We aimed to use the aforementioned scores to quantify the risk of 30-day mortality, major vascular and bleeding events (MVASC/BARC), and cerebrovascular insults (CVI) in patients undergoing different access routes in transcatheter aortic valve replacement (TAVR).  High CHA2DS2-VASC, HAS-BLED, and combined “CHADS-BLED” scores were calculated and compared to the predictive value of the established EuroSCORE and STS score.  Out of 1329 patients, 980 transfemoral (TF) TAVR (73.7%) and 349 transapical (TA) TAVR (26.3%) were included. CHAp < 0.0001). Concerning 30-day mortality, only the STS score for TF TAVR  and EuroSCORE for TA TAVR  could show some predictive value. High CHADS-BLED was associated with enhanced CVI  and more frequent MVASC/BARC  in the all-comers TAVR cohort. All risk models failed in the prediction of CVI and MVASC/BARC for TA TAVR patients.  In all-comers TF TAVR patients, the applied risk models showed only poor association with 30-day mortality while, in patients with concomitant AF, a strong association was observed using the combined CHADS-BLED score . The combined CHADS-BLED score was a strong predictor for 30-day mortality in TF TAVR patients with AF. A high CHADS-BLED score showed a good predictive value for major vascular and bleeding events as well as CVI in TF TAVR patients. This study is registered at clinical trials ( Transcatheter aortic valve replacement (TAVR) is an established therapeutic option in patients with symptomatic severe aortic stenosis (AS). While the STS score and the EuroSCORE are two appropriate risk score models to ascertain patients' individual short-term mortality and morbidity after cardiac surgery , 2, ther2DS2-VASC score has been demonstrated to predict the risk of cerebrovascular events in patients with [The CHAnts with  and withnts with ,7. Additnts with , even innts with . Althougnts with –13 and nnts with , 7, 9. Tn = 980, 73.7%) or TA  TAVR, CHA2DS2-VASC and HAS-BLED scores were calculated as well as the logistic EuroSCORE I and STS-PROM scores. The combined CHADS-BLED score was calculated by adding the values of the single CHA2DS2-VASC and HAS-BLED scores. All procedures were performed according to the current guidelines between 2009 and 2019, respectively, and under local anesthesia for TF access and general anesthesia for TA access. TF TAVR was performed with different generations of either the self-expandable CoreValve System  or the balloon-expandable SAPIEN System . TA TAVR was predominantly performed by using the SAPIEN System  or in very few cases the Engager System .From 2009 to 2019, out of 1329 patients with either TF (NCT01805739).All patients provided written informed consent for TAVR and the use of clinical, procedural, and follow-up data for research. The study procedures were in accordance with the Declaration of Helsinki. The Institutional Ethics Committee of the Heinrich-Heine University approved the study protocol (4080). The study is registered at clinical trials  analysis and the c-index (area under the curve (AUC)) were used to identify the sensitivity and specificity of the CHA2DS2-VASC, HAS-BLED, and combined CHADS-BLED cutoff points for 30-day mortality, MVASC/BARC, and CVI. The optimal cutoff values were defined by Youden's index, the point at which the value of “sensitivity + specificity − 1” was maximum.The collected data included patients' characteristics, imaging findings, periprocedural in-hospital data, laboratory results, and follow-up data. Continuous data were described by mean and standard deviation, median, or upper and lower 95% confidence interval (interquartile ranges). Categorical variables were characterized by frequencies and percentages. Continuous variables were compared using Student's p < 0.05 was considered statistically significant.The data analysis was performed using the statistical software SPSS  and GraphPad Prism . All statistical tests were 2-tailed, and a value of p < 0.0001) and predominantly male . In summary, general atherosclerosis in the meaning of concomitant coronary artery disease (CAD), peripheral artery disease (PAD), cerebrovascular disease (CVD), and porcelain aorta (PAo) were more frequent in patients undergoing TA TAVR. The logistic EuroSCORE I (logES-I) was higher in TA TAVR patients according to the predescribed risk profile . No difference was observed concerning mono or dual antiplatelet therapy, usage of (new) oral anticoagulants, or triple therapy at admission. A full overview of baseline clinical and functional characteristics is displayed in Supplemental Baseline patients' characteristics differed in consequence of the particular risk profile and selection bias of TF versus TA assigned approaches: TA patients were younger  in the TF TAVR and 7.7% (n = 27) in the TA TAVR cohort (p = 0.0012%). Cardiovascular death was documented in 39 cases with higher amount in patients undergoing TA approach . Further causes of overall 30-day death were cerebrovascular accidents , infection/sepsis , and unknown reasons .Periprocedural death within the first 30 days was recorded in 59 cases (4.4%) in the overall cohort with a mortality distribution of 3.3% (n = 52) in the TF TAVR and 4.6% (n = 16) in the TA TAVR cohort. There were no significant differences in both cohorts regarding the overall distribution and subclassifications. Transient ischemic attacks counted with 19 events (1.4%), ischemic strokes with 33 events (2.5%), hemorrhagic CVI with 4 events (0.3%), and undetermined CVI with 6 events (0.5%). MVASC/BARC events recorded in 146 cases (11.0%) were significantly higher in the TA TAVR cohort . Except for conversion to surgery and need for permanent pacemaker therapy, all secondary outcomes were more unfavorable in TA TAVR patients. A complete overview of primary and secondary outcomes according to VARC-2 is displayed in Supplemental Cerebrovascular events were recorded in 68 cases (5.1%) in the overall cohort with a distribution of 5.3%  and TA TAVR patients . Concerning 30-day mortality in all-comers TF and TA TAVR patients, only the STS score for TF TAVR  and the EuroSCORE for TA TAVR  could show a predictive value.The risk model discrimination performance is reported in p < 0.0001) and CHADS-BLED  performed best concerning prediction of CVI.HAS-BLED  and CHADS-BLED  were superior to CHA2DS2-VASC  in TF TAVR patients, while the best prediction of MVASC/BARC for TF TAVR was reached by the STS score . Indeed, all risk models failed in the prediction of the primary endpoints in patients undergoing TA access.Regarding the prediction of MVASC/BARC, HAS-BLED  analysis and the c-index  were used to identify the sensitivity and specificity of the CHADS-BLED cutoff points for 30-day mortality, CVI, and MVASC/BARC. The optimal cutoff values were defined by Youden's index. >7 points turned out to be the cutoff with “sensitivity + specificity − 1” becoming the maximum regarding the combined CHADS-BLED calculation  and less obese . Concomitant CAD, PAD, and PAo were also more frequent in patients undergoing TA TAVR. While all other risk models were comparable in both groups, only the HAS-BLED was higher in TF TAVR patients . No difference was documented concerning mono or dual antiplatelet therapy, usage of (new) oral anticoagulants, or triple therapy following TAVR. A full overview of differing characteristics between TF and TA TAVR patients with CVI and/or MVASC/BARC positive profile is displayed in Supplemental As mentioned before, TA patients were predominantly male , followed by CHA2DS2-VASC  and HAS-BLED , and STS score . Again, no association at all could be found for risk models in TA TAVR patients.Risk model discrimination performance is reported in p=0.019), while the STS score  and EuroSCORE  were superior to HAS-BLED  in terms of MVASC/BARC, similar to the results for all-comers TF TAVR cohort.HAS-BLED performed best concerning prediction of CVI  was associated with enhanced CVI and more frequent MVASC/BARC, but only to the same extent as HAS-BLEDThe risk models failed even with higher score thresholds to predict primary endpoints in TA TAVR patients, except for EuroSCORE and STS score in terms of 30-day mortalityCVI and MVASC/BARC events were not linked to different antithrombotic and/or antiplatelet regimesThe present study evaluated the risk of 30-day mortality, major vascular and bleeding events, and the incidence of CVI in dependence from the aforementioned scores in patients undergoing different access routes in TAVR and revealed several findings:2DS2-VASC  and HAS-BLED  are well-established and routinely used scores for therapeutic decision support concerning patients with AF. In the past few years, these risk models were also stratified for conditions other than AF [2CHA2DS2-VASC score and 30-day mortality. Patients with a baseline CHA2DS2-VASC score ≥6 or a modified score ≥7 appeared to have increased short-term mortality [2DS2-VASC score [Accurate risk assessment for TAVR patients remains challenging and simple risk scores for bedside use are still lacking. Both the CHA than AF –13. Hamiortality . In compSC score . Honda e2DS2-VASC nor HAS-BLED score was associated with 30-day mortality in all-comers TF and TA TAVR patients, and all risk models failed in the prediction of the primary endpoints in patients undergoing TA access. Hence, only in patients with AF, the CHADS-BLED was strongly associated with 30-day mortality. This is in close relation to the original use of the scores, namely, prediction of outcome only in patients with AF, refusing former results about their usefulness in risk prediction regardless of the presence of AF [However, bedside score-derived prediction of mortality and adverse events in the context of different access routes comparing TF and TA TAVR has not been investigated so far. Our study is in line with former reports that TA patients show higher mortality and periprocedural adverse events than TF patients , 4. Surpce of AF , 12, 13.2DS2-VASC is able to provide strong correlations for in-hospital stroke but with low accuracy [Looking at the several event rates, high CHADS-BLED >7 points was associated with enhanced CVI, MVASC/BARC, and combined events in TF TAVR patients, but only in the all-comers cohort (with and without AF). No association could be found in low vs high CHADS-BLED scoring and incidence of the primary endpoint events in TA TAVR patients. Former studies could show that single use of CHAaccuracy , comparaaccuracy , 16 or aaccuracy . HoweverThe more traditional risk scoring systems STS score and logistic EuroSCORE I have been developed and validated for the prediction of 30-day mortality and major comorbidity rates in surgical populations. They are commonly used to assess risk in patients considered for TAVR as well due to lack of proper alternatives. As expected, the STS score and EuroSCORE turned out to be most predictive for 30-day mortality in all-comers TF and TA TAVR in the present study.Surprisingly, the STS score was superior to HAS-BLED in the prediction of MVASC/BARC events although the STS score does not contain any bleeding-specific features as HAS-BLED does. As reported in former studies, there is an effect of post-TAVI bleeding on short-term mortality. Wang et al. showed by meta-analysis that there is an about 3-fold increase in 30-day mortality associated with bleeding events, which could explain the good predictive value of the STS score in this field .However, while these operative risk scores are derived from surgical aortic valve replacement, they tend to overestimate TAVR mortality because of many procedural confounders . Furthermore, many important noncardiac factors such as frailty, malignancy, and neurological status are not part of these risk models. This is in line with our result that one-third of the deaths in our study were due to infection or sepsis, supposing various influencing noncardiac factors not being reflected by the parameters included in the current risk scores. In addition, none of the risk scores take into account anatomical factors such as vessel calcification and procedural aspects, both variables that strongly impact short-term outcome in TAVR.These findings highlight the need of more precise scoring systems regarding the complex clinical situation of patients with severe aortic valve stenosis. This is why the current guidelines acknowledge the deficient character of the risk scores and recommend a multidisciplinary heart team-based decision for TAVR after detailed clinical evaluation with participation of patients and their families .The current study is a hypothesis-generating retrospective single-center analysis designed to test the association between single and combined use of risk scores regarding periprocedural adverse events and 30-day mortality. Due to the long time period between 2009 and 2019, there is a high variability in devices and generation of devices. With advances in technique, device, and expansion of TAVR to lower risks groups, the mortality and CVI rates have improved over time. In the present analysis, the measured outcomes were not linked to different antithrombotic and/or antiplatelet regimes. The authors cannot exclude that this absent effect is due to patients' noncompliance in medication intake although all patients had comprehensible medication plans before and after TAVR.Further studies are warranted to validate our findings, taking other procedural factors and different risk profiles—high, intermediate, and low risk—into account. A propensity-matched score analysis should be considered to clarify the observed differences in risk model-derived prediction of adverse events in TA and TF patients.The combined CHADS-BLED score was a strong predictor for 30-day mortality in TF TAVR patients with AF but failed to predict any adverse event in TA TAVR. Neither a single unified scale or scoring system nor a combination of established risk scores seems to be able to adequately predict both short-term mortality and occurrence of adverse events like MVASC/BARC or CVI in patients with aortic valve stenosis. The complex clinical situation of these patients and different access routes for TAVR require more accurate risk assessment tools, especially regarding noncardiac factors like functional status and frailty as well as anatomical and procedural factors.


Effective primary care interventions for multimorbidity are needed.To evaluate the effectiveness of a group-based, 6-week, occupational therapy-led self-management support programme (OPTIMAL) for patients with multimorbidity.A pragmatic parallel randomised controlled trial across eight primary care teams in Eastern Ireland with 149 patients with multimorbidity, from November 2015 to December 2018. Intervention was OPTIMAL with a usual care comparison.Primary outcomes were health-related quality of life (EQ-5D-3L) and frequency of activity participation (Frenchay Activities Index [FAI]). Secondary outcomes included independence in activities of daily living, occupational performance and satisfaction, anxiety and depression, self-efficacy, and healthcare utilisation. Complete case linear regression analyses were conducted. Age (<65/≥65 years) and the number of chronic conditions (<4/≥4) were explored further.A total of 124 (83.2%) and 121 (81.2%) participants had complete data at immediate and 6-month post-intervention follow-up, respectively. Intervention participants had significant improvement in EQ-VAS  at immediate follow-up  but no difference in index score  or FAI . At 6-month follow-up there were no differences in primary outcomes and mixed results for secondary outcomes. Pre-planned subgroup analyses suggested participants aged <65 years were more likely to benefit.OPTIMAL was found to be ineffective in improving health-related quality of life or activity participation at 6-month follow-up. Existing multimorbidity interventions tend to focus on older adults; preplanned subgroup analyses results in the present study suggest that future research should target younger adults (<65 years) with multimorbidity. Included interventions predominately centred on care organisation such as case management or patient-oriented interventions, for example, patient education interventions. The review concluded that previous interventions focused predominantly on people with defined comorbid conditions or on multimorbidity in patients aged >65 years, and recommended a focus on risk factors common across comorbid conditions or generic outcomes such as daily functioning.3 In 2018, the largest randomised controlled trial (RCT) of a multimorbidity intervention, the 3D study,4 examined the effect of general practice-based 6-monthly patient multidisciplinary reviews of the dimensions of drugs, depression, and health, based on multimorbidity guidelines. It found no effect on health-related quality of life (HRQoL) but did report significant improvements in patients’ experience of care.Individuals with multimorbidity, the presence of ≥2 chronic conditions, have poorer health outcomes, higher health service utilisation, and higher healthcare costs.5,6 The clinical and cost-effectiveness of such interventions for multimorbidity remain unclear.7 Studies of the popular peer-led Stanford chronic disease self-management programme have produced modest effects when delivered in settings outside of the US.8 The Medical Research Council framework for complex interventions9 was used to develop and pilot OPTIMAL, a professionally-led 6-week group self-management support programme for multimorbidity.10,11 The OPTIMAL programme is underpinned by self-efficacy theory, focusing on topics of concern to those with multimorbidity, and is professionally led by primary care occupational therapists, because of the profession’s focus on function, with input from a physiotherapist and pharmacist. Further details of the OPTIMAL programme content and delivery are outlined in 11Self-management interventions aim to maximise physical and psychosocial functioning by providing individuals with skills to manage symptoms, treatments, and the psychosocial consequences of living with a chronic condition.9The aim of the present study was to conduct a definitive RCT to evaluate the effectiveness of the OPTIMAL programme in improving HRQoL and frequency of activity participation, and to test its sustainability after programme completion, as per Stage III of the Medical Research Council framework.12A pragmatic parallel two-arm RCT was reported following CONSORT guidelines for the design, conduct, and analysis of RCTs.13–15 a process evaluation was also conducted alongside the OPTIMAL trial to evaluate its implementation within existing primary care services in Ireland and will be reported separately.The study was carried out in the Irish primary care health system in the greater Dublin region. Ireland has a mixed public and private primary healthcare system, with one-third of the population entitled to free primary care based on low income through the General Medical Services Scheme. Primary care teams typically include GPs and practice nurses who are independent contractors, and allied health practitioners including community nurses, occupational therapists, physiotherapists, dietitians, and social workers, all of whom are employees of the public health system, that is, the Health Service Executive. Primary care in Ireland remains underdeveloped and fragmented;Participants with multimorbidity were recruited through primary care team members and self-referral across eight Health Service Executive primary care areas in which participating occupational therapists were based. Recruitment and intervention delivery was conducted over four sequential time blocks, with two primary care team areas in each time block. Referring clinicians were informed about the study via post, email, and presentations at primary care team meetings. Referrals were forwarded to a gatekeeper in each area’s occupational therapy department who contacted referred patients 7–10 days after referral to confirm their participation.11 The age limit of ≥40 years was chosen because multimorbidity is relatively uncommon in patients younger than this and to facilitate targeted recruitment.16,17 A broad, inclusive, and commonly used definition of ≥2 chronic conditions was used. The World Health Organization’s definition of chronic diseases as health problems that require ongoing management over a period of years or decades was used.18 The inclusion criteria of ≥4 repeat medications was included to identify a group in the broader multimorbidity population that is at increased risk of poor health outcomes and more likely to benefit from an intervention.3 Written informed consent was obtained from all trial participants. The trial ran from November 2015 to December 2018.The following participant inclusion criteria were applied: age ≥40 years; ≥2 chronic conditions; ≥4 repeat medications; and an ability to travel to the centre where the intervention would be delivered. These criteria were the same as those used in the exploratory RCT, which proved to be effective, with the exception of age.Following baseline data collection for each site (area), participants were individually randomised into intervention (OPTIMAL programme) or control  groups. Randomisation and allocation was carried out remotely by an independent statistician. Randomisation was performed using Stata (version 14), was stratified by sex, and random permutated blocks of size 2 and 4 were used. Couples who were recruited were randomised as a unit to avoid contamination. The independent statistician informed therapists at each site of participant allocation and therapists in turn informed participants of their allocation by telephone 1–2 weeks before the intervention began.10,11 Before programme delivery, occupational therapists received a half-day of training and a facilitator manual to standardise programme delivery and maintain intervention fidelity.Full details of the OPTIMAL programme have been published previously and the programme content is summarised in 10,11 Outcomes were collected immediately post-intervention (primary outcome measures only) and 6 months post-intervention.Outcomes were chosen to reflect the intervention’s theoretical underpinnings and based on the previous OPTIMAL pilot studies.Baseline assessments were conducted via interview with occupational therapists in each site. Immediately post-intervention, intervention and control participants self-completed primary outcomes by postal survey (in a 3-week period of intervention completion). All 6-month follow-up (post-intervention completion) assessments were collected via interviews, with a researcher blinded to participant allocation and a record of broken blinding was maintained. Because of the nature of the intervention, it was not possible to blind participants to their group allocation. Data collection for control participants was matched to intervention participants in the same time block to ensure an equal length of follow-up.19,20 The EQ-5D-3L comprises two parts, the descriptive system and a visual analogue scale (EQ-VAS). The descriptive system consists of five dimensions including mobility, self-care, usual activities, pain/discomfort, and anxiety/depression, each of which is rated on three severity levels. The EQ-VAS is a vertical visual analogue scale whereby participants rate their perceived health status from 0  to 100 . The EQ-5D-3L descriptive system can be converted to an index score based on societal preferences for health states.21 Secondary outcome measures included the Nottingham Extended Activities of Daily Living scale,22 the Stanford Chronic Disease Self-Efficacy 6-item Scale (SEMCD),23 the Hospital Anxiety and Depression Scale (HADS),24 and the Canadian Occupational Performance Measure (COPM).25 Self-reported healthcare utilisation, including GP visits, emergency department visits, outpatient appointments, hospital admissions, and nights spent in hospital was collected for 6 months before baseline data collection and 6 months post-intervention .Two primary outcome measures were used: HRQoL (measured using the EQ-5D-3L) and frequency of activity participation (measured using the Frenchay Actvities Index [FAI]).n = 108) and 80% power. Furthermore, retention was 10% higher at follow-up than originally anticipated. In the EQ-VAS, improvements of 14 points have been reported as representing a large effect size.26 Interim mean EQ-VAS baseline scores were 59.1 (standard deviation [SD] 20.3). To improve a baseline EQ-VAS score of 59.1 by 14 points, with 80% power, required a total sample size of 68 (n = 34 per group). Improvements of 4 points in FAI total scores have been reported as clinically significant.27 Interim mean FAI baseline scores were 25.3 (SD 7.5). To improve a baseline FAI score by 4 points, with 80% power, a sample size of 114 in total (n = 57 per group) was required. The revised sample size calculation indicated that the study required 144 participants, which incorporated a 20% loss to follow-up.A sample size of 200 participants was calculated, using pilot trial data, based on 90% power to detect a clinically relevant change in both primary outcome measures at a 0.05 significance level and to allow for 30% loss to follow-up (see Supplementary Box S1 for details). However, difficulties with recruitment resulted in revision of the sample size using interim trial baseline data , baseline scores, area, number of conditions, and age. The intervention was group based (by area). Given the small number of areas, however, all models were adjusted for area by including area as a fixed effect. Further analyses were explored by adjusting for differences in marital status at baseline but no differences were seen.28All results were analysed using Stata (version 14). For primary and secondary outcomes, the primary analysis was intention to treat, including all randomised participants, all retained in the group to which they were allocated, and using complete case analyses. All analyses used multiple linear regression models, with results presented as point estimates (mean differences [MD]), 95% confidence intervals (CI), and 29 Furthermore, pre-planned subgroup analyses based on previous literature3 evaluated the effects of age (<65 and ≥65 years) and the number of chronic conditions present (<4 and ≥4) by adding interactions with allocation to the models. Such analyses can be useful in individualising patient care.30 A sensitivity analysis was conducted excluding the couples recruited (see Supplementary Tables S2 and S3 for details).A pre-planned secondary per protocol analysis was conducted, excluding those randomised who did not receive the intervention (non-adherence was defined as attending <3 OPTIMAL sessions), based on previous studies.n = 59; 75.6%). Participants were recruited between February 2016 and February 2018. In total, 149 participants consented and completed baseline data collection . A totalP = 0.027). However, there were no differences between intervention and control groups in the EQ-5D-3L index  and frequency of activity participation  at immediate follow-up. There were no differences in primary outcomes at 6-month follow-up (For HRQoL (EQ-VAS), significant differences were seen in favour of the intervention group at immediate follow-up .Pre-planned subgroup analyses examined the effect of age (<65 and ≥65 years) and number of conditions (<4 and ≥4) on primary outcomes . ImmediaP = 0.008).At 6-month follow-up, in those aged <65 years, compared with those aged ≥65 years, the effect of the intervention compared with usual care was significant for HRQoL (EQ-VAS) only . While no differences were found in other elements of self-reported healthcare utilisation, a significant difference was seen in favour of the intervention group in hospital outpatient appointments .Secondary outcomes were examined at 6 months and no evidence of significant differences were seen in activities of daily living , self-efficacy (SEMCD), or anxiety and depression (HADS-A and HADS-D) . While tAdjusted per protocol analyses found significant differences in favour of the intervention in self-efficacy (SEMCD), satisfaction with and ability to perform activities (COPM-S and COPM-P), self-reported outpatient appointments, and nights spent in hospital (see Supplementary Table S4 for details).3 The programme was effective in improving HRQoL, as measured by the EQ-VAS, at immediate follow-up, although this effect was not maintained at 6-month follow-up, and there was no effect on the EQ-5D index at either time point. The EQ-VAS provides data presenting participant’s self-assessment of their health, while the index score is based on societal preferences for health states.19 There was evidence, based on pre-planned subgroup analyses, that the programme was effective for younger participants, aged <65 years, in improving HRQoL. Regarding secondary outcomes, OPTIMAL showed an effect on occupational satisfaction and self-reported hospital outpatient appointments at 6-month followup, but had no effect on all other outcomes.This study is the first definitive RCT of OPTIMAL, an occupational therapy self-management support programme for individuals with multimorbidity in primary care.10,11 The pragmatic nature of the RCT, conducted in Irish primary care settings and using referral processes similar to those used in practice, was designed to include participants representative of those with multimorbidity in primary care. Study retention was high.This RCT, which investigated OPTIMAL, was based on previous feasibility and pilot studies, as per the Medical Research Council framework for complex interventions, which was a strength, and it was reported following CONSORT guidelines for parallel trials.3,4Data regarding the number of patients approached by GPs and primary care team clinicians were not collected given the pragmatic nature of the study, reflecting routine service referral pathways. It is therefore not possible to determine an overall response rate or draw definitive conclusions about the programme’s generalisability to individuals with multimorbidity in primary care. However, the participants were a fairly representative group of individuals with complex multimorbidity having an average of four conditions, eight to nine regular medications, and a mean age of 65 years, which is younger than previous multimorbidity trials.31 One researcher conducted all 6-month follow-up assessments via interview, blinded to allocation. The original power calculation was revised downwards from 90% to 80% because of recruitment difficulties. This limitation increases the possibility of making a type II error due to an inadequate sample size, that is, a ‘false negative’ finding. Bias as a result of selective outcome reporting is a concern in trial reporting.12 The Goal Attainment Scale32 was included as a secondary outcome measure in the trial registry. However, in both the pilot trial and the present study, the Goal Attainment Scale was used with the intervention group participants only during intervention to assist in goal setting.11 There was no comparison with control group participants.A further limitation is that multiple assessors conducted baseline assessments, however, training was provided in outcome measure administration to minimise the risk of rater bias.In the present study there were some inconsistencies in use of the Goal Attainment Scale to guide programme goal setting across sites. As the measure was a secondary outcome measure with no comparison, these results are not reported.3 While such analyses can be useful in individualising patient care and provide evidence that can guide targeting of interventions, these should be interpreted with caution given the study was not powered to detect these subgroup differences.The subgroup analyses were preplanned based on the literature, which recommends targeting of multimorbidity interventions across the age range and evidence suggesting that those with higher levels of morbidity are at risk of poorer outcomes.4 included 14 studies with a range of interventions, but found little or no benefit. A 2019 study of a clinical medication review for patients with multimorbidity found no improvements in the EQ-5D index score but, like OPTIMAL, the present study did find improvements in the EQ-VAS.33 It is not clear why these interventions have shown significant improvements in the EQ-VAS but not in the index, and there is a need for further consideration of measures used to detect changes in HRQoL .The updated 2018 meta-analysis of HRQoL from the Cochrane review conducted by the 3D team34 or may have less capacity to adopt new behaviours. Approaches such as OPTIMAL may be more effective for adults aged 40–65 years with multimorbidity to develop self-management strategies, thus enhancing elements of HRQoL.Core OPTIMAL programme components including information provision, problem solving, and goal setting, targeted risk factors and health behaviours. Those with multimorbidity may require more intensive or ongoing interventions to improve outcomes such as HRQoL. While there was an improvement in HRQoL at immediate follow-up, as measured by the EQ-VAS, this was not sustained at 6-month follow-up. The immediate effect on HRQoL may reflect the short-term benefit gained from the programme’s social interaction. Subgroup analyses for patients aged <65 years suggested HRQoL improvements at 6-month follow-up and improved frequency of activity participation immediately post-intervention. Older individuals with multimorbidity may have developed coping strategies over the years,3,35,36 While the OPTIMAL programme focuses on improving activity participation and is led by occupational therapists, it covers a wide range of topics, and may not have sufficiently targeted participants’ functional concerns. While subgroup analyses results should be interpreted with caution, it is possible that younger participants with multimorbidity were initially less active in social and community activities, and had more scope for improvement. However, it does not appear that this effect was sustained after programme completion.The OPTIMAL programme did not have an effect on frequency of activity participation. Previous studies have suggested that interventions may be effective if they target functioning.37There were mixed effects on secondary outcomes relating to function. The significant difference in satisfaction with occupational (activity) performance (COPM-S) is consistent with previous research, suggesting that satisfaction with participation for those with multimorbidity had a greater effect on wellbeing than performance or activity accomplishment.38 While intention-to-treat analysis found no significant differences in self-efficacy, as measured by the SEMCD, the per protocol analysis found a statistically significant difference for intervention participants at 6-month follow-up. The original RCT of the Stanford chronic disease self-management programme39 also reported improved self-efficacy that was associated with improved health status and reduced healthcare utilisation. However, these findings have not been replicated in settings outside the US, and the present study does not provide clear evidence of an impact on self-efficacy.40,41The OPTIMAL programme was guided by self-efficacy, a concept developed from social cognitive theory by Bandura.42–45 No differences were found in anxiety and depression at 6-month follow-up. Only one of the OPTIMAL programme sessions specifically addressed mental wellbeing, which may not sufficiently address anxiety and depression.Patients with multimorbidity have higher levels of mental health problems, which are associated with increased healthcare utilisation, cost, and activity limitations.7Previous studies of chronic disease self-management programmes have found minimal improvements in depression, despite emotional management being a core aspect of effective self-management.46,47 OPTIMAL includes strategies for improving communication with healthcare providers and managing multiple medications, and it is possible that the programme resulted in more effective use of healthcare providers. OPTIMAL may have improved the patient’s experience of care like the 3D study,4 but this was not measured. However, self-management programmes alone may be insufficient to reduce healthcare utilisation in multimorbidity. Self-reported healthcare utilisation was used because of logistical difficulties in accessing clinical records across multiple sites, and under-reporting can be a limitation.2,48,49 The authors of the present study plan to publish articles on the process evaluation and economic evaluation, which will add further to interpretation of trial results.A significant difference was found in self-reported hospital outpatient appointments but not in other elements of healthcare utilisation. Previous studies of self-management programmes produced inconsistent results regarding healthcare utilisation.While this trial of an occupational therapy-led self-management support programme found no effect at 6-month follow-up, subgroup analyses suggested a benefit for younger participants with multimorbidity. There remains a need to develop effective interventions targeting both HRQoL and function for patients with multimorbidity in primary care.3Future research should evaluate in more detail the effectiveness of the OPTIMAL programme in younger individuals with multimorbidity, given that patients in this subgroup appear to be at risk of poorer outcomes and existing multimorbidity interventions tend to focus on older adults.


Are dance-based mind-motor activities associated with benefits for fall prevention and better physical functions, such as balance, mobility, and strength, in healthy adults 65 years and older?This systematic review and meta-analysis of 29 randomized clinical trials found that dance-based mind-motor interventions were associated with a statistically significant reduction (37%) in fall risk and a statistically significant reduced rate (31%) of falls. There was a statistically significant association between favorable physical function outcomes and dance-based mind-motor activities for participants in the dance-based mind-motor intervention groups compared with those in the control groups.Dance-based mind-motor activities may help fall prevention efforts in healthy older adults. Falls increase morbidity and mortality in adults 65 years and older. The role of dance-based mind-motor activities in preventing falls among healthy older adults is not well established.To assess the effectiveness of dance-based mind-motor activities in preventing falls.aged 65 and older, accidental falls, and dancing.Systematic search included the PubMed, Embase, Cochrane Library, Web of Science, CINAHL, PsychINFO, Abstracts in Social Gerontology, AgeLine, AMED, and Scopus databases from database inception to February 18, 2018, using the Medical Subject Headings This systematic review and meta-analysis included 29 randomized clinical trials that evaluated a dance-based mind-motor activity in healthy older adults with regard to fall risk, fall rate, or well-established measures of physical function in the domains of balance, mobility, and strength. The included studies targeted participants without comorbidities associated with higher fall risk. Dance-based mind-motor activities were defined as coordinated upright mind-motor movements that emphasize dynamic balance, structured through music or an inner rhythm  and distinctive instructions or choreography, and that involve social interaction.Standardized independent screening, data extraction, and bias assessment were performed. Data were pooled using random-effects models. The study followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guideline.g).Primary outcomes were risk of falling and rate of falls. For the secondary end points of physical function , standardized mean differences (SMDs) were estimated and pooled  risk of falling  and a significantly reduced (31%) rate of falls . In addition, dance-based mind-motor activities were significantly associated with improved physical function in the domains of balance , mobility , and lower body strength  but not upper body strength .Among healthy older adults, dance-based mind-motor activities were associated with decreased risk of falling and rate of falls and improved balance, mobility, and lower body strength. This type of activity may be useful in preventing falls in this population. This systematic review and meta-analysis assesses the association of dance-based mind-motor activities with falls and physical function among healthy older adults. Therefore, we conducted a systematic review and meta-analysis to summarize the current evidence from RCTs among healthy adults 65 years and older.For this systematic review and meta-analysis, we conducted a systematic search in PubMed, Embase, the Cochrane Library, Web of Science, CINAHL, PsychINFO, Abstracts in Social Gerontology, AgeLine, AMED, and Scopus databases from database inception to February 18, 2018, using thesaurus terms and key words (eAppendix in the PRISMA) reporting guideline.21 At every stage, 2 independent researchers  screened each article. Disagreements were solved by consensus and, if necessary, by the consultation of a third independent reviewer (P.O.B.-C.). We included only RCTs that analyzed the effect of dance-based mind-motor activities on the number of persons who fell  or on the number of falls  as primary outcomes or at least 1 of the selected secondary outcomes related to physical function  compared with an active (exercise not related to dance-based mind-motor activities) or a passive control group.Screening and data extraction were predefined and standardized and followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses  and distinctive instructions or choreography, and involve social interaction. Mind-motor activities are activities that combine cognitive and physical tasks  that involve working memory and deliberate motor control. Dance-based mind-motor activities can be performed solo, in a pair, or in group formations. In addition to several styles of well-known dance-based mind-motor activities, such as folk or ballroom dancing, tai chi fulfills the above definition. Contrarily, most yoga styles focus on static body postures rather than dynamic movements and therefore were excluded. Furthermore, we excluded stepping exercises on so-called dance platforms because of the lack of social interaction.22 All included studies had been approved by an ethics committee and informed consent of participants had been obtained.We only considered RCTs that included healthy and ambulatory older adults  living in the community or in independent living facilities. We excluded studies involving participants with comorbidities that directly affect the risk of falling . Only articles in the English language fulfilled the eligibility criteria. Two reviewers  independently assessed potential sources of bias using the Cochrane Collaboration’s tool for assessing risk of bias.To determine the strength of associations between a dance-based mind-motor activity and risk of falling and rate of falls, we pooled risk ratios (RRs) for risk of falling and Poisson-derived incidence rate ratios (IRRs) for the rate of falls using random-effects models based on intention-to-treat analysis. We estimated the IRRs based on reported incident falls and person-years in trials that did not directly report IRRs.24 the 1-leg test,26 and the functional reach test27 for balance; Timed Up and Go test28 for mobility; and sit to stand tests30 for lower body strength and the handgrip strength test31 for upper body strength. To determine the strength of associations between a dance-based mind-motor activity and physical function measures, we calculated a pooled Hedges g standardized mean difference (SMD) using a random-effects model.32 If a study reported outcomes stratified or by 2 tests that assessed the same outcome, we pooled the reported effect sizes before entering them into the meta-analysis. We weighted stratified outcomes by the inverse of the variance, assumed dependency between the assessments, and used a conservative correlation coefficient of 0.8.32 To interpret the effect of the intervention, we followed the guideline suggested by Cohen: equivalent effect size (SMD of 0), small effect size (SMD of 0.2), medium effect size (SMD of 0.5), and large effect size (SMD of 0.8).33For the secondary outcomes, only trials that reported standardized, validated, and clinically used tests to measure balance, mobility, or strength were included: the Berg Balance Scale,I2 statistic as suggested by Borenstein et al.32 We performed prespecified subgroup analyses by type of dance-based mind-motor intervention (non–tai chi vs tai chi), frequency (<3 vs ≥3 times per week), duration (<12 vs ≥12 weeks), type of dwelling (community dwelling vs independent living facilities), and type of randomization (participants randomized vs cluster randomization). In addition, we performed a random-effects meta-regression by intervention frequency and intervention duration if more than 10 RCTs were included.Heterogeneity was quantified by the 34 and the Egger test for balance, mobility, and lower body strength.32 We used the Egger publication bias plot and funnel plots to visually assess indication of publication bias.35 We used Stata software, version 15 (StataCorp LLC) for data analysis.36 Statistical assessment was 2 sided and considered statistically significant at P < .05.Small-study effects were assessed for each outcome if more than 10 RCTs were available. We conducted the Harbord modified test for the primary outcome of RR of falls44 reported on the risk of falling, 7 trials47 reported on the rate of falls, and 4 trials reported on both. A total of 15 trials56 reported on balance, 13 trials62 reported on mobility, 13 trials65 reported on lower body strength, and 4 trials59 reported on upper body strength. From 4627 screened publications, 29 RCTs that fulfilled the inclusion criteria were identified  or Asia63 . Five trials each were from Europe58 and Oceania,65 and 2 trials57 were from South America.More than half of the trials were conducted in North America44 of 1579 participants, with a weighted mean (SD) age of 73.2 (4.9) years, a median sample size of 127 , and median intervention duration of 22 weeks .Dance-based mind-motor activities were associated with 37% reduction in risk of falling  A based oPrespecified subgroup analyses revealed a significant association with reduction of risk of falling for trials that conducted interventions 3 times per week or more  and for trials with durations between 12 and 24 weeks   B based oSubgroup analyses suggested this association was most pronounced in trials that conducted the intervention 3 times per week or more  and for trials that lasted between 12 and 24 weeks   age of 72.3 (4.3) years, a median sample size of 77 , and a median intervention duration of 16 weeks . Subgroup analyses by intervention type found a higher SMD for non–tai chi  activities. Interventions performed 3 times per week or more were associated with a higher SMD . Sensitivity analyses excluding the trial by Hopkins et al50 (published much earlier than the others) or the trial by Serrano-Guzman et al54 (driving the result) also found an association between dance-based mind-motor and improved balance .An association was found between dance-based mind-motor activities and improved balance  A in 15 t62 of 1379 participants, with a weighted mean (SD) age of 73.0 (3.3 years), a median sample size of 54 , and a median intervention duration of 16 weeks . For mobility, decreased completion time indicated improvement in Timed Up and Go test results. Hence, negative values would favor the intervention groups over control groups. Subgroup analyses found positive associations for non–tai chi activities  and for activities with 3 sessions or more per week . The results remained positively associated within sensitivity analyses that excluded the trial by Hopkins et al50 or the trial driving the results by Cepeda et al57 .An association was also found between dance-based mind-motor activities and improved mobility  B based o65 with 1613 participants with a weighted mean (SD) age of 73.1 (3.3) years, a median sample size of 54 , and a median intervention duration of 12 weeks . Subgroup analyses supported a positive association with improved lower body strength for non–tai chi activities  and intervention frequencies of 3 times per week or more . In a sensitivity analysis excluding the early trial by Hopkins et al,50 the SMD remained significant . No significant association was found between dance-based mind-motor activities and upper body strength   A, based to 0.38) B based o22 For balance, visual inspection of the funnel plot suggested asymmetry, but there was no evidence of small-study effects based on the Egger test . For mobility and lower body strength, the funnel plots showed asymmetry, and the Egger test result was statistically significant , suggesting that small-study effects cannot be ruled out.The limited number of trials did not allow for the assessment of small-study effects for the risk of falling, rate of falls, and upper body strength.I2 = 54.8%, P = .03; rate of falls: I2 = 73.0%, P = .001). For secondary outcomes, substantial heterogeneity was found in all domains except upper body strength .Moderate heterogeneity was found for the primary outcomes  and a duration of the intervention between 12 and 24 weeks are associated with greater benefits from these interventions with regard to fall and functional outcomes.68 of multicomponent physical exercise interventions or tai chi alone. Although the analyses for the primary outcomes included more trials that investigated tai chi than non–tai chi dance-based mind-motor activities, the results by subgroups of type of dance-based mind-motor activities suggest that non–tai chi trials might have similar associations with risk of falls and fall rate reductions. In particular, there was a consistently stronger association with improvement of function and non–tai chi dance-based mind-motor interventions for balance, mobility, and lower body strength, and the results therefore extend and strengthen the evidence base of exercise trials that focus on multitasking skills for fall prevention among healthy older adults.For fall prevention, the analysis of dance-based mind-motor interventions are in alignment with findings of prior meta-analyses69 or included tai chi interventions only.68 Other meta-analyses on the effect of dance-based mind-motor activities preselected on cardiovascular risk,70 Parkinson disease,71 or cognitive72 function. Sheldon et al67 report subgroup analyses only for 1 trial in the category dance, whereas the present study additionally included 2 trials that investigated non–tai chi activities for each outcome .To our knowledge, this meta-analysis is the first that aims to summarize the associations of dance-based mind-motor activities beyond tai chi with the risk of falling and the rate of falls among healthy older adults. Earlier systematic reviews that investigated dance-based mind-motor activities among healthy older adults suggested a beneficial effect for fall prevention solely based on the improvement of balance or strengthFurthermore, the present meta-analysis supports benefits of dance-based mind-motor activities for several dimensions of physical function, including balance, mobility, and lower body strength. The observed nonsignificant association on upper body strength may be explained by the fact that the upper extremities in dance-based mind-motor activities are used for expression and partnering rather than building strength or body weight support.38 and was built on the conceptual framework established by the Prevention of Falls Network Europe (ProFaNE).67 In addition to ProFaNE’s definition of 3-dimensional training, this study combined mind and motor abilities, as well as the domain of social interaction. In this meta-analysis, 13 of 29 trials65 tested dance styles.This study has strengths and limitations. Strengths include the comprehensive search strategy within 10 different databases, which incorporated unpublished information from authors67This study found consistent associations between dance-based mind-motor activities and improvement for falls and physical function, and the associations remained significant for all sensitivity analyses performed with influential studies excluded. Finally, relevant to implementation of dance-based mind-motor activities into public health strategies, most trials included in this meta-analysis reported at least 80% adherence, which may be better than previously reported adherence rates for other structured exercise interventions.51This meta-analysis also has limitations. Because of the limited number of non–tai chi trials, several of these trials had to be excluded, giving a stronger weight to the tai chi interventions. However, subgroup analyses excluding the tai chi trials support a similar positive association of non–tai chi activities with balance, mobility, and lower body strength as tai chi. Although data for risk of falling and fall rate were collected with sufficient quality, aspects of physical function, such as balance and strength, were evaluated in part with nonstandardized and noncomparable assessment devices, such as balance platforms or isokinetic machines, which led to the exclusion of several trials that reported on physical function. Finally, these results might not be generalizable to older men because all but 1 trial included mainly women.68 However, additional high-quality trials investigating other types of dance-based mind-motor activities are needed to evaluate options for populations who do not have a tradition of tai chi practice but do have strong cultural bonds to activities such as folk and ballroom dancing or eurhythmics. Additional trials are needed to investigate dance-based mind-motor activities, considering optimal duration and frequency for most effective fall prevention among healthy older adults.Although these results found significant positive associations across risk of falling, rate of falls, and 3 of 4 investigated functional measures and are therefore promising in their consistency and effect size for fall prevention, the study also documented limitations in the quality of individual trials. This is true especially for the non–tai chi interventions. Tai chi is among the best-studied activities for older adults.


This approach highlighted a subgroup of genes, which value as promising candidate therapeutic targets was further confirmed by protein expression analyses. The in vitro activity of 15 inhibitor drugs against either these genes or their pathways was then analyzed, in order to identify the most active ones in terms of inherent activity and ability to overcome cisplatin resistance. NSC130813  and triptolide, both targeting NER factors, proved to be the two most active agents, without evidence of cross-resistance with cisplatin. Combined in vitro treatments showed that NSC130813 and triptolide, when administered together with cisplatin, were able to improve its efficacy in both drug-sensitive and resistant osteosarcoma cells. This evidence may indicate an interesting therapeutic future option for treatment of osteosarcoma patients who present reduced responsiveness to cisplatin, even if possible effects of additive collateral toxicities must be carefully considered. Moreover, our study also showed that targeting protein kinases belonging to the mitogen-activated protein kinase (MAPK) or fibroblast growth factor receptor (FGFR) pathways might indicate new promising therapeutic perspectives in osteosarcoma, demanding for additional investigation.Treatment of high-grade osteosarcoma, the most common malignant tumor of bone, is largely based on administration of cisplatin and other DNA damaging drugs. Altered DNA repair mechanisms may thus significantly impact on either response or resistance to chemotherapy. In this study, by using a panel of human osteosarcoma cell lines, either sensitive or resistant to cisplatin, we assessed the value as candidate therapeutic targets of DNA repair-related factors belonging to the nucleotide excision repair (NER) or base excision repair (BER) pathways, as well as of a group of 18 kinases, which expression was higher in cisplatin-resistant variants compared to their parental cell lines and may be indirectly involved in DNA repair. The causal involvement of these factors in cisplatin resistance of human osteosarcoma cells was validated through gene silencing approaches and  Osteosarcoma (OS) is the most common malignant tumor of bone, which accounts for about 5% of childhood and adolescence neoplasms. High-grade OS is usually treated with neoadjuvant chemotherapy protocols based on cisplatin (CDDP), doxorubicin, methotrexate, and ifosfamide. However, despite this aggressive approach, 35–45% of patients still recur and experience an unfavorable outcome –5.Three out of the four conventional drugs, which are most commonly used in first-line chemotherapy for high-grade OS, induce DNA damages either directly (CDDP and ifosfamide) or indirectly (doxorubicin). Therefore, resistance mechanisms related to DNA damage response can significantly impact on OS chemotherapy unresponsiveness. Among these drugs, CDDP is the agent which has most extensively been studied in relation to DNA repair. A consistent body of evidence is showing that the onset of clinical unresponsiveness to CDDP usually creates further therapeutic complications, because patients can also become cross-resistant to the other DNA damaging chemotherapeutic drugs used in first- or rescue treatment protocols , 6.ERCC excision repair 1 (ERCC1) negatively impacts on the clinical responsiveness to CDDP-based treatments and on patients' outcome  , 7, 8. W outcome . HoweverIn addition to NER, other DNA repair pathways, first of all the base excision repair (BER), have been indicated or proved to be implicated in CDDP resistance of several human tumors –12, but Cellular response to CDDP-induced DNA damage is also mediated by downstream effects on cell cycle and mitosis regulation , 11. TheBased on our previously (unpublished) gene expression analyses, we observed that CDDP-resistant human OS cell lines showed increased expression of several kinases in comparison with their corresponding parental cells. Among these kinases, 18 can be targeted by inhibitor drugs of which some have already entered clinical trials or have shown promising preclinical activities in human cancers different from OS.In this study, we first confirmed the expression level of these 18 kinases in human OS CDDP-resistant variants in comparison with their parental cell lines.Moreover, the role of genes belonging to NER or BER pathways and of the aforementioned 18 kinases for CDDP resistance in human OS cells was estimated, in order to indicate new candidate markers, which may be considered to overcome resistance to CDDP in OS patients.in vitro efficacy of drugs targeting the most significantly emerged genes or pathways has been assessed.Finally, the in vitro studies were performed on the U-2OS and Saos-2 human OS cell lines and a panel of variants resistant to CDDP .The The U-2OS and Saos-2 cell lines were purchased from the American Type Culture Collection . Variants resistant to CDDP were established by exposing the drug-sensitive U-2OS and Saos-2 parental cell lines to stepwise increasing concentrations of CDDP and characterized as previously described .DNA fingerprint analyses of 17 polymorphic short tandem repeat sequences were performed for all cell lines, confirming their identity.2 atmosphere. Drug resistant variants were continuously cultured in presence of CDDP at the concentration used for their selection.All cell lines were cultured in Iscove's modified Dulbecco's medium (IMDM), supplemented with penicillin (20 U/ml)/streptomycin (20 U/ml)  and 10% heat-inactivated fetal bovine serum , and maintained at 37°C in a humidified 5% COAnalyses focused on genes belonging to the NER and BER pathways, which are known to play key roles for CDDP resistance in several human cancers, and on the 18 druggable protein kinases selected on the basis of our previous observations, which indicated their increased expression in U-2OS- and/or Saos-2-derived CDDP-resistant variants in comparison with their parental cells . ExpressIn a first set of experiments, each gene was silenced by transfecting cells with three different siRNAs specific for different regions of the same gene  for 24 h, whereas controls were cultured in presence of scrambled siRNAs. Transfection was performed by using each siRNA at a final concentration of 5 nM and 0.3 or 1.25 μl lipofectamin RNAiMAX  per well in a 96-well or 24-well plate according to the manufacturer's protocol. After 24 h, medium was changed and cells were maintained in siRNA-free medium for additional 48 h. The extent of gene silencing was estimated at 72 h for each siRNA by qRT-PCR on the ViiA 7 instrument (Thermo Fisher Scientific) in order to identify the siRNA with the strongest effect on mRNA down-regulation. Gene expression analysis was performed using the TaqMan® Gene Expression Cells-to-CT™ Kit  and appropriate TaqMan® Gene Expression Assays (Applied Biosystems) listed in in vitro sensitivity to CDDP was estimated on the basis of drug dosage response curves, assessed by using the 3--2,5-dephenyltetrazolium bromide (MTT) assay kit . For all cell lines, the IC50 value (CDDP concentration inducing 50% growth inhibition) was determined inside each experimental condition. To quantify the extent of the increased CDDP sensitivity after gene knock-down, ratios between the IC50 values of cells incubated with scrambled siRNAs and those of silenced cells were calculated.The siRNAs producing the highest mRNA down-regulation were then selected to verify whether the inhibition of a specific gene expression was related to a corresponding increase in CDDP sensitivity. For this second set of experiments, 48 h after seeding and transfection, the cells were incubated with different dosages of CDDP for additional 48 h. Controls were incubated with scrambled siRNAs. The Cells were cultured in petri dishes until confluence, harvested by scraping and lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific) and Benzonase . Protein concentrations were determined by the Bradford Protein Assay . Equal amounts of cell lysates (80 μg) were separated by SDS-PAGE on 4–20% gradient gels (Thermo Fisher Scientific) and then transferred onto nitrocellulose membranes . Then, membranes were blocked in 5% BSA in 1 X TBS containing 0.1% Tween-20  and incubated in primary antibodies  overnighProtein loading was assessed by coomassie R-250 staining (Bio-Rad). Fold changes in protein expression level were determined by densitometric analysis of western blots and autoradiographs using the publicly available ImageJ software .in vitro efficacy, are listed in the The drugs targeting the prioritized genes/pathways, which have been tested for their In vitro drug efficacy was assessed in terms of in vitro growth inhibition activity estimated with the MTT assay (as described above) on the two parental cell lines (U-2OS and Saos-2) and their CDDP-resistant variants with, respectively, the lowest and the highest resistance level . For three drugs, X80, quercetin and SSR128129E, the CellTiter-FluorTM Cell Viability assay  was used according to the manufacturer's protocol. For each cell line, the drug IC50 value was determined, in order to highlight the possible presence of cross-resistance due to the mechanisms present in CDDP-resistant variants.in vitro association of each prioritized drug with CDDP was determined after 96 h of combined treatment with the IC50 dosage of each drug. In drug sequence experiments, cell lines were sequentially exposed for 48 h to their corresponding IC50 dosage of CDDP and then to the IC50 dosage of each prioritized drug for additional 48 h. These combinations were then repeated with the opposite sequence. The type of interaction in terms of synergism, antagonism or additivity, was defined on the basis of the combination index (CI) of each two-drugs combination, which was calculated with the equation of Chou-Talalay by using the CalcuSyn software . By following the CalcuSyn software indications, the drug–drug interaction was classified as synergistic (SYN) when CI was lower than 0.90, additive (ADD) when CI ranged between 0.90 and 1.10, or antagonistic (ANT) when CI was higher than 1.10.The two most active drugs which emerged from these analyses were prioritized for further evaluations. The efficacy of the CDK3, FLT4, MAP2K3, MAP2K5, MAPK1, MAPK3, PIK3C3.Expression levels of genes listed in RNA interference was used to determine the causal involvement in CDDP resistance of the genes listed in in vitro CDDP sensitivity. Cell lines silenced with the siRNAs listed in the ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, and XPA increased CDDP sensitivity in the U-2OS/CDDP300 and U-2OS/CDDP1 μg resistant variants. Silencing of ERCC1, ERCC2/XPD, and ERCC4/XPF increased CDDP sensitivity also in U-2OS parental cellsSilencing of FGFR1 silencing increased CDDP sensitivity in the U-2OS/CDDP1 μg and in parental cellsMAP2K3, MAPK3, and PIK3CB was associated with an increase of CDDP sensitivity in the U-2OS/CDDP4 μg resistant variant.Silencing of Additional reversal activity of CDDP resistance, with IC50 ratios close to the 2.0 cut-off value, was observed for the knock-down of these and other genes , even ifERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XPA, MAP2K3, MAPK3, and PIK3CB. FGFR1 was also selected based on its ability to reverse CDDP resistance also if its expression was found to be moderately higher than in parental cells. All these genes showed evidence of increased expression in CDDP resistant variants and their knock-down proved to be associated with an enhancement of CDDP sensitivity.By coupling the results derived from the assessment of gene expression level in association with CDDP resistance  and the To further validate these genes as candidate therapeutic targets to overcome CDDP-resistance, their expression at protein level was assessed by western blot in both U-2OS and Saos-2 parental cell lines and all their CDDP-resistant variants. All proteins encoded by these prioritized genes proved to be expressed in all cell lines. In the U-2OS series , there wWhen considered together, these results further supported the indication of all these genes as candidate drug targets.in vitro activity of drugs listed in The As shown in In U-2OS variants , higher By considering together the findings obtained in both U-2OS and Saos-2 cell line series, the most active DNA repair-targeting agents, without evidence of cross-resistance, proved to be NSC130813  and triptolide (targeting ERCC3/XPB).The most active kinase-targeting drugs, without evidence of cross-resistance with CDDP, were ulixertinib , hypotemycin (targeting the MAP2K pathway), PD173074 and FIIN-2 (both targeting FGFR1).In order to verify whether treatment with each inhibitor drug was able to increase sensitivity to CDDP, the same group of cell lines were incubated with increasing CDDP concentrations in the absence (control) or presence of the IC20 dosage of each inhibitor drug. A ratio ≥ 2.0 (meaning a decrease of at least 2-fold of CDDP-IC50 in presence of the inhibitor drug) was considered as indication of a drug-induced CDDP sensitization.NSC130813  and triptolide proved to be the two drugs with the most relevant activity, being able to increase CDDP sensitivity for more than 2-fold in all the U-2OS and Saos-2 cell lines .For all these reasons, NSC130813  and triptolide were prioritized for evaluation in combination experiments with CDDP.NSC130813  and triptolide were tested in combination with CDDP, in order to verify whether these treatments lead to positive interactions. As shown in Sequential drug exposure experiments  mainly rMany chemotherapeutic drugs, including several agents used in first-line and rescue chemotherapy protocols for OS exert their activity by directly or indirectly damaging DNA. Consequently, the ability of tumor cells to repair drug-induced DNA damages significantly impacts upon efficacy of these compounds , 10, 11.Our study focused on a group of DNA repair genes and kinases, which we found to be upregulated in our panel of CDDP-resistant human OS cell lines, in order to verify whether they could be considered as new candidate therapeutic targets. In particular, analyses focused on genes belonging to the NER and BER pathways and on 18 druggable protein kinases, which resulted to be overexpressed in association with the development of CDDP resistance.in vitro CDDP sensitivity, confirming its involvement in reduced sensitivity to this drug. The genes that emerged to be most strictly related to CDDP unresponsiveness, were prioritized as candidate targets for the second phase of the study, in which their increased protein expression was confirmed in CDDP-resistant cells, justifying the subsequent in vitro studies of drugs interacting with these markers or their pathways.After a screening to identify the most effective siRNA to knock-down each prioritized gene, drug-sensitive and resistant cell lines were silenced and treated with CDDP, in order to identify those genes which down-regulation produced a corresponding increase of the The impact of these genes for DNA repair activity in our experimental models was further confirmed by functional analyses, in which the cells' capability to repair CDDP-induced DNA damages was assessed after silencing of each prioritized gene by the COMET assay . This evin vitro activity in CDDP-resistant variants compared to parental cell lines, suggesting the presence of cross-resistance mechanisms. The possible reasons for this apparent cross-resistance were not further explored because they were beyond the aims of this study. However, it can be hypothesized that cross-resistance might be due either to differential expression of transporters that recognize these drugs as substrate and efflux them out of the cells or to detoxification processes that are more active in CDDP resistant cells and inactivate these agents. Other reasons may be the activation of alternative or redundant pathways, which replace the function of the targeted pathway in CDDP resistant cells, which consequently become less sensitive to these drugs.Drugs targeting the prioritized targets or pathways were selected on the basis of their reported promising activity in other experimental models and/or their use in clinical trials for human tumors. Among the 15 evaluated agents, TGX221, AZD6482, FR180204, AZD4547, GDC0994 and, at a lower extent, quercetin showed a reduced NSC130813  and triptolide (targeting ERCC3/XPB) proved to be the two agents with the most relevant activity on both CDDP-sensitive and -resistant cell lines. Moreover, these two drugs did not show evidence of cross-resistance with CDDP and proved to reverse CDDP resistance in all drug-sensitive and -resistant cell lines. For these reasons, they were further tested in combined treatments  with CDDP, in order to verify whether these combinations may lead to positive interactions.When considered together, results obtained by the combined treatments indicated that NSC130813  and triptolide have to be administered together with CDDP, in order to improve its efficacy in both drug-sensitive and resistant OS cells. If transferred to a clinical setting, this association has to be considered regarding possible effects of additive collateral toxicities.NSC130813 , also known as [4-[(6-chloro-2-methoxy-9-acridinyl)amino]-2-[(4-methyl-1-piperazinyl)methyl]], is a compound which was shown to act synergistically with CDDP and mitomycin C by interfering DNA repair through the disruption of the interaction between ERCC1 and ERCC4/XPF . TargetiTriptolide is a diterpene triepoxide isolated from a traditional Chinese medicinal plant with anti-inflammatory, immunosuppressive, contraceptive and antitumor activities . In the It has been demonstrated that triptolide covalently binds to human ERCC3/XPB, inhibiting its DNA repair-related activity , 24, 25.On the basis of this body of evidence, we have explored whether in OS cells triptolide-mediated inhibition of NER may improve CDDP activity. Our findings indicated that inhibiting DNA repair through the simultaneous administration of CDDP and triptolide may be a new interesting treatment avenue to overcome CDDP resistance in OS.ClinicalTrials.gov Identifier: NCT03117920), acute myeloid leukemia , and different advanced solid tumors , but results of these regimens are presently not available. Moreover, other triptolide derivatives and analogs have been used in clinical studies aimed to test their efficacy and safety  and PD173074 (targeting FGFR1) showed some promising activity, without evidence of cross-resistance with CDDP. Although these drugs were not further analyzed in combined treatments with CDDP, the obtained findings suggest that targeting protein kinases that influence DNA repair activities may indicate new promising therapeutic perspectives in OS, demanding for additional investigation. This perspective is particularly interesting because there are many protein kinase inhibitors in various stages of clinical development worldwide and the majority of them are used for cancer treatment  models may further support their clinical use. All these activities are presently planned and will focus on the drugs screened and highlighted by this study.Our results can also be the basis for further In high-grade OS, when patients fail to respond to first-line treatment and relapse, therapeutic options and drugs effective for rescue chemotherapy protocols are scarce, also because resistance mechanisms developed against first-line chemotherapeutic drugs can also be responsible for reduced responsiveness to the agents used in the subsequent regimens.Inhibition of DNA repair can be considered as a promising treatment strategy to enhance the efficacy of currently available DNA damaging drugs.There are several genes and proteins involved in modulating the cellular response to DNA damage, each one may serve as target to enhance the efficacy of conventional therapeutic modalities. The current efforts in the development and deployment of several classes of DNA repair targeting compounds justify the hope to achieve new tailored treatment approaches through the use of these inhibitor drugs, which may ultimately drive toward innovative regimens aimed to improve patient outcomes.The evidence emerged within this study about the possibility of successfully combining CDDP with drugs targeting DNA repair factors or protein kinases involved in these processes may indeed indicate new therapeutic options for specific OS patient cohorts, who have reduced cure probabilities.Datasets generated and analyzed in this study will be made available by the authors to qualified researchers, upon justified request.in vitro drug testing. ET, MF, SV, and AF-R performed the gene silencing screening. ET and MF carried out the DNA damage evaluations. ET, MF, CH, and SL took part in the molecular biology and western blot studies. ET, MF, CH, MP, and MS performed the statistical analyses. All authors have made a substantial, direct, and intellectual contribution to the work.ET, MF, CH, and MS designed the study. MS coordinated the study and drafted the manuscript, which was revised by all authors, who approved it for publication. ET, MF, AF-R, MP, and FM carried out the cell culture-based experiments and the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. CH is Associate Editor of the section Pharmacogenetics and Pharmacogenomics of Frontiers in Pharmacology.


Studies examining incident heart failure (HF) have been limited to select populations. To examine incident HF with broader generalizability, there is need to assemble a HF-free cohort using a geographically-diverse sample. We aimed to develop and validate a simple medication-based strategy for assembling a HF-free cohort from the REasons for Geographic And Racial Differences in Stroke (REGARDS) study.We examined REGARDS participants with ≥6 months of Medicare inpatient and outpatient claims data at the time of the baseline in-home study examination. To assemble a HF-free cohort, we identified and excluded participants taking HF-specific medications. To validate this approach, we evaluated event rates among this cohort and assessed diagnostic performance using Medicare claims-based definitions of HF as the referent standard.Among 28,884 eligible participants, 3125 were excluded from the proposed HF-free cohort, leaving a total of 25,759 (89%) participants. Depending on the Medicare definition used as the referent, the negative predictive value of this approach ranged from 95.0–99.2%. Negative predictive value was stable across age, sex, and race strata.The approach to assemble a HF-free cohort in REGARDS can serve as the basis for future studies to examine incident HF in REGARDS and similar studies. Approximately 6 million people live with heart failure (HF) in the United States . UnderstThe REasons for Geographic And Racial Differences in Stroke (REGARDS) study includes 30,239 African American and white men and women recruited from the 48 contiguous United States between 2003 and 2007 . All REGREGARDS participants completed a computer-assisted telephone interview which collected information on sociodemographics and medical history. Following the interview, a trained health technician performed an in-home examination to collect blood and urine specimens, measure blood pressure, conduct an electrocardiogram, and record an inventory of medications used by the participants. Age, race, sex, need to sleep on multiple pillows (orthopnea), and waking at night because of breathlessness  were self-reported. Atrial fibrillation was assessed based on self-reported diagnosis or atrial fibrillation recorded during the electrocardiogram. Hypertension was assessed based on self-reported use of medications to control blood pressure or measured blood pressure greater than 140 mmHg systolic or 90 mmHg diastolic.Among the 30,239 eligible participants in REGARDS, we excluded 56 participants with anomalous data and 469 with no follow-up beyond the baseline assessment. We also excluded 686 participants with missing data on self-reported atrial fibrillation, 135 participants with missing data on baseline medication use, and 9 participants who had adjudicated HF hospitalizations between the baseline telephone interview and the in-home study visit. The remaining cohort included 28,884 eligible participants  [To validate our medication-based assembly of a HF-free cohort, we identified a subset of REGARDS participants who had at least six months of available Medicare fee-for-service inpatient and outpatient claims data at the time of the baseline in-home study examination  10,340) . Medicar 10,340) . The Med0,340 . MInternational Classification of Diseases, 9th revision, Clinical Modification (ICD-9-CM). We defined HF according to ICD-9-CM diagnosis codes 402.01, 402.11, 402.91, 404.01, 404.03, 404.11, 404.13, 404.91, 404.93, and 428.xx, as used by the Centers for Medicare & Medicaid Services for publicly reporting HF quality measures [To assess the validity of our approach to assemble a HF-free cohort, we created 3 Medicare claims-based definitions of HF to serve as referent comparators. First, we defined a HF hospitalization as at least one inpatient claim for a principal diagnosis of HF . Secondly, we defined a principal diagnosis of HF as at least one inpatient claim with a principal discharge diagnosis of HF or at least two claims for outpatient physician visits (including emergency department visits) with principal diagnoses of HF . Finally, we defined “any diagnosis” of HF as at least one inpatient claim or two claims for outpatient physician visits where HF was a principal or secondary diagnosis (“Any diagnosis of HF”). During the period when REGARDS participants were enrolled (2003–2007), the United States used the We first compared the characteristics of participants included and excluded from the HF-free cohort using means and standard deviations for continuous variables and numbers and percentages for categorical variables. Among the population with linked Medicare claims, we calculated negative predictive value using each of the 3 Medicare claims-based definitions of HF as the referent. The negative predictive value represents the proportion of the HF-free cohort who did not have evidence of HF based on Medicare claims, and is thus the most important diagnostic performance parameter for assembling a HF-free cohort. We also calculated sensitivity, specificity, and positive predictive values for each of the 3 Medicare claims-based definitions. We calculated 95% confidence intervals using the normal approximation. In addition, we examined the performance of the HF-free cohort stratified by subgroups chosen a priori. These subgroups were based on age (≥75 versus 65–75 years of age), sex, and race. Analyses were conducted using SAS 9.4 .We also conducted sensitivity analyses to determine the incremental value of additionally excluding participants with self-reported orthopnea and/or PND from the HF-free cohort, as well as the incremental value of additionally excluding participants with adjudicated HF hospitalization within one year of follow-up.The study sample was comprised of 28,884 eligible REGARDS participants. Among them, 25,759 participants (89%) did not take HF-specific medications and were therefore included in the HF-free cohort. Characteristics of the HF-free cohort and the 3125 who were excluded due to suspected HF are shown in Table The validation cohort was comprised of 10,059 eligible REGARDS participants. Compared to the broader REGARDS cohort, patients in the validation cohort were older and had a slightly higher prevalence of several comorbid conditions. Among the validation cohort, 8492 participants (84%) did not take HF-specific medications and were therefore included in the HF-free cohort. Characteristics of the HF-free cohort and those who were excluded due to suspected HF based on diagnosis codes are shown in Table Depending on the diagnostic-code definition used as the referent, the negative predictive value of the medication based approach to identifying a HF-free cohort ranged from 95.0–99.2% Table . The negSpecificity ranged from 86.3–90.4%, and sensitivity ranged from 62.5–78.2%  to assemble a HF-free cohort was limited; negative predictive value improved slightly, but at a sacrifice of excluding an additional thousand participants Table A. SimilaBy excluding participants based on their use of HF-specific medications, we successfully assembled a cohort within REGARDS where very few participants had a history of HF. This HF-free cohort can be examined in the future to study incident HF among a contemporary geographically-diverse study sample. Notably, HF hospitalization rate for the HF-free cohort was one-sixth the rate for individuals excluded due to possible baseline HF. The HF hospitalization rate for the HF-free cohort was much lower than the rates observed in major HF trials, lending additional support for our approach of identifying a cohort free of HF . We furtPrior observational studies of HF-free cohorts used to identify and describe incident HF have included those derived from communities in Olmstead County, Minnesota  and FramGiven the large number of pharmacologic agents that are indicated for use in HF, many of which are specific to HF, , 18 we sOur approach of excluding participants with suspected HF based on their medication use optimized negative predictive value, the diagnostic performance parameter most important to assembling a HF-free cohort. In developing our strategy to assemble a HF-free cohort, we also considered the potential consequence of excessively excluding participants, which would limit the size and generalizability of the cohort, and could significantly hamper statistical power for future studies. Accordingly, our approach preserved a moderately-sized cohort of 25,759 participants. In sum, our strategy maximized negative predictive value without significantly sacrificing the size of the cohort.HF affects a diverse population that includes varying age, men and women, and whites and African Americans , 20. PriWhen screening for HF, symptomatology like orthopnea and PND are frequently assessed. We therefore examined the impact of incorporating these self-reported symptoms in our medication-based strategy. Our results revealed that orthopnea and/or PND did not substantively improve negative predictive value. This is consistent with limitations of performance observed for the diagnosis of HF in the clinical context . Thus, wThere were some limitations to our study that should be noted. Sensitivity and positive predictive value were not particularly high; thus this strategy may be more appropriate for developing a HF-free cohort than for detecting HF. Although a medication inventory was collected for nearly all REGARDS participants, we cannot be certain that these were complete in all cases. Finally, Medicare is an imperfect referent due to limitations in the accuracy of claims-based diagnoses . FurtherOur investigation has assembled and validated a HF-free cohort in REGARDS, which can serve as the basis for future studies to examine incident HF in REGARDS and perhaps similar study samples. Our medication-based approach may be worth examining in other cohorts that have rigorous methods of data collection on medications to ascertain its generalizability. REGARDS in particular offers an excellent cohort for studying incident HF because it is a contemporary geographically diverse cohort with a large number of African American and white participants. Accordingly, through this HF-free cohort, REGARDS has the potential to provide insights on incident HF.Additional file 1. Performance of approach to assemble a heart failure-free cohort in the REasons for Geographic And Racial Differences in Stroke (REGARDS) study population compared to Medicare referent standards, according to ageAdditional file 2. Performance of approach to assemble a heart failure-free cohort in the REasons for Geographic And Racial Differences in Stroke (REGARDS) study population compared to Medicare referent standards, according to sexAdditional file 3. Performance of approach to assemble a heart failure-free cohort in the REasons for Geographic And Racial Differences in Stroke (REGARDS) study population compared to Medicare referent standards, according to race