal disease (OR 1. physical activity. 95% CI 1. SES. Other measures Smoking status. syncope and seizures (OR 2. 95% CI 0. Among older men (≥60 years).9%) mood disorder due to a GMC and 2 (1. 95% CI 1.48 to 3. State College. was also based on self-report.002) and liver disorders (OR 2. Models were adjusted for age (model I) and then for age. those with mood disorder due to GMC were included in all analyses. Tobacco smoking was documented and grouped as current or not.77. anti-arrhythmic. ischaemic cardiomyopathy. Statistical analysis Differences in characteristics between those with and without mood and anxiety disorders were compared using chi-square analyses for discrete variables and Mann–Whitney for non-parametric continuous variables. therefore. These values inform the Index of Relative Socioeconomic Advantage/Disadvantage (IRSAD).008).6%) minor depression. 3 (1. alcohol consumption and smoking status (Table 2). In order to determine socioeconomic status (SES).47.007).43. Interactions between exposure variables were examined. Participants with a history of mood or anxiety disorder were younger than those without.69.3%) dysthymia. alcohol consumption and smoking status (model II).3%) obsessive compulsive disorder and 3 (5%) anxiety disorder not otherwise specified. cardiac inotropic agents. Self-reported migraine and other recurrent headaches were grouped together. 95% CI 0.9%) were identified with major depressive disorder. USA). ABS software was used to determine the Socio-Economic Index for Areas (SEIFA) from the 2006 ABS Census data. no differences in outcome were detected.45 to 5. Lifetime pulmonary disease included self-reported asthma. residential addresses were matched to the corresponding Australian Bureau of Statistics (ABS) Census Collection District (CCD). thereby providing a single measure to rank the level of disadvantage at the area level. High medical burden was classified as a diagnosis of three or more conditions listed above. P = 0.05 were accepted as significant (including interaction terms).80. Minitab. Values ofP <0.3%) substance induced mood disorder. P = 0. blackouts. diuretics.3%) met criteria for panic disorder. Alcohol consumption (average grams consumed daily over a 12 month period) was determined by a validated food frequency questionnaire [55].16 to 2. These relationships remained significant after further adjustment for BMI. Of the 60 participants (12. Psoriasis was defined by self-report. hepatitis.003). P = 0.41.21. a lifetime history of mood disorder was associated with increased odds of pulmonary disease (age-adjusted OR 2.93 to 2. Results A total of 160 men (17%) had a lifetime history of mood disorder. 7 (11. Each analysis was performed with and without participants with mood disorders due to GMC.31 to 3. or number of bedrooms in a dwelling). SEIFA values indicate the level of advantage or disadvantage within each CCD. Characteristics for the total group and men with or without lifetime history of mood disorder and anxiety disorder Mood disorder After adjustments for age. of which 139 (86. not of the individual subject. other cardiovascular agents) stroke.9%) bipolar disorder. small geographic areas that incorporate approximately 250 houses [56].98. 95% CI 1.detailed above) or a combination of self-reported or medicated (beta-adrenergic blocking agents. Men were classified as active if they participated in light to vigorous activity on a regular basis. 6 (10%) social phobia. BMI. the type of occupation from unskilled employment to professional positions.09. educational attainment ranging from no qualifications to a tertiary degree or higher. P = 0. mood disorder was associated with increased odds of GORD (OR 2. 95% CI 1. cirrhosis and liver failure. SEIFA values summarize the characteristics of subjects within an area. a history of mood disorder was associated with increased odds of high medical burden (age-adjusted OR 1. 15 (25%) post-traumatic stress disorder. Logistic regression was used to calculate odds ratios (OR) with 95% confidence interval (95% CI) of lifetime medical conditions for those with lifetime mood/anxiety disorders in comparison to those with no mood/anxiety disorder.41.3%) generalized anxiety disorder.40 to 4. no association was detected for those younger than 60 years ( P >0. 95% CI 1. medication use and physical activity level were obtained by self-report [51]. Furthermore. No .104) and gastrointestinal disorders (OR 1. Liver disease diagnosis. P = 0. physical activity. otherwise the groups were similar (Table 1). transient ischaemic accident (TIA). recurrent headaches (OR 2. antiangina.93 to 2.21.7%) specific phobia. Syncope and seizures included self-reported epilepsy.00. including fatty liver. emphysema and chronic bronchitis. P = 0. Cancer was defined as any malignant tumour including non-melanoma skin cancer.2%) with a history of anxiety disorder. P = 0. Table 1.

P = 0. SES.53. cancer and psoriasis in this sample of men.20 to 4. gastrointestinal disease (OR 2.77.67.24. 95% CI 2. physical activity. BMI. such as recurrent headaches and syncope and seizures. had an age and gender adjusted odds ratio of 2. [24] utilising data pooled from 18 general population surveys reported those identified with either non-comorbid depression or comorbid depression and anxiety. self-reported severe symptoms of recurrent heartburn or acid regurgitation [62]. Table 2. 95% CI 0.005). 95% CI 1. the association has been viewed according to the diathesis-stress model. thyroid disorders. for which there is no adequate physical explanation. focus site) to low mood [68]. neurological features. 95% CI 1. Scott et al. the relationship was not sustained following adjustment for demographic and behavioural confounders including drinking behaviour.5 and 4. respectively).032 and OR 2.association was observed between mood disorders and thyroid disorders. alcohol consumption and smoking status did not affect these outcomes (Table 2). Discussion Our population-based study reports associations between mood and anxiety disorders and physical illness.96 to 3. cardiovascular disease.83. blackouts and dizziness. with as many as 46% of the adult population reporting headaches. cardiovascular disease. Recently. physical activity. SES. mood and anxiety disorders were both associated with high medical burden. those identified with depressive. as there is little evidence linking specific epilepsy related factors (for example. respectively.63]. and psoriasis were more common among individuals with anxiety disorder.066). Anxiety disorder also tended to be associated with metabolic risk factors (OR 1.001). P = 0. GORD (OR 3. as measured by the Hospital Anxiety and Depression Scale. Depression has also been shown to co-occur with epilepsy. Importantly. Anxiety disorders presented a different profile. chronic bronchitis and emphysema are more common among older people and most likely contribute to the different pattern observed for older and younger men.43 to 5. were associated with undiagnosed psychiatric disorders.61]. the association persisted following correction for alcohol in our study.84. P= 0. a factor we could not explore[70]. metabolic risk factors.33. liver disorders and pulmonary diseases in older men). metabolic risk factors. with depression resulting from the chronic stress associated with the threat of recurring seizures. is well documented [22. metabolic risk factors.63 to 7. cardiovascular disease. pulmonary disease. anxiety disorder was associated with increased likelihood of thyroid disease (OR 5. cancer and psoriasis. In our sample.049. however. Our results confirm other population-based data investigating the association between mood disorder and GORD [60. only older men with a mood disorder were at increased risk of pulmonary disease.92.0. 11% migraines and 42% tension headaches [64]. P = 0. pulmonary disease can be considered in stages reflecting severity. our results support Morgan et al. We hypothesize . we did not detect a relationship between mood disorders and thyroid disorders. in particular. The relationship between lifetime anxiety disorder and metabolic risk factors and lifetime anxiety disorders and psoriasis reached significance following adjustment for age. as measured by the Composite International Diagnostic Interview. P = 0.14. Furthermore.003).82. In a sample of over 60. Mood disorders were associated with increased risk of many of the disease groups (GORD. although we speculate that the heterogeneous groupings we employed may have diluted associations in our sample population.22. 95% CI 1.to three-fold increased risk of reflux symptoms.20.30]. recurrent headaches.53. In regard to syncope and seizures. syncope and seizures. The link between mood disorders and cardiovascular disease. however.01 to 7. Mood disorders and symptomology have previously been shown to be associated with low BMD and subsequent fracture. mood disorders have been shown to be associated with gastrointestinal disorders in both clinical and population-based samples [72-74]. 95% CI 1. mood disorders have also been shown consistently to be more prevalent in patients with GORD in clinical practice[15.66]. for chronic headaches. thyroid disorders and metabolic risk factors. 95% CI 1. Similarly. Musculoskeletal and gastrointestinal disorders tended to be associated with mood disorders. P = 0.29. Wilhelm and colleagues [69] reported an association between depression and liver pathology in a populationbased survey of 10. gastrointestinal disease.00 to 7. GORD. Excessive alcohol consumption is known to be highly correlated with liver disease [21].000 participants residing in Norway. Age-adjusted (model I) and fully-adjusted (model II) odds ratios for medical comorbidities in men with lifetime history of mood and anxiety disorders Anxiety disorder After adjustment for age. alcohol consumption and smoking status (OR 2.10. anxiety and comorbid symptoms. liver disorders or cancer. where both biological and medication related processes are thought to play a role [71]. There were no associations detected between anxiety disorders and musculoskeletal disease. Akin to our results. had a two. Further adjustment for BMI.011) and high medical burden (OR 2. Similar to our findings and other population-based studies [65.641 adults. In contrast to other population-based studies.07 to 4. P<0. Chronic headaches are high prevalence disorders. Although the association between asthma and mood disorder is evident across the full adult age range [23]. Moreover. [67] who reported fainting.

Abbreviations ABS: Australian Bureau of Statistics. as previously discussed. including psoriasis. BMI: Body mass index. potentially reducing healthcare utilisation. DXA: Dual energy X-ray absorptiometry. a common issue where study participants are required to be healthy enough to attend the research centre. However. which have been shown to have a negative impact on clinical outcomes [77]. our observations must be considered with caution. SCID-I/NP: Structured clinical interview for DSM-IV-TR research version. hypercholesterolemia and obesity. in contrast to previous studies that have mainly focused on older patients. BMD: Bone mineral density. which have been demonstrated to generally agree with medical record data [78]. which may impact differently on psychological status. and the consideration of several possible confounding factors. diabetes. and Sharon L Brennan have no conflicts of interest. where possible this was confirmed by medical records. SES: Socioeconomic status. Finally. The cross-sectional nature of the study does not allow verification of a cause-effect relationship between mental and physical illness. SEIFA: Socioeconomic Index for Areas. [35] reported an increased risk of thyroid disorders in patients with generalised anxiety disorder. IRSAD: Index of Relative Socioeconomic Advantage/Disadvantage. disease grouping and data collection limited our ability to identify the degree of illness severity (that is. as well as poorer self-care and treatment adherence. Furthermore.that these results may be influenced by a healthy participant bias. Similarly. CI: Confidence interval. Thyroid disorders and metabolic risk factors have also been associated with anxiety disorders. Conclusion Our study provides further population-based evidence supporting the link between mental and physical illness in men. These findings are consistent with previous studies showing both depression and anxiety to be associated with an increased number of somatic conditions [5. nonpatient edition. Anxiety has been previously considered to be a non-disease related factor that impacts negatively on quality of life associated with GORD [75] and. GMC: General medical condition. In our study. Although many of these disorders were based on self-report of a physician‟s diagnosis. which in turn increase medical burden [36]. longitudinal studies are needed to determine the directionality of this relationship. 4th edition. medication use or clinical data. GORD and gastrointestinal disorders were also associated with anxiety disorders. GORD: Gastro oesophageal reflux disease. On the other hand. An age interaction was only evident when exploring the association between mood disorders and pulmonary disease. social phobia or panic disorder and recommended screening for thyroid dysfunction in patients with anxiety disorders. A major strength of our study is that the sample population spans the full adult age range. diagnoses were made utilising semistructured clinical interviews (SCID-IV).76]. As with mood disorders. or the presence of physical pathologies due to predisposing psychiatric disorders. Why this association is seen with anxiety disorders only in our sample population is unclear. Further strengths of the study include the measurement of mood and anxiety disorders. Competing interests Livia Sanna. DSM-IV-TR: Diagnostic and Statistical Manual for Mental Disorders. data from the present study concur with previous studies indicating that those with anxiety disorder have higher rates of gastrointestinal disorders [34]. Paolo Girardi has in . we were unable to make a distinction between diseases with an episodic course and those with ongoing symptoms. Furthermore. text revision. including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript. Harter and colleagues [34] demonstrated that patients with a lifetime history of generalised anxiety disorder or panic disorder reported significantly higher lifetime prevalence rates for dermatological disorders. TIA: Transient ischaemic accident. It is plausible that this relationship may be due to mood and anxiety disorders causing physiological changes. thus the relationship between mental illness and each of the physical conditions was consistent across all ages. a gold standard tool. Psoriasis has been previously associated with anxiety disorders. Recognising the link between these illnesses is important in order to manage the development of mood and anxiety disorders in response to a medical condition. Amanda L Stuart. severe stroke. which Culpepper and colleagues reported to be all likely due to a prolonged stress response in patients with untreated anxiety disorder [2]. Integrating treatment approaches and monitoring comorbidity of mental and physical illnesses is likely to improve disease course and outcome as well as enhance patients‟ functional and health status. is commonly observed within population-based samples reporting GORD symptoms [62]. causing functional impairment. GOS: Geelong Osteoporosis Study. Mark A Kotowicz. such as hypertension. Simon et al. an increased physical burden. anxiety was linked to a combination of the most common risk factors for metabolic disorders. TIA and medically controlled hypertension were grouped) or time since onset of the physical condition. Ascertaining medical conditions by a self-report checklist may be compromised by imperfect recall and response bias error. may result in the development of an anxiety and/or mood disorder. Our data suggest that both mood and anxiety disorders are associated with overall medical burden.

The University of Melbourne.9%. Sanofi Synthelabo. Results A total of 3. LJW took part in the conception and design of the study. Finally.4% with the AQ.1%. Glaxo SmithKline. ANZ Charitable Trust. P = 0. Conclusions The use of illicit and prescription drugs for CE or ME is an underestimated phenomenon among surgeons which is generally attributable to high workload.266.000 to 1.the past three years received research support from Lilly and Janssen.9% of all surveyed surgeons confessed to having used a prescription or illicit drug exclusively for CE at least once during lifetime. Pfizer. 95% CI. 95% confidence interval (CI). However.640. Bristol Myers Squibb. Organon. Lundbeck and Pfizer and a paid speaker for Astra Zeneca. CRC for Mental Health.543. Eli Lilly. were positively associated with the use of drugs for CE or ME. 15. The RRT guarantees a high degree of anonymity and confidentiality when a person is asked about stigmatizing issues.005). Pfizer. Michael Berk has received Grant/Research Support from the NIH.306 questionnaires were distributed and 1. which can put patients at risk. Stanley Medical Research Institute. Janssen Cilag. Eli Lilly and Sanofi-Aventis and funding from the Geelong Region Medical Research Foundation. 95% CI.105). Servier and Astra Zeneca. such as drug abuse.4%).666.099) as compared to 2. Pfizer.5-fold higher than that of the AQ (19. interpretation of the analysis and critically revised the manuscript. Novartis. Authors’ contributions LS and ALS took part in the conception and design of the study. Coping strategies should be taught during medical education. P = 0. He has been a paid consultant for Astra Zeneca. 1. Amgen (Europe) GmBH and the NHMRC. P = 0. participated in advisory boards for Lilly. 1. burnout or symptoms of depression. Methods Surgeons who attended five international conferences in 2011 were surveyed with an anonymous self-report questionnaire (AQ) regarding the use of prescription or illicit drugs for CE and ME and factors associated with their use. Simons Foundation.290. the Ronald Geoffrey Arnott Foundation.3% to 19. 95% CI.05) or in private life (OR: 1. Such intake of drugs is associated with attempts to counteract fatigue and loss of concentration. Eli Lilly. Mayne Pharma. and private stress.9%. N = 1. the Dairy Research and Development Corporation. Lundbeck. data cleaning and statistical analysis. The Randomized Response Technique (RRT) was used in addition.337.091 to 1. perceived workload. 8. acquisition of the data. concentration deficits. Janssen Cilag. Keywords: . MBF. 95% CI. and gross income (OR: 1. drug use for CE may lead to addiction and to overestimation of one‟s own capabili ties. Deakin University and the NHMRC. Beyond Blue. Organon.9% to 23. and Schering. As one would expect. Geelong Medical Research Foundation. 11. NHMRC. Solvay and Wyeth. All authors read and approved the final manuscript. Julie A Pasco has received speaker fees from Amgen.0%.145 entered statistical analysis (response rate: 36. An even larger discrepancy between the RRT and AQ was observed for the use of antidepressants with a 6-fold higher prevalence (15. Bristol Myers Squibb. distress. MB. The University of Melbourne. The prevalence of surgeons taking pharmacological cognitive enhancement (CE) or mood enhancement (ME) drugs has not been systematically assessed so far. the American Society for Bone and Mineral Research. Barwon Health. Eli Lilly. Acknowledgments The study was funded by Background Surgeons are usually exposed to high workloads leading to fatigue and stress. Lana J Williams has received Grant/Research support from Eli Lilly. Perpetual Trustees. and received honoraria from Lilly and Organon. the prevalence rate assessed by RRT was approximately 2. This not only increases the likelihood of mistakes during surgery but also puts pressure on surgeons to use drugs to counteract fatigue. interpretation of the analysis. N = 1.02). Glaxo SmithKline. Bristol Myers Squibb. critically revised and supervised the writing of the manuscript. Organon. Glaxo SmithKline. According to the AQ. PG and SLB took part in the interpretation of data and critically revised the manuscript. JAP and MAK took part in the conception and design of the study.038 to 1. 1. logistic regression analysis revealed that pressure to perform at work (odds ratio (OR): 1. interpretation of the analysis and took primary responsibility for writing the manuscript.

321.27]. sleep deprivation (especially in shift work). Furthermore. We estimated prevalence rates of CE and ME among surgeons using AQ and RRT. Participants were asked to drop the questionnaire into black boxes after having filled in the questionnaire anonymously.). for example. gender. methylphenidate (MPH)).11].31]. the use of prescription and illicit drugs could be a coping strategy to manage poor working conditions. This present survey reports the first data on prescription and illicit drug use for CE and ME among surgeons. burnout or symptoms of depression by the use of enhancing substances. surgeons can come under pressure to counteract fatigue. A recent study using the Randomized Response Technique (RRT) shows prevalence rates of even 20% for the use of prescription and illicit drugs among university students for CE [33]. together with usage association factors. Warren and colleagues discussed possible reasons for surgeons to consider the use of substances for cognitive enhancement (CE) or mood enhancement (ME). (ADHD)) who had physicians‟ prescriptions for any drug were excluded. an AQ about the use of prescription and illicit drugs for enhancing cognitive functions or mood was developed and distributed to the participants during the conferences. Furthermore. depression. although previous studies showed no immediate or delayed mood enhancing effect [18. napping. concentration deficits. Thus. Beyond that. age of first use.306 German-speaking surgeons who attended five international conferences of the German Society of Surgery (Deutsche Gesellschaft für Chirurgie). Therefore. The study was carried out according to the Principles for Medical Research Involving Human Subjects according to the Declaration of Helsinki. Based on previous research about prescription and illicit drugs for CE.5% decided to use caffeine tablets for CE [27.29]. antidepressants such as selective serotonin reuptake inhibitors (SSRI) are known to be used for ME. Participants gave informed consent by returning the questionnaire and were informed about this procedure in the introduction section of the questionnaire. Nevertheless. However. (psycho-) stimulants (amphetamines (AMPH). The study was approved by the local Ethics Committee (Landesärztekammer Rheinland-Pfalz) (No. Furthermore.9% for illicit stimulants (AMPH) whereas 10. 837. and so on) or the use of coffee which may cause tachycardia and worsening tremor [10. we asked for the age of first use. Previous studies among high school and university students using anonymous questionnaires (AQ) have shown prevalence rates for the use of prescription stimulants (MPH) to be 0. reduction of cognitive performance due to sleep deprivation is a common problem in shift work. last month and last week (frequency). methamphetamine. the AQ included questions about potential risk factors associated with usage of drugs and questions about biometrical parameters (for example. and confidentiality when a person is prompted to answer sensitive questions about socially undesirable or illicit behavior [34-38]. the data of participants with self-reported psychiatric disorders (for example. last year. to some extent. Prescription drug(s). and so on.8% and 2. patient safety or surgeons‟ well-being [10]. Nevertheless. those who had been were excluded from a second participation. In order to maintain high cognitive performance. and even to higher rates of suicide [1-5]. Stimulants and modafinil have been demonstrated to attenuate disruption in cognitive performance and mood during night-shift work and sleep deprivation [20-25]. there is no scientific evidence so far that either external or internal pressure leads to the consumption of prescription or illicit drugs to cope with these stressors. Beyond that. We were mainly interested in healthy participants using drugs specifically for CE or ME. anonymity.Cognitive enhancement. This increases the likelihood of mistakes during surgery [6-9]. Methods The data for this study resulted from a survey conducted in 2011 among 3. distress.08 (6318)). Surgeon(s). Previous studies revealed the use of stimulants and modafinil for CE purposes among students having at least an immediate provigilant effect [14. . Studies using RRT guarantee an especially high degree of privacy. fatigue. relaxation techniques. it is unclear whether ME affects only mood or also other aspects such as self-esteem or self-representation. After the first conference. performance enhancing drugs may be especially attractive to surgeons due to the fact that they appear to be a time-saving and. However. modafinil as well as antidementives and antidepressants have no consistent effects for CE or ME in healthy non sleep-deprived subjects [12-20].30. an effective and easy alternative to more time-consuming coping strategies (for example. modafinil or beta blockers for CE purposes among participating academics[32]. an online poll conducted by the journal Nature depicts a lifetime prevalence rate of 20% for stimulants. this procedure was approved by the above mentioned local Ethics Committee. including such issues as internal (to maximize one´s own potential) and external pressure (by the employer or the public).28. potential participants were asked if they had been assessed before.26. burnout. attention deficit/hyperactivity disorder. The AQ asked about the „non-medical use of stimulants with the particular intention of CE‟ and „the non-medical use of antidepressants with the particular intention of ME‟ during lifetime. sleep. age. Stimulant(s) Background Surgeons are often exposed to an excessive workload leading to mental and physical exhaustion.

25). employment status.25≈0.25/365.1993=19.1% of all participants received this question).25/365. pressure to perform subjectively evaluated as burdensome and pressure to perform subjectively evaluated as harmful to health. For the regression. Forexamplefora=328/1105≈0.25) received the sensitive question (B) and 32. family status. 11 for having a physician`s prescription for drugs because of mental disorders and 39 (pair wise) for giving . Statistical analyses were performed with SPSS for Windows.05%. pressure in private life. hours of work. The probability of answering the non-sensitive question (A) with „yes‟ is πn = 49.25/365. gross income. 67. age.).25≈0.0205=2.p=245.25) the non-sensitive question (A). πˆs=a−(1−p)πNp the proportion of „yes‟ responses with respect to the sensitive questions can be estimated from proportion a of total „yes‟ responses in the sample.91%to23. 61 had to be excluded: 9 for not being a physician. AQ questions were analyzed using a multiple logistic regression analysis. Ordinal variables with five or more categories (pressure to perform at work. Therefore. Is this birthday in the first third of a month (1st to 10th day)? If yes.93%. if no. p denotes the probability of receiving the sensitive question (Question B. questions were presented to participants as follows: Please consider a certain birthday (yours.thisy ieldsπˆs=328/1105−(1−245.93±1. The regression has been analyzed by referring to cases without missing values (complete case analysis). Table 1 shows the variables included for the regression after forward selection.25/365.25/356.05.6% (181.25245.25)181.9% (120/365. etc.thatis. living with children. Means are given with their corresponding standard deviation (SD) (mean ± SE) and Clopper-Pearson confidence intervals (95% CI). illicit Amphetamines. your mother‟s. The variables which were analyzed as potential multivariable predictors of the use of prescription or illicit drugs for CE before forward selection were (available parameters): pressure to perform at work. Risk factors for the use of prescription or illicit drugs for CE among surgeons by multivariable analysis Results Participants´ characteristics A total of 1. self-presentation?‟ The interviewers are not able to know which question the respondent has to answer.SE(πˆs)=0. A 95% confidence interval (CI) for the unknown prevalence can be computed from the sampling variance Var(πˆs)=a(1−a)np2 where n denotes the sample size [33. we modified Question B as follows: „Did you ever use antidepressants without medical need for enhancing your mood and/or self-esteem.000419−−−−−−−√≈0.0.25≈0. pressure in private life. and so on) without a medical need for cognitive enhancement? Note that only you know which of the two questions you will answer o Yes o No For assessing the use of antidepressants for mood enhancement.30.39]. the variable selection procedure was performed by using stepwise forward selection with a selection level of 0.25)2≈0.306 distributed questionnaires were returned. Table 1.Thusthe95%confidencein tervalis19.The AQ contained questions about the use of drugs for CE and ME as well as questions using RRT. gender.g. participants can reply honestly without compromising themselves. There are no further variables for which we adjust.204 (36.50.andn=1105.1% (245. evaluation of career opportunities.962. After a brief introduction about the RRT stressing the anonymity of this technique. all variables which significantly influence the drug use for CE/ ME are listed in this table. satisfaction with professional success.25/365.67. Of all participants. please proceed to Question B.05. pressure to perform subjectively evaluated as burdensome and pressure to perform subjectively evaluated as harmful to health) were treated as continuous variables.rangesfrom15. Version 17. Of the participants. p = 67. The results are presented as odds ratio (ORs) with confidence limits and P-values. gross income. Methylphenidate.Var(πˆs)=328/1105(1−328/1105)1 105(245.25/365.000419. please proceed to Question A. type of employer. Modafinil.95%.4%) of 3.πN=181.25/365. Question A: Is this birthday in the first half of the year (prior to the first of July)? Question B: Did you ever use prescription and/or illicit drugs (e.

DAK.9%. Of the respondents. On the other hand.incomplete answers. there exists a severe paucity of data about drug use for CE among employed adults.145 surgeons entered the final statistical analysis. see Table 2.5% senior surgeons and 22. online surveyed via e-mail 20.32. Logistic regression analysis suggests that other factors play no role in the use of prescription and illicit drugs: gender ( P = 0.to 50-years old) with a response rate of 15%.3% to 19.151). prescription or illicit drug use for CE or ME was associated with the pressure to perform at work or in private life and with gross income. Thus.252.133 to 1.9% of all surveyed surgeons used prescription or illicit drugs with the particular intention of CE by AQ.010 to 1. 95% CI: 1.543. living with children (P= 0.1%.2 on a 6-point Likert scale. the data of 1. the non-representative DAK study showed a lifetime prevalence rate of 5%. 8. it is useful to consider several factors as follows: With the exception of the present study.042) or in private life (OR: 1. Table 2.9% of the surgeons confessed to having used a prescription or illicit drug exclusively for CE at least once during their lifetime. as well as for age of first use using the AQ. An even larger discrepancy between the RRT and AQ was observed for the use of antidepressants with a 6-fold higher prevalence rate.4% were surgeons in training. In contrast.099) as compared to 2.3) and moderate to severely harmful (2. 2.8 ± 13.327.406. Table 5. there are substantial differences regarding the prevalence rate in the present study.015 to 1. Age of first use did not differ significantly between prevalence rates. AQ results for prevalence rates of the use of antidepressants for ME Prevalence rates of CE and ME measured by AQ compared to the randomized response technique (RRT) Prevalence rates measured by the RRT are considerably higher than prevalence rates measured by AQ. 95% CI: 1. 15. Differences between last-year and last-month prevalence rates for CE and ME are small.3). The pressure to perform optimally at the job was estimated to be severe (3. For further details. whereas the difference between lifetime prevalence and last-year prevalence rate is remarkably higher. satisfaction with professional success (P = 0. a logistic regression analysis revealed that pressure to perform at work (OR: 1. 0 = not at all. 95% CI: 1.743.5-fold higher than the AQ estimate.7 ± 1.4% answered that they had already used antidepressants for ME whereas the RRT revealed a prevalence of 15.0%.9% (95% CI.809).9%. type of employer (P = 0. the corresponding RRT estimate was approximately 2.002) were positively associated with the use of drugs for CE or ME (see Table 4).4% with the AQ.6 ± 1. On the one hand. P = 0. Table 3. .744.698). Furthermore. which in turn is higher than last-month and last-week prevalence rates (see Table 3 and 4). there are significant differences compared with previous studies of drug use for performance enhancement. By contrast.620).9% to 23. 56.720). the RRT results showed a higher prevalence of 19. family status (P = 0. AQ results for prevalence rates of the use of prescription drugs + illicit drugs for CE Table 4. Without accurately distinguishing prescription and over-the-counter drugs. see Tables 3 and 4. n = 1. Table 5 shows that with AQ.27. Participants` characteristics Lifetime-prevalence of CE and ME is higher than last-year prevalence. judged to be moderate to severely burdensome (2. 15. AQ and RRT results for lifetime prevalence rates of prescription or illicit drug use for cognitive enhancement (CE) or mood enhancement (ME) Factors associated with the use of prescription and illicit drugs for CE/ ME Finally. 11. „cognitive enhancement‟ or „pharmacological neuroenhancement‟ regarding cognition (for example. Furthermore.036) and gross income (OR: 1. For more details on lifetime-. P = 0.40-42]).1% directors or deputy directors.26.105). 5 = very much). P = 0. last-month and last-week prevalence rates. that is. [10. employment status (P = 0. a German health insurance company.0 hours. using AQ.820). Regarding prevalence rates and associated factors. There are an increasing number of studies dealing with „academic performance enhancement‟. last-year. 19. age (P = 0.000 employed members (20. n= 1. Discussion The AQ results of this study indicate that 8. hours of work (P= 0. 21.366). The mean weekly workload was estimated to be 56. that is.822).3 ± 1.1% (95% CI. Participants were asked about their use of various substances for CE and mental well-being without medical need [43].829) and evaluation of career opportunities (P= 0.

MPH and modafinil are also the most prevalently used drugs in our survey. This is almost 5 to 6 years younger than among surgeons. followed by modafinil and beta blockers [32]. However. together with gross income. These rationales seem to be the same as among surgeons [1-5]. although. both studies excluded participants with ADHD or other psychiatric disorders where prescribed psychiatric medications were involved. Interestingly. Many other studies did not exclude these „patients‟. the use of CE drugs started substantially later in life. this association is not tenable for professional life in general. This may be one of the most important aspects when assessing pharmacological CE or ME. This may be associated with the older age of surgeons. Both surveys. We were able to show that pressure to perform at work or in private life. are positively associated with the use of prescription or illicit drugs for CE or ME and are the only factors associated with drug use for this purpose.0 years. „uneasiness‟. Such factors should be addressed in detail in further studies. The present study allows us for the first time to compare AQ questions with RRT questions in one single integrated survey about drug use.1% for CE and ME compared to the lower prevalence rates using direct questions. suggest higher risk behaviors in male compared to female subjects [33. In this respect. „to concentrate‟ and „improve alertness‟ have been salient participants‟ reas ons for misuse of stimulants.6% for illicit stimulants [27]. these results match the RRT results of our study. However. While we were not able to show a significant influence of gender on the use of potential CE. The literature is somewhat inconsistent on this subject. „anxiety‟.Stated reasons for usage were: „depressed mood‟. we found that prevalence rates for stimulants in the present study are slightly higher than in our earlier studen ts‟ survey. demonstrated that 20% of participants had already used prescription drugs for non-medical reasons to improve concentration and improve their focus for a specific task.9% for CE and 15. „nervousness‟. we cannot interpret this finding as a general proof of a direct causal relationship between feeling pressure and the use of CE substances. a non-random online poll by the journal Nature which unfortunately did not specify respondents. AMPH) and 2. MPH was the most popular substance. „fatigue‟. Beyond that.9% and 15. only students‟ substance use for academic performance enhancement has been surveyed. This strengthens the validity of the RRT prevalence rates of 19. the surveyed groups are not directly comparable.or ME-substances. For the illicit use of prescription ADHD medications among college students. and „p roblems of concentration‟ [43]. our previous study among 1. decades before. a previous meta-analysis of 38 RRT validation studies by Lensvelt-Mulders and colleagues reported that RRT provides more valid data than other survey methods. Further hypothesized factors were revealed to play no role in the use of prescription or illicit drugs for CE. revealing higher prevalence rates including those where participants misuse their own prescribed medication [40]. Regarding lifetime prevalence. However. However. admittedly. Outside of these particular studies. This agrees with the results of our study. DeSantis and colleagues found a significantly higher prevalence rate in male than in female students [46].29% for prescription stimulants (MPH. These results show a comparable prevalence to those of the present study. study participants are 43 years old (mean) which may imply that two decades ago.500 high school and university students (over 18 years) using AQ. However. whereas Teter and colleagues found no gender differences regarding prescription stimulant use among college students [47]. Our results do not confirm this finding. both potentially highly stigmatizing subjects.000 participants revealing a past-year prevalence rate of 5% to 9% in grade and high schools and 5% to 35% in college-age individuals [40]. who themselves had been medical students and later trainee surgeons. Furthermore. The most recent study about CE among 2.45]. However. . For this important meta-analysis which included many significant studies about stimulant misuse among students. This underlines the relevance of the survey method in general.1% for ME [49]. Surveyed surgeons answered that their age of first use of prescription or illicit drugs for CE was 24. A previous study by our research group among 1. studies focusing upon this particular association in the context of a different surveyed group from that of the present study. all these studies only allow an indirect comparison of different survey methods. Furthermore. Partridge and colleagues revealed that a high percentage of the public media portrayed CE as common which accords with our high prevalence rate for CE [42].500 students revealed 17 to 18 years to be the age of first use of prescription or illicit drugs for CE [27]. In particular. Dietz and colleagues revealed that significantly more male than female students used prescribed or illicit drugs for CE. guarantee a relatively high level of anonymity. assessed lifetime prevalence rates of 1. „memory deficits‟. A meta-analysis by Wilens and colleagues examining prevalence rates of stimulant misuse included 21 US studies with 113. online polls as well as the present RRT study. We found a positive association of pressure to perform at work or in private life and gross income with the use of drugs for CE. CE is only a side aspect of the study. At least to our present knowledge. This explains the substantially higher (past-year) prevalence rate compared to the present study. Beyond that.48]. Methodologically.600 students using the RRT shows a one-year prevalence rate of 20% for the non-medical use of prescription and illicit drugs for CE [33]. this finding contrasts with that of our survey study of the same group among university students leading to the assumption of a „phantom debate‟ [44. first use of antidepressants for ME was 39 years (mean) compared to an average of 24-yearold participants using CE drugs for the first time. there are no data from empirical studies which allow meaningful comparison with our data: studies of stude nts‟ drug use focusing on CE as well as the previously cited Nature poll did not examine these factors. it strengthens the validity and reliability of the higher RRT results of 19. Furthermore.

On the other hand. for example. A survey study by Partridge and colleagues showed that university students already seem to have a realistic idea of the effects and side-effects [44]. Thus. Conclusions The use of illicit and prescription drugs for CE or ME is an underestimated phenomenon among surgeons. vigilance. The present results indicate that about 15% to 20% of surgeons have used drugs for CE or ME at least once during their lifetimes. Furthermore. misuse. One would expect surgeons to know these limited effects and to avoid the use of these prescription and illicit drugs for CE. stimulants and modafinil have enhancing effects on simple cognitive domains. However. together with careful studies of drug (side) effects to substantiate discussions of ethical and legislative aspects. atomoxetine. beyond that. such as fatigue. We can only speculate about the reasons. We obtained a response rate of 36. According to randomized controlled trials (RCTs). surgeons may not know the missing pro-cognitive effects or overestimate the effects of such drugs. Both factors may be harmful for users. 15% have already used antidepressants for ME. On the one hand. at least regarding prescription drugs.[27. antidementive drugs and antidepressants. for example. we asked surgeons for the non-medical use of stimulants for CE and antidepressants for ME. reviews and meta-analyses there are almost no pro-cognitive effects regarding normal healthy non-sleep-deprived subjects on simple and higher cognitive domains [12-17]. Beyond that. knowledge – and even overestimation – about pro-cognitive effects in sleep-deprived subjects only confirms that sleep deprivation is a common phenomenon among surgeons. stimulants have subjective „pro-effects‟ [12. reviews and meta-analyses (for example. Since many more male subjects participated in our study. we did not specifically ask for the context of use. a response rate of 36. so that we were hardly able to control potential participants‟ behavior concerning the return of the questionnaires to the black boxes provided. I) information about the restricted usefulness and risks of the use should be . substance use – or even misuse – can be considered a highly stigmatizing subject leading to low response rates. addiction) of such drugs seem to be underestimated by users. Beyond that. Also. amphetamine tablets (for example. A number of limitations of the present study are worth identifying here. Nevertheless. The fact that sleep deprivation leads to clearer results supports this hypothesis [12]. Furthermore. However. the side effects and effects of long-term use (for example. furthermore. pro-cognitive effects on higher cognitive domains are very limited. the response rate of 36. This may be attributed to high workload and perceived work-related and private stress. Adderall®).4% may be considered relatively high and comparable to other studies assessing stigmatizing subjects with anonymous questionnaires [36].19-25].18]. cardiologic side effects. One can presume that the effects on higher cognitive skills are indirect effects which are mediated via simple cognitive skills. we do not know in particular whether subjects with more positive attitudes or more negative attitudes on the topic participated disproportionately which may have caused a response bias. gastro-intestinal side effects.4% which is a low response rate compared to previous studies using RRT (for example. Beyond severe side-effects which are described in package-inserts accompanying these drugs and the results of RCTs. which may indicate missing knowledge about the effects of antidepressants in normal healthy subjects or overestimation of the putative effects. This may explain why we did not find gender differences in prevalence rates whereas earlier studies including our own have partly shown that male subjects more often used drugs for CE than female subjects. stimulants can cause addiction and further addictive behavior. vigilance. and so on). questionnaires were distributed during conference breaks in huge buildings. whether surgeons had used it directly prior to surgical interventions. jitteriness. antidepressants (such as SSRI) have no mood enhancing effect in normal healthy subjects at all [12. agitation. stimulant use may put patients at risk given the fact that previous research has shown that stimulants may lead to overestimation of one‟s own capabilities. However. modafinil. Another important factor is the likelihood of a participation bias: Since the response rate is only one third. hypertension. every fifth surgeon has already used these drugs. such as stomach ache. a potential gender bias exists. Another important factor is the side-effect profile and safety risks of amphetamines which have to be considered. such as methylphenidate.33]).Surgeons should know about the effects and side-effects of the substances used for CE or ME. Substances such as modafinil seem to counteract fatigue and loss of concentration and thus may provide simple pharmacological help for stressed surgeons. there are slightly pro-cognitive effects on higher cognitive domains and. such as tachycardia. diarrhea.4% together with the non-random sample limits the generalizability of our findings. However. the misuse of illicit drugs and prescription drugs without prescription is a federal offense. Nevertheless. Beyond that. However. The contemporary debate on cognition -enhancing drugs requires a broader data base on consumption rates in populations at risk. Therefore.14. psychomotor skills and reaction time in sleep-deprived subjects.

provided. to test the performance of our gene signature for patient outcome prediction. relevant coping strategies has to become an integral part of medical education. a genome-wide portrait on the contributing molecular factors to invasion heterogeneity is lacking. he is trained in mathematical psychology and s tatistics and is a member of WADA‟s doping prevalence research group. Media and Sports. However. Germany. Acknowledgements Caroline McFarlane and Teresina Tassone-Steiger. both medical students. CE: Pharmacological cognitive enhancement. William Keenan for proofreading the manuscript. expert in sports medicine and an active member of the National and World Anti Doping Agency (NADA and WADA). and III) information about. CB and KL belong to the Department of Psychiatry and Psychotherapy. we collected data from four independent drug-testing experiments to validate our findings on compound response prediction. We identified invasion-associated (IA) genes by correlating our invasion profiling data with the Affymetrix gene expression data on NCI-60. drafting. and the development of. KL and PS monitored data collection. IH is a mathematician and expert in statistics belonging to the Institute of Medical Biostatistics. KL and AGF are psychiatrists. Abbreviations AMPH: Amphetamine(s). ME: Pharmacological mood enhancement. Germany. CB is a sociologist. and revising the manuscript. MPH: Methylphenidate. PS and RU analyzed the data of the Randomized Response Technique (RRT) which was cross-checked by IH. Johannes Gutenberg-University Mainz. Faculty of Social Science. The BMBF had no influence on the content of this manuscript. Authors’ information AGF. Cancer cells also show a broad range of invasion ability. Methods Our lab performed an invasion assay on the NCI-60 panel. KL and PS participated in the conception and design of the study. CB and in particular IH analyzed and checked the AQ data. Authors’ contributions AGF. focusing on the IA genes. PD. CI: Confidence interval. we obtained published clinical and molecular data from two recent adjuvant chemotherapy cohorts. We then employed the recently released chemosensitivity data of 99 anti-cancer drugs of known mechanism to investigate the gene-drug correlation. Afterwards. II) guidelines on how to deal with drug use among employees who have contact with patients have to be provided. All authors read and approved the final manuscript. RU belongs to the Department of Psychology. AGF. one on lung cancer and one on breast cancer. Epidemiology and Informatics (IMBEI) of the University Medical Center of the Johannes-Gutenberg University Mainz. RRT: Randomized Response Technique. University of Tübingen. Finally. one powerful approach is to employ the panel of 60 human cancer cell lines developed by the National Cancer Institute (NCI). Dr. PD and PS belong to the Department of Sports Medicine. To gain insight on the genetic mechanism of drug sensitivity. Germany. University Medical Centre Mainz. OR: Odds ratio. ADHD: Attention deficit/ hyperactivity disorder. All authors participated in data interpretation. Chemosensitivity and tumor metastasis are two primary issues in cancer management. The authors want to thank Prof. PS is trained in internal medicine. Financial project funding: German ministry of Research and Education (BMBF) No. Results . Cancer cells often exhibit a wide range of sensitivity to anti-cancer compounds. AGF. AQ: Anonymous questionnaire. KL. 01GP0807 (2009 – 2011). Rehabilitation and Disease Prevention. RCT: Randomized controlled trial. SD: Standard deviation. contributed to the study by distributing questionnaires during the conferences. SSRI: Selective serotonin reuptake inhibitor Competing interests The authors declare that they have no competing interests.

(b) Drug sensitivity prediction by tumor-invasion markers: to evaluate how the expression levels of IA genes in a tumor are likely to be indicative of the tumor‟s resistance or responsiveness to an anti-cancer drug. The eight-gene signature predicted relapse-free survival for the lung and breast cancer patients (log-rank P = 0. Both drug sensitivity and gene expression profiles of the NCI-60 panel are available from the public domain. the biological mechanism of the genes involved in such cohort-initiated genomic predictors may not be easy to elucidate. Despite this. (c) Drug discovery with predictable sensitivity: to find anti-cancer drugs whose efficacies correlate with tumor-invasion potential and can be predicted by tumor-invasion markers. Like the drug-sensitivity heterogeneity. Invasion. USA) and spotted cDNA array technology [2]. a characterization of genes associated with both the invasion potential and drug-sensitivity heterogeneity is lacking. the more challenging problem of how to translate the elucidated relationship for clinical outcome prediction still awaits more studies. are helpful in elucidating the complex relationship between drug responsiveness and gene expression. More subtly. The eight-gene signature features the cancer hallmark epidermal growth factor receptor (EGFR) and genes involved in cell adhesion.1) and 1. The molecular characteristics of these cell lines have been subjected to various DNA microarray studies using both Affymetrix (Santa Clara.76) respectively. Keywords: NCI-60. renal cancer.81 (95% CI = 1. 0. However. we obtained a subset of IA genes whose expression levels correlated with drug-sensitivity profiles. metastasis is another major issue in studying treatment efficacy for many cancers. NCI-60 is a diverse panel of 60 cell lines used by the Development Therapeutics Program (DTP) of the National Cancer Institute (NCI) to screen more than 100. Metastasis. breast cancer and prostate cancers.00021). central nervous system cancer. we found 633 IA genes from the invasion-gene expression correlation study. Chemotherapy Background Gene-expression profiling of tumors from patient cohorts has been used to develop gene signatures for clinical outcome prediction. This eight-gene signature (derived from NCI-60) for chemosensitivity prediction was validated by a total of 107 independent drug tests on 78 tumor cell lines. a signature combining estrogen receptor (ER) status and predicted chemo/endocrine responsiveness succeeded in identifying patients with high probability of survival following taxane and anthracycline chemotherapy [1]. However. in vitro chemosensitivity experiments on cancer cell lines. ITGA3. it is possible that the clinical outcome of chemotherapy may hinge on the growthinhibition of the more invasive cells rather than the less invasive cells in the neoplasm. ovarian cancer. RAI14. melanoma. for each of the 99 drugs.First. however. such as the NCI-60 cell line panel. We report the finding of a unique signature that predicts chemotherapy survival for both lung and breast cancer. We identified a set of eight genes ( EGFR.0263.000 compounds since 1990 [2-5].76 to 16. migration. AHNAK. colon cancer. In addition to chemosensitivity. docetaxel. Then.33 (95% CI = 1. So we conducted the invasion assay for 53 solid tumor cell lines from the NCI-60 panel in our lab. there is no public invasion phenotype data for NCI-60. lung cancer. tumor growth and progression. including invasive breast and lung cancer. invasion. erlotinib. There are four aims in this study: (a) Molecular markers of tumor invasion potential: to identify the set of IA genes whose expression levels are likely to be indicative of the invasion potential of a tumor. GLS. CA. . IL32 and NNMT) showing significant gene-drug correlation with paclitaxel. Conclusions Our study sheds light on the intricate three-way interplay among gene expression. MYLK. These human cancer cell lines are derived from patients with leukemia. tumor cells often exhibit a wide range of invasion ability. (d) Clinical validation: to demonstrate the use of the IA gene signature for predicting clinical outcome. Such invasion heterogeneity may exist not only between different cancer types. Multivariate Cox regression yielded a hazard ratio of our signature of 5. Augmenting the NCI-60 model with in vitrocharacterization of important phenotype-like invasion potential is a cost-effective approach to power the genomic chemosensitivity analysis. most of which were outside of the NCI-60 panel. but also among the individual cells from the same malignant neoplasm of a patient. everolimus and dasatinib. On the other hand.19 to 2. Microarray. Recently. invasion and compound-sensitivity.

and GSE4127 (n = 29. we studied the genedrug correlation to identify IA genes that can be predictive of a cell‟s responsiveness to the compound. OR. and U133plus2. all from Invitrogen. For each of the 99 anticancer compounds of known mechanism. For each cell line. ON. The invasion capacities for cell lines were examined by using membrane invasion culture system. Becton Dickinson. To show the clinical relevance of our finding. GSE9633 (n = 16. . Franklin Lakes.The correlation matrix for (A) anti-microtubule and for (B) targeted therapy drugs. Table S4. The heatmap for the correlations between 744 IA gene expression and 99 drug respons e (−logGI50) in NCI-60 cell lines. Histogram of invaded cell counts (ICC) after subtracting the tissue-group means. The independent gene expression data of 78 cell lines used in the drug response validation were obtained either from the GEO website. Cell adhesion: Integrin-mediated cell adhesion and migration pathway. Supplementary information. we reported the average invasion cell count after three repeats (n = 3). Kaplan–Meier survival curves for survival analysis of the four-gene signature in lung cancer and breast cancer cohorts. Plots of the eight-gene risk scores between drug sensitive and drug resistant groups of cell lines after removing the nine cell lines that came from the NCI60 panel. II. were downloaded from the DTP [6]..) [10]. The suspension leukemia cancer cell lines were excluded. Figure S6. we searched for published chemotherapy clinical cohorts with related regimens to test the performance of our gene signature.Genes having significant gene-drug correlation with everolimus. The number of cells in each blot was counted under a microscope with Analytical Imaging Station system (Imaging Research Inc. Canada). Kaplan–Meier survival curves for survival analysis of the eight-gene signature in breast and lung cancer patients who did not receive systemic treatment. Figure S4. The expression level of eight signature genes in the nine NCI-60 cell lines. Eugene. Probe mapping between U133plus2 and U95 was provided by Affymetrix. Figure 1. NJ) were coated 30 μg matrigel (BD Biosciences. We identified a set of 633 IA genes as the likely marker candidates of tumor invasion potential. one on lung cancer and the other on breast cancer. MD. GSE6569 (n = 23. A schematic diagram illustrating the design of this study. Procedures for determining the invasion-associated (IA) genes. Table S6. Balko et al. The cells were suspended in RPMI containing 10% FBS and seeded into the upper wells of the chamber (2. Combination use of anti-microtubule and targeted therapy agents. the membrane of the transwell was fixed for 10 minutes at room temperature with methanol and stained for 30 minutes with 50 μg/ml propidium iodide (Sigma.Figure S2. Table S1.5 × 104 cells/well). Figure S5.05) is shown in bold face. dasatinib. The HTS FluoroBlok inserts containing 8-μm pores (Falcon. respectively. All but nine cell lines were outside of the NCI-60 panel (Additional file 1: Table S1). breast cancer) [7]. The final list of eight IA-genes is shown in bold face. CA. Table S3. erlotinib. A final set of eight IA genes for chemosensitivity prediction on five selected compounds was obtained. lung. gene expression and compoundsensitivity profiling. The dotted line indicated the mean of each group. The significant correlation (P <0. and 10% fetal bovine serum.We then conducted a series of statistical analysis to combine information from invasion profiling. prostate cancer) [8]. Catharines. Methods Matrigel invasion assay We purchased the panel of NCI human cancer cell lines (NCI-60) to conduct tumor invasion assays. lung cancer) [9] or by e-mail request (n = 10. USA) using Affymetrix U95. Enrichment analysis of 633 invasion-associated genes by functional ontology enrichment tool in MetaCore. Validation of microarray gene expression data with qPCR.Correlation between the invasion profile and each of the 99 drug sensitivity profiles of NCI60 cell lines. Our signature succeeded in predicting the clinical outcome for both cohorts. USA). St. We then validated the gene signature with additional cell lines. St. We found two recent studies containing anti-microtubule chemotherapy. All cells were grown in tissue culture flasks at 37°C in 5% CO 2 in RPMI 1640 with 2 mM L-glutamine. Cell line gene expression data Two gene expression datasets for NCI-60 cell lines.TaqMan probes ID for eight gene expression validation. Figure S3. Additional file 1. Table S5. After incubating for 24 hours at 37°C. produced by Gene Logic (Gaithersburg. Table S2. Louis.Figure S1. Figure 1 outlines how we approached each of our four aims. MO). paclitaxel and docetaxel profiles. Figure S7. San Jose.

The invasion-associated genes were obtained by computing the Pearson's correlation coefficients between gene expression profiles and invasion ability profile in NCI-60 cell lines. with the invaded cell counts (ICC) ranging from 129 to 5. Control cohorts Four cohorts where the patients were systemically untreated after surgery were used. ovarian cancer (OV).10. The drug sensitivity in the 78 independent cell lines was measured either by -logGI50 value [9] or by the negative of log10 half maximum inhibitory concentration (IC50) value [7.048). conducted at the M. We used the adjuvant cisplatin/vinorelbine therapy arm where after surgery (n = 71). tumor stage nodal status and histological grade. a randomized controlled trial of adjuvant vinorelbine/cisplatin versus observation alone [12]. there are substantial differences between the ICCs of different cell lines. The real-time reverse transcription quantitative PCR analysis The expressions of eight signature genes and a control gene TBP in nine lung cancer cell lines of NCI-60 were measured by reverse transcription qPCR(RT-qPCR) with specific Taqman probes and primer sets (Additional file 1: Table S2). A total of 99 drugs were analyzed. All statistical analyses were performed in the R language environment [11]. Seven compounds. Even within the same tissue group. We calculated the eight-gene risk score for each patient via simple averaging: Risk score=(GEGFR+GITGA3+GNNMT+GMYLK+GIL32+GGLS+GAHNAK+GRAI14)/8. breast cancer (BR) and prostate cancer (PR) [16-18].10]. inactive in all cell lines.8. of which the patients were those with newly diagnosed Human Epidermal Growth Factor Receptor 2 (HER2)–negative breast cancer under chemotherapy containing sequential taxane and anthracycline–based regimens (followed by endocrine therapy if ER positive) [1]. Student‟s t-test was used in validating the drug sensitivity prediction of gene signatures.514 (Figure 2). the 53 cancer cell lines are classified into eight groups: melanoma (ME). a lung cancer cohort. Multivariate Cox proportional hazard regression analysis was used to evaluate independent prognostic factors. We tested the equality of the ICC group means by ANOVA and found that the significance barely passes the 5% mark ( P = 0. Gene expression was quantified in relation to the expression of the control gene with the use of sequence detector software and the relative quantification method (Applied Biosystems).6MB Download file This file can be viewed with: Microsoft Word Viewer Chemosensitivity data We downloaded the NCI-60 chemosensitivity data for anti-cancer drugs with known molecular mechanisms. The lung cancer cohort came from JBR. Kaplan-Meier survival curves were obtained and compared by log-rank tests. According to the tissue origins. The breast cancer cohort of 508 patients came from a prospective multicenter study. Cancer chemotherapy cohorts Two chemotherapy cohorts were used in this study. colon cancer (CO). D. GSE7390 n = 198 [14] and GSE11121 n = 200 [15]). gender. were excluded. The fourth. We applied the normal score transformation to preprocess the gene expression data before computing the Pearson correlations. The transcripts were amplified with Taqman One-Step RT-PCR Master Mix Reagent (Applied Biosystems) and a detection system (ABI Prism 7900HT. . renal cancer (RE). The wide range of this distribution sh ows that the within-group variation means are greater than the between-group variation means. Survival analysis We calculated the patients‟ risk scores from the eight IA genes and classified them into the high -risk or the low-risk groups with the mean of risk score as the threshold value. Additional file 1: Figure S1 gives the overall distribution for the deviation of an individual cell line‟s ICC from its group mean. such as age. Statistical analysis The comparison of the tumor invasion ability between tissue types was performed by ANOVA. was the OBS arm where patients were under observation after surgery (n = 62). central nervous system cancer (CNS). Results Invasion heterogeneity The matrigel invasion assay of the 53 NCI-60 solid tumor cell lines shows a great variation between different cell lines. Applied Biosystems). including those used in targeted therapy [5]. Anderson Cancer Center. This suggests that the tissue of origin may not be an essential factor in characterizing invasion heterogeneity between cancer cell lines. Three of them were the breast cancer cohorts (GSE2034 n = 286 [13]. Clustering was used to order the IA probes. lung cancer (LC). Drug sensitivity was measured by the negative of log10 GI50 (−logGI50).Format: DOC Size: 1.

JAK1. ITGB1. were obtained. We show the expression levels of the 744 probes (Affy U133plus2) in the 53 cell lines with a heat map (Figure  3). c-Src and ERK2-mediated phosphorylation of FAK1 promotes its release from focal contacts and ERK2-mediated phosphorylation of paxillin promotes the association of nonphosphorylated FAK1 with paxillin at new or growing focal contact sites [22].PAK1 phosphorylation leads to the activation of LIM-kinase 1 (LIMK1) [23]. activation of myosin regulatory light chains (MRLC) [24]. We input the set of 633 IA genes (744 probes) and used the Functional Ontology Enrichment tool with the default settings. the higher ones being on the left.Figure 2. EGFR. The Integrin-mediated cell adhesion and migration pathway shows that ITGA3 promotes focal adhesion kinase ( FAK) autophosphorylation and creates a binding site for c-Src.025)/744. The results indicate the involvement of cell adhesion and cytoskeleton remolding actin cytoskeleton network ( ACTN1. Functional enrichment analysis of IA genes We use the MetaCore™ (Thomson Reuters.MYL6 and MYH9). Heat map for the expression of IA genes. Notably. ITGA3. LIMK1. The results show that our IA genes were enriched in cell adhesion and cytoskeleton remodeling pathways (Additional file 1: Table S3). The ordering of the genes is the output of the hierarchical clustering algorithm applied to the 744 expression profiles. which features three IA genes. NY). ATCB. The top panel of genes contains 341 probes that correlate negatively with the invasive ability. SMAD1and SMAD3). Supplementary information). VCL and CFL). MYLK. and (b) the sign of correlation must be consistent in both datasets (see the Additional file 1. Invasion profiling of NCI60 cancer cell lines. inhibition of MYLK. We estimated the false discovery rate (FDR) at the confirmation stage to be 0.20]. FAK signaling is important for integrin regulated cell adhesion and migration. CD44. Activation of MYLK together with inactivation of PAK1 contributes to cell-matrix adhesion dynamics. ZYX. which represented 633 distinct genes.49E-8. A positive correlation means that cell lines . A total of 744 probes.417*0. A simplified version of the Integrin-mediated cell adhesion and migration pathway is shown Additional file 1: Figure S2. ACTG1. Figure 3. and myosin signaling (MYLK1. EGFR signaling also activates the FAK/Src pathway [19. microtubule cytoskeleton (TUBB and TUBB6). Dotted lines indicate the mean of invasion cell counts for all cell lines in each tissue group. to conduct functional enrichment analysis of IA genes. ACTN4.417 = total number of probes considered at the confirmation stage and 0. matrix metalloproteinase signaling (CDH2. Association of drug sensitivity with IA gene expression To evaluate how the abundance of the IA gene transcripts in a cancer cell correlates with the cell‟s response to each of the 99 anticancer drugs for which the drug-sensitivity profiles on NCI-60 were available.08 (FDR = (2.CAV1 and CAV2). (P = 5. Integrin recruits FAK and a cytoskeletal protein vinculin and alpha-actin to focal contacts. which has a much higher ratio of root to total nodes and significant P-value. Additional file 1: Table S3 list the significantly enriched pathways and networks ( P <0. The genes in the top panel have negative correlations with invasion while the genes in the bottom panel have positive correlations. FAK is a tyrosine kinase which interacts with the important oncogene c-Src. Identification of 633 IA genes We identified the IA genes from Affymetrix U133plus2 NCI-60. The ordering of cell lines in this figure is based on their invasion abilities.025 = the P-value cutoff with the sign consistency criterion). a web-based computational platform designed for system biology and drug discovery. FAK activation regulates theERK-mediated phosphorylation and activation of Myosin light chain kinase (MYLK) contributes to cell-matrix adhesion dynamics [21]. We further examined the Integrin-mediated cell adhesion and migration pathway. Each dot in each tissue group gives the invaded cell counts by the matrigel invasion assay for one cell line (n = 3). a drug we address further in this paper. we computed Pearson's correlation between the gene expression profile of each IA gene and the chemosensitivity profile of each compound. integrin signaling (ITGA3. this pathway is the target of dasatinib [25].0001). New York. input/total nodes = 11/48). while the bottom panel of 403 probes correlates positively with the invasion ability. Cell lines are divided into groups by their tissue origin. where 2. and U95 NCI-60 microarray gene expression datasets by a twostage procedure which required that (a) the gene expression profile must be significantly correlated with the invasion profile in both datasets. TGFB2. Cell lines are ordered according to the invasion ability (measured by ICC) with the highest ICC placed leftmost. TGFBR2.

Gene-drug heat map for 17 drugs We represent the 744 by 17 gene-drug correlations as a heat map after clustering the genes (Figure 5A).27]. (A) Heatmap showing the gene-drug correlations for tubulinbinding and targeted therapy agents. red. (B) The specific pattern for the eight-gene signature enlarged from A. Similarly. Drugs with higher counts are this study‟s primary interest. The dotted line gives the mean of the probe counts in each group. cells with lower expression of genes. (A) The number of IA probes with significant (P <0. Heatmap of gene-drug correlation. we present each drug‟s count of the drug-sensitivity-correlated IA probes by grouping according to the drug‟s action mechanism (Figure 4A). the compound with the highest count is zoledronic acid. dasatinib and erlotinib are much higher than other correlations in the target agents. we were unable to find the drug sensitivity validation and gene expression data outside the NCI panel for zoledronic acid. The clustering pattern in Figure 5A can also be easily detected from the column-to-column correlations (Additional file 1: Table S4). negative correlation. the efficacy of the two groups of compounds tends to be in the opposite direction. In other words. A higher count indicates the availability of more IA genes for the sensitivity prediction of that drug. positive correlation. Interestingly. To investigate Additional file 1: Figure S3B further. tend to be more sensitive to tubulinbinding agents but they also tend to be more resistant to the three-target therapy drugs. while a negative correlation indicates the opposite. such as EGFR andITGA3. Figure 5. a bisphosphonate drug used to prevent skeletal fractures in cancer patients and to treat osteoporosis. showed a color pattern almost completely opposite to the tubulin-binding agents (Figure 5). Our functional enrichment analysis of IA genes shows that the Src signaling pathway played an important role in integrin-mediated cell adhesion and migration pathway (Additional file 1: Figure . tend to be more resistant to tubulin-binding agents but they also tend to be more sensitive to the three-target therapy drugs. The correlations among everolimus. Figure 4. we found that the top three compounds in the group of target therapy agents. dasatinib and erlotinib. We find that on average. An IA probe showing a significant correlation with a drug would be called a drug-sensitivity-correlated probe for that drug. We identified all the significant correlations ( P <0. Dasatinib is a clinically studied SRC inhibitor for cancer therapy [28].05). We continued our study on the 17 drugs from these two groups.05) gene-drug correlation with each anti-cancer compound is plotted according to the grouping of the drug mechanism. Interestingly enough. the group of tubulin-binding agents ranks the highest. such as MYB and TOB1. the correlation between paclitaxel and docetaxel is much higher than other correlations in the tubulin-binding agents. Blue. recent studies indicate zoledronic acid can prevent skeletal metastases through inhibition of invasion and angiogenesis of cancer cells [26. everolimus. This means that cells with higher expressions of genes. (B) The numbers of significant IA probes in tubulinbinding agents and targeted therapy agents. However. and displayed the findings in Additional file 1: Figure S3B.with higher gene expression are more sensitive to the drug. The distribution of drug-sensitivity-correlated IA probes. Individually speaking. followed by the targeted therapy. The count for each compound in these two groups is given in Figure 4B. We counted the number of drug-sensitivity-correlated IA probes for each compound. We kept the ordering of compounds to be the same as in Figure 5B. Such a drug would be more likely to have differential anti-cancer effects among the tumors showing differential invasion potential. Likewise. The gene-drug correlation results are shown in a heat map (Additional file 1: Figure S3A).

we also noticed that the numbers of sensitivity-correlated IA genes that these five compounds had are higher than other drugs in the respective mechanism groups (Figur e 4A). Glutaminase (GLS) was shown to be up-regulated in MYC induction cancer cells and inhibition of GLS decreased the cancer cell growth[35. Plots of the eight-gene risk scores between drug-sensitive and drug-resistant groups of cell lines. It should be noted. which were then divided into drug-resistant and drug-sensitive groups according to the IC50 value of the experiment outcome. were applied separately to 29 lung cell lines. 19 probes are in the Affy HG U-133A chip and 7 probes are in the HG U-133B chip. We searched the public domain extensively for drug response experiments involving any of the five featured compounds.009 in paclitaxel-treated group. we exclude the seven U133-B probes.36]. This prompted us to ask if these five compounds might share any drug-sensitivity-correlated IA genes that could be used for drugsensitivity prediction. The fourth experiment applied erlotinib to 10 lung cancer cell lines. MYLK. Retinoic acid induced 14 (RAI14) is an actin cytoskeleton protein regulated by retinoic acid[37].0044 for breast cancer and P = 0. Erlotinib inhibits the intracellular phosphorylation of tyrosine kinase associated with the EGFR [29]. and FAK-Src signaling (ITGA3. Finally. Biological functions of eight invasion-associated genes The eight IA genes we identified are associated with cellular cytoskeleton.013 in docetaxel-treated group. Four experiments were found. Selection of drug-sensitivity-correlated IA genes for five compounds In addition to the mechanism sharing and the clustering pattern observed. Again. cell invasion and oncogenetic signaling. From the gene expression data. NNMT. the difference between erlotinib-sensitive and erlotinib-resistant was significant (t. MYLK is a key target of FAK–Src signaling [34]. AHNAK nucleoprotein was reported as pseudopod-specific proteins in different metastatic human tumor cell lines [39]. which were then divided into dasatinib-sensitive and dasatinib-resistant groups according to the experiment outcome. A total of 72 data points are plotted. and P = 0. By inspecting the drug-sensitivity-correlated IA gene list for these five compounds. including the well-known cancer driver EGFR. Figure 6.129e-11) is observed. alpha 3) mediates cell survival and invasion through FAK-Src signaling[31-33]. each dot representing the expression level of a gene measured by qPCR (horizontal axis) and the microarray (vertical axis). In the third experiment [8]. 26 common probes are identified (Additional file1: Table S5). High expression of interleukin 32 (IL32) was shown to cause a worse clinical outcome in lung cancer patients [38]. AHNAK. A final set of eight IA genes (EGFR. GLS. This indicates that the eight gene biomarkers still cannot accurately classify cell lines into a resistant group versus a sensitive group. Validating gene signature with independent cell lines We used the simple averaging to combine the expressions of eight IA genes into an eight-gene score. MYLK). Figure 6). and RAI14) are obtained.025.test P = 0. The activity of nicotinamide N-methyltransferase (NNMT) in catalyzing the Nmethylation of nicotinamide is important for biotransformation of drug and xenobiotic compounds [40]. . dasatinib was applied to 16 prostate and 23 breast cancer cell lines [7]. however. Figure 6). Because most microarray data are available in the public domain used only the U-133A chip.69. Only those probes with SD ranking within the top 10% are retained. ITGA3 (integrin. promoting cell migration [44] and cell invasion [45]. The dotted line indicated the mean of each group. that these plots also showed an overlapping pattern in the distributions between the drug-resistant group and drug-sensitive group. we check the sign of correlation between gene expression and drug sensitivity to ensure the consistency in predicting sensitivity or resistance. A significant positive trend (Pearson correlation = 0. In the anti-tubulin group. The eight-gene scores show a significant difference between the dasatinib-sensitive group and dasatinib-resistant group (t-test P = 0. ITGA3. IL32. docetaxel is a semisynthetic side chain analog of paclitaxel. NNMT was identified as a tumor biomarker [41-43]. We confirm the quality of the microarray gene expression data by performing the qPCR assay for these eight genes on nine lung cancer cell lines in NCI-60 (Additional file 1: Figure S4). confirming the consistency of the two assays. 2.0044 for prostate cancer. Figure 6).S2). In the first two experiments [9]. Everolimus is an mTOR inhibitor [30]. paclitaxel and docetaxel. We use the standard deviation (SD) of the expression across all 53 cell lines to further exclude probes showing low expression variation. we calculated the eight-gene score for each cell line and found a significant difference between the drugresistant group and the drug-sensitive group (t-test P = 0. two standard anti-microtubule agents. Among them.

This shows that our eight-gene signature is an independent predictor for patient outcome. Kaplan–Meier survival curves for survival analysis of the eight-predicted genes in (A) lung cancer and (B) breast cancer cohorts.33 (P = 0. and 1.003) for the lung cancer cohort. We use the simple average expression of the eight-genes as the risk score to dichotomize patients into two groups. All significant differences in the first three experiments were still valid. Figure 7B). Two recently published studies on the vinorelbine containing regimen for lung cancer [12] and the taxane-containing regimen for treating invasive breast cancer [1] were obtained. For the fourth experiment. we found nine cell lines from the NCI-60 panels. We hypothesize the potential overlap between the set for invasion ability phenotype and the set for drug responsiveness phenotype. mainly because the sample size (n = 8) was too small. We removed these cell lines from the validation data and conducted the comparison again (Additional file 1: Figure S5). We found that the low-risk group has a significantly longer relapse-free survival time than the high-risk group in the lung cancer cohorts (P = 0. we applied it to four control cohorts (three for breast cancer and one for lung cancer) of which the patients were systemically untreated after surgery.In these validation experiments.81 (P = 0. Each phenotype is naturally associated with its own set of molecular determinants. Clinical outcome prediction in adjuvant chemotherapy lung and breast cancer patients To test if the eight genes have the predictive power in clinical outcome evaluation. The adjusted hazard ratio (HR) is 5. then we may use these shared determinants to estimate the overall invasion potential of the cancer cells in a tumor and also use it to predict the drug response at the same time. The result shows that the effect of our gene signature is still significant. While cell line models have been used to predict treatment response or patient survival by genes associated with drug sensitivity in the cancer cell lines [46-49]. To weigh in the interplay of both factors directly. we did not pursue this line of analysis further because the phenotye-phenotype . However. Figure 7. the low-risk group shows significantly longer distant relapse-free survival in the breast cancer cohorts (P = 0. these studies have not considered the varying metastasis potential of a tumor. Figure 7A) and similarly. To demonstrate that the predicative capacity of the eight-gene signature is specific to chemotherapy. Our approach is based on the observation that invasion ability and drug sensitivity are both phenotypes of the cell lines available for study.006) for the breast cancer patients (Table 1). Multivariate Cox regression analysis of the eight-gene signature for predicting relapse-free survival in cancer patients Discussion The invasive or metastatic potential of a malignant neoplasm and the growth-inhibition of tumor cells by a therapeutic agent are two common denominators of patient survival in cancer systemic therapy. By identifying these common determinants. We further conducted multivariate Cox proportional hazard regression analysis with our gene signature and other prognostic factors (including age and tumor stage) as the predictors.00021. our pre-clinical gene signature discovery method features the co-integration of invasion phenotypes and compound-sensitity profiles with gene expression at the full genome scale. because the tumor microenvironment in a patient is different from the growth environment of cancer cell lines monitored in a lab. then genes correlated with response to these drugs can be used to predict the drug sensitivity. gene expression and drug response would be to correlate invasion with drug response first.0263.0788. The eight gene signature failed to predict clinical outcome (Additional file 1: Figure S6). we searched the public domain for chemotherapy cohort studies involving any of the two anti-microtubule compounds we used in obtaining the eight genes. Table 1. the robustness of an invasion molecular marker becomes an important factor for increasing the chance of success in clinical applications. the P-value increased to 0. Once the most correlated drugs are identified. However. An alternative strategy of analyzing the three-way interaction of invasion.

We report the identification of a unique eight-gene signature for both lung and breast cancer. which predicted the relapse-free survival of adjuvant chemotherapy patients. but the correlation is weak (Additional file 1: Table S6. the correlation between the eight-gene score and the drug sensitivity is stronger (−0. but may be more likely to succumb to the said targeted therapy. There have been several studies on augmenting the anticancer effect of chemotherapy with targeted therapy. which is again stronger than the correlation between invasion and drug sensitivity (0. 0. such as DNA copy number. of which the patients received a regimen containing an anti-microtubule agent. The eight-gene signature showed a positive correlation with the sensitivity of targeted therapy compounds and a negative correlation with the sensitivity of anti-microtubule compounds (Figure 5B). The discovery of prognostic biomarkers for chemotherapy patients remains critical toward improving the efficacy of cancer treatment. We find that among the four genes ANKRD49 and LPHN1 are in the IA gene list and only ANKRD49has a significant correlation with paclitaxel and docetaxel. The eight-gene signature obtained here may be useful for the development of individualized cancer therapy. 0. On the other hand. dasatinib and everolimus is 0. which are harder to eradicate by antimicrotubule compounds. despite the weak correlation between the invasion phenotype and the drug sensitivity phenotype. Similarly. the eight-gene signature provides an averaged profile of the gene expression by individual cells with varying invasion potential.26.correlation is often weaker than phenotype-genomic determinant correlation.54.52. only four drugs pass the statistical significance and the best two correlations are only 0. To gain robustness of our gene signature. In addition.41. The signature features the cancer hallmark EGFR and genes involved in cell adhesion cell migration. Then a series of statistical analyses were designed to increase the robustness of the final eight-gene signature in predicting drug sensitivity for the selected compounds.09. respectively). respectively). without considering drug sensitivity. cell invasion. Abbreviations . The four-gene signature failed to predict the survival outcome for the two validation cohorts receiving adjuvant chemotherapy (Additional file 1: Figure S7). Because high values of the eight-gene signature correlate with high invasion potential in cancer cells. cancer cell‟s invasion potential and cancer cell‟s responsiveness to an anti -cancer compound. methylation and microRNA. Other genomic components. When applying to the patient‟s tumor specimen.24. Previously. our data show that most druginvasion correlations appear weak. This helps improve the robustness of the genetic marker thus obtained. -0. A low eight-gene score indicates that the overall invasion potential of the tumor is low and the chance of the patient‟s favorable response to regimens containing anti -microtubule compounds increases. Conclusions We have shown that augmenting the NCI-60 model with in vitro characterization of important phenotypes like invasion potential is a cost-effective approach to power the genomic chemosensitivity analysis. a high eight-gene score predicts the abundance of the cells of higher invasion potential.39 and −0. We succeeded in applying our gene signature to one lung cancer cohort and one breast cancer cohort. This is indeed the case.35 (Additional file 1: Table S6).16. respectively). this suggests that the direct correlation between the invasion profile and the sensitivity profiles of anti-MT drugs may be negative. the correlation between the eight-gene score and the sensitivity for erlotinib. 0. we used the invasion data from all 53 NCI60 solid tumor cell lines and obtained 633 IA genes. The overlap in distribution between the drugsensitivity group and the drug-resistant group (Figure 6) suggests that drug response in cell lines is a very complex phenotype which is not fully characterized by our gene signature. Erlotinib was shown to be more sensitive in the doxorubicin-resistant human breast cancer cell lines and paclitaxel-resistant human ovarian cancer cell lines[51] and the sensitivity was positively correlated with EGFR expression. instead of using different panels of tissue origins in NCI-60 to obtain different sets of IA genes for different types of cancer. single-nucleotide polymorphisms. Our method of gene discovery may be applicable in studying other cancers. Our analysis delineates the complex three-way interplay of gene expression. respectively.44. the eight-gene signature is an effective genomic marker for invasion potential and it can be used to predict the drug‟s differential growth-inhibition efficacy that varies between cancer cells of higher invasion potential and those of lower invasion potential. Our approach overcomes the limitations of weak phenotype-phenotype correlation by looking for statistical evidence of correlations directly from the genomic determinant. correlation = −0. This suggests the combined use of targeted therapy like dasatinib or erlotinib with anti-microtubule agents to increase the regimen efficacy of chemotherapy alone. 0. The eight-gene score differed between drug-resistant and drug-sensitive cell lines (Figure 6). our team performed invasion profiling for the nine lung cancer cell lines of NCI-60 to obtain a four gene signature for clinical outcome prediction [50]. -0. As a matter of fact. The success in using the same signature to predict patient outcome for different types of cancer showed the robustness of this gene signature. differences in lab environment may also contribute to the variations observed in the data.46. More references were provided in Additional file 1. Supplementary information Text II. tumor growth and tumor progression. Therefore. On the other hand.28 for paclitaxel and docetaxel. have not been considered in our study. There is room to improve our eight-gene signature for drug-sensitivity prediction.

IA: Invasion-associated. LC: Lung cancer. RE: Renal cancer. MYLK: Myosin light chain kinase. FAK: Focal adhesion kinase.AHNAK: AHNAK nucleoprotein. MRLC: Myosin regulatory light chains. CO: Colon cancer. PCY and KCL provided the study materials and reagents. SLY participated in setting up the experiment platform for the cell line invasion and qPCR validation. GLS: Glutaminase. conceived the drug sensitivity and clinical prediction study design. DTP: Development therapeutics program. GW and KCL collected public gene expression and statistical data analysis. alpha 3. ER: Estrogen receptor. EGFR: Epidermal growth factor receptor. Acknowledgments This research is supported by NSC 98-2314-B-001-001-M . YCH. IL32: Interleukin 32. Br: Breast cancer. ME: Melanoma. SLY. FDR: False discovery rate. KCL and YCH set up the study aims and rationalized the approach. PCY and KCL helped chart the study strategy and were involved in drafting and finalizing the manuscript. ICC: Invaded cell counts. OV: Ovarian cancer. LIMK1: LIM-kinase 1. ITGA3: Integrin. Competing interests The authors declare that they have no competing interests. PR: Prostate cancer. SY. Authors’ contributions YCH carried out the invasion experiments. CNS: Central nervous system. HR: Hazard ratio. CHL. and wrote the paper. NNMT: N-methyltransferase. KCL and YCH proposed the conceptual framework to link gene expression variation with heterogeneity of invasion potential and drug sensitivity in cancer cells. NCI: National cancer institute. HYC. All authors read and approved the final manuscript for publication.