Independent positive associations between sdLDL and pPON1 within HDL3a and between malondialdehyde (MDA) and pPON1 within HDL2b were shown in the STEMI group. The increased oxidative stress and increased proportion of sdLDL in STEMI are closely related to the compromised antioxidative function of small HDL3 particles and the altered pPON1 within HDL.The aim of this multicentric study was to assess the impacts of oxidative stress, inflammation, and the presence of small, dense, low-density lipoproteins (sdLDL) on the antioxidative function of high-density lipoprotein (HDL) subclasses and the distribution of paraoxonase-1 (PON1) activity within HDL in patients with ST-segment elevation acute myocardial infarction (STEMI). In 69 STEMI patients and 67 healthy control subjects, the lipoproteins’ subclasses were separated using polyacrylamide gradient (3–31%) gel electrophoresis. The relative proportion of sdLDL and each HDL subclass was evaluated by measuring the areas under the peaks of densitometric scans. The distribution of the relative proportion of PON1 activity within the HDL subclasses (pPON1 within HDL) was estimated using the zymogram method. The STEMI patients had significantly lower proportions of HDL2a and HDL3a subclasses ( One of the clinical manifestations of atherosclerosis is acute myocardial infarction with two entities: ST-segment elevation acute myocardial infarction (STEMI) and non-ST-segment elevation acute myocardial infarction (NSTEMI). It is one of the most common causes of emergency admission and sudden death in developed countries and, in recent decades, in developing ones . It has It is now accepted that dyslipidemia, inflammation, oxidative stress, and immune processes play important roles in the pathogenesis of atherosclerosis, but the mechanisms of their interactions are still unknown ,4,5,6. AOn the other hand, high-density lipoprotein (HDL), well known for its antiatherosclerotic effects, constitutes a highly heterogeneous group of particles with different structures and functionalities ,12,13. IThe crucial mechanism of atheroprotective HDL properties is reverse cholesterol transport, but the enzyme paraoxonase-1 (PON1) presented on HDL particles (PON1 within HDL) also plays a significant role in cardiovascular protection. The antioxidative role of PON1 is primarily reflected in its ability to hydrolyse lipid peroxides, thus protecting LDL particles and cell membranes from oxidation . It is wThe difference in the HDL subclasses and pPON1 within the HDL proportions between the control and STEMI groups was evaluated using an analysis of covariance (ANCOVA), with age, BMI, HT, and smoking status included as covariates. The adjusted means for the HDL subclasses and pPON1 within the HDL proportions are shown in Furthermore, we examined the proportions of the HDL subclasses  and pPONThe STEMI patients and controls with sdLDL proportions of > 50% had a higher proportion of HDL3a particles than their counterparts without increased sdLDL. Although no difference in the HDL3 subclasses between the STEMI patients and controls was observed, an sdLDL proportion of > 50% had a significant effect on the proportion of HDL3 particles. In particular, the HDL3 subclasses were more prevalent in individuals with sdLDL proportions of > 50%. The results are shown in The pPON1 within HDL2a showed a lower trend in subjects with an sdLDL proportion of > 50%, as shown in p = 0.007) and positively with PON1 within HDL 3a (10.30 (0.34–20.29), p = 0.04) in the STEMI patients. On the other hand, higher logMDA concentrations are related to higher PON1 activity in the HDL2b subclasses (9.65 (1.64–17.66) p = 0.02), although only in STEMI patients. Multicollinearity was not detected; the tolerance values for all the domains were lower than 1.The current study demonstrates that STEMI is accompanied by modifications in the distribution of the HDL subclasses and changes in the functionality of HDL particles, reflected by alterations in the pPON1 within HDL. For the first time, our study analysed the distribution of pPON1 among the distinct HDL subclasses in STEMI patients.Our study provided an intriguing finding, which was that elevated levels of total cholesterol (TC), LDL-C, and sdLDL were not observed in STEMI patients in comparison to healthy controls, as would be expected. The possible mechanisms for this phenomenon include the acute phase response associated with the up-regulation of LDL-C receptor activity , so the We found that our STEMI patients had increased proportions of small HDL3b and HDL3c subclasses and diminished pPON1 within HDL3. Therefore, the results of the present study indicate that the antioxidative ability of HDL3 particles in STEMI patients is reduced, and such findings can be explained by changes in the composition and maturation of HDL particles . BearingA high level of inflammation, which is present in STEMI, can affect HDL particles’ structures and functions, as well as PON1 activity. The increased hsCRP concentrations and decreased pPON1 within the HDL3 subclasses in our STEMI patients, in comparison to the control group, support the hypothesis that HDL subclasses’ changes during the intensive inflammatory period are associated with a decreased antioxidative capacity, as proposed by Bains et al. . AdditioRegarding sdLDLs, it is generally expected that healthy subjects have a lower percentage of sdLDLs than cardiovascular disease (CVD) patients, but the results of the current study did not confirm this. There are several reasons for this finding: individual variations (genetic factors and lifestyle), as well as subclinical risk factors without signs of developed CVD . All of these factors can contribute to differences in the presence of sdLDLs, which would require further investigation, including comprehensive lipid profiling with a subclass analysis and long- term follow-up to evaluate the actual risk associated with sdLDLs in a specific population.Therefore, we further investigated the influence of STEMI and an increased sdLDL proportion on the distribution of the HDL subclasses and pPON1 within HDL. The obtained results showed that the STEMI patients and control subjects with increased sdLDLs had an increased proportion of HDL3 particles. In the control subjects, such a redistribution of HDL particles may be a protective response to the abundance of sdLDL particles. However, our STEMI patients were characterised by elevated TG and reduced HDL-C concentrations, with an abundance of small-sized HDL particles. In atherogenic dyslipidemia, increased CETP enhances HDL remodelling from large to small HDL subclasses and accelerates the catabolism of HDL. Namely, the transport of cholesteryl ester from cholesterol-rich lipoproteins to TG-rich lipoproteins is mediated by CETP, which results in the enrichment of HDL particles with TGs . TG-enriThe analysis of pPON1 within the HDL subclasses showed that the control subjects with increased sdLDLs had higher pPON1 within HDL2b than their counterparts without increased sdLDLs. These data are consistent with the results of our previous study on women with polycystic ovary syndrome, which demonstrated a high proportion of PON1 activity within the HDL2b subclasses in women with LDL phenotype B . HoweverIn contrast, the finding of increased pPON1 within HDL3a in the STEMI patients with an sdLDL proportion of >50%, in comparison to their counterparts with an sdLDL proportion of ≤50%, might be a consequence of the preponderance of the HDL3a subclasses in the STEMI group with increased sdLDLs. Furthermore, the multivariable regression analysis confirmed an independent association between the increased proportion of sdLDL and pPON1 within the HDL3a particles in the STEMI patients. Such association was not observed in the control subjects. Moreover, our finding that a higher pPON1 within HDL2a was inversely associated with sdLDL in the control group suggests that a preponderance of sdLDL particles diminished the PON1 within the HDL2a subclasses, even in the absence of cardiovascular disease. Since there is little known about the distribution of pPON1 within HDL subclasses, additional studies are needed to verify such presumptions.Finally, some study limitations and strengths should be mentioned. The first limitation is undoubtedly the small number of study participants, as well as the need to determine some apolipoproteins , so further investigations with more subjects and more parameters should verify and expand our findings. Moreover, our research was conducted only on STEMI patients, so there is a need to include other groups of patients with CVD to more comprehensively assess the association between the HDL subclass distributions and pPON1 within HDL, especially in the presence of increased sdLDLs. On the other hand, our study’s importance is reflected in the fact that it provides additional insight into the mechanisms responsible for HDL dysfunction and provides data on the PON1 distribution within the distinct HDL subclasses in STEMI patients.This study, designed as a multicenter pilot study, included a total of 136 participants and was performed on STEMI patients, much like our recent previous study . The stuMedical documentation was used to obtain data about the treatment and health status of the study participants. The essential data concerning each control subject included in the study, such as age, gender, weight, height, blood pressure, and smoking status, etc., were collected through interviews. In addition, the study protocol included a BMI calculation and evaluation of HT.Every participant was informed about the study’s aims and provided written consent before entering the study. The whole research was planned and conducted following the principles of the Helsinki Declaration . The stuPlasma and serum were separated using centrifugation at 1500 rpm for 10 min and divided into multiple aliquots. The aliquots of each sample were stored at −80 °C until they were assayed. The samples were thawed immediately before the analyses.® Xpand Plus biochemical analyser . The Friedewald equation was used to calculate the LDL-C concentration , the formed phenol reacted with 4-aminoantipyrine, creating a pink complex that correlated with the PON1 activity in a particular HDL subclass. The areas under the peaks of the densitometric scans of the samples reflected the pPON1 within HDL, as previously described [After the electrophoretic separation of the HDL particles, we used the zymogram method described by Gugliucci et al.  to assesescribed .The enrolled participants were divided into two groups according to their proportions of sdLDL and the impact of increased sdLDL on the distribution of the HDL subclasses and pPON1 within HDL. An increased proportion of sdLDL was considered in subjects with sdLDL of >50% .2 test. ANCOVA was carried out to investigate the differences in the HDL subclasses and pPON1 within HDL between the patients and controls. An adjustment was performed for the demographic variables that were significantly different between the examined groups. A two-way analysis of variance (ANOVA) was used to evaluate the effects of an sdLDL proportion of > 50% in the STEMI group on the distribution of the HDL subclasses and pPON1 within HDL. The correlations were investigated using a Pearson correlation analysis. A multivariable linear regression analysis with forward selection was performed to examine the independent contributions of the lipid status, lipid peroxidation, and inflammatory parameters to the pPON1 within HDLs. To provide a linear relationship between the dependent and independent variables, we performed a logarithm transformation of the sdLDL, LDL, HDL, TG, hsCRP, and MDA values. To avoid multicollinearity, the VIF scores were below ten and a tolerance score above 0.2 was used.Normality assumptions were analysed using the Kolmogorov–Smirnov test. Student’s t-test was used to compare the variables with normal distributions. If the normality assumption was violated, the Mann–Whitney U test was used. Differences among the categorical variables were estimated using an χp < 0.05 for the 2-tailed test.The normally distributed continuous variables were presented as mean ± SD and skewed variables were expressed as median (low and high quartile) values, while the categorical variables were shown as relative frequencies. All the analyses were performed using PASW Statistics 27 . All the tests were considered significant at Our data showed that STEMI patients are characterised by an impaired maturation and functionality of HDL particles and alterations in the distribution of pPON1 within HDL. Moreover, the antioxidative dysfunction of small HDL3 subclasses is intimately associated with oxidative stress and dyslipidemia in STEMI patients. Further research should investigate the potential underlying mechanisms for the observed changes in pPON1 within HDL related to other risk factors.


The incidence of dengue is increasing rapidly and is a challenging health issue in the Philippines. Epidemiological data are largely based on a passive-surveillance reporting system, which leads to substantial under-reporting of cases.To estimate dengue infection and disease incidence prospectively at the community level in an endemic area of the Philippines using an active surveillance strategy.We implemented active surveillance in the highly endemic community of Alaminos, Laguna. The study consisted of a 1-year follow-up with 2 visits scheduled at the start and end of the study, as well as regular active surveillance in between and unscheduled visits for suspected cases. Blood samples were collected and analyzed to detect dengue during the first scheduled visit and all unscheduled visits, and clinical examination was performed at all visits .We enrolled 500 participants, aged from 6 months to 50 years; 76.2% were found positive for immunoglobulin G , with 92.0% among those aged 9–17 years. Active (weekly) surveillance identified 4 virologically confirmed cases of dengue ; all in participants aged ≤14 years.Routine surveillance programs such as sentinel sites are needed to characterize the entire clinical spectrum of symptomatic dengue, disease incidence, and transmission in the community. Dengue is a mosquito-borne disease caused by the 4 serotypes of the single-stranded RNA dengue virus  [Worldwide, the incidence of dengue is rapidly increasing, 15-fold in the past 20 years; half of the global population remains at risk, and the World Health Organization estimates 100–400 million worldwide infections every year . A studyThe global increase in dengue infections is believed to be due to population growth, increasing worldwide mobility, urbanization, climate change, and the inability to sustain effective vector control , 10. TheDengvaxia, Sanofi Pasteur), is currently available and is licensed in about 20 countries [Only one vaccine, a tetravalent live-attenuated chimeric yellow fever dengue vaccine, .The study was conducted following Good Clinical Practice and all applicable regulatory requirements including the contemporary revision of the Declaration of Helsinki and was approved by the Institutional Review Board of the Research Institute for Tropical Medicine of the Department of Health at the Muntinlupa City, on February 16, 2016. The study was registered at Study researchers used public announcements and community meetings to inform the community about the study. Eligible individuals were aged from 6 months to 50 years, living in households reachable by phone who planned to remain at the same residence throughout the study, and who agreed to go to the study site for visit(s) in case of acute febrile illness. They were also required to observe and document the signs and symptoms of dengue, and required to understand how to take and report body temperature. Informed consent to participate was obtained from all eligible individuals or their legal guardians and documented.A maximum of 2 people per household was considered sufficient to capture the prevalence of infection among household members in the community. The recruitment was a collaboration between the study team and the Municipal Health Office. Basic information about the study  was given by the health center staff to visiting parents of possible participants, and posters containing basic study information had been displayed at collaborating health centers. Parents were invited to visit the study site if interested in additional information.Figure 1): the first at enrollment (Visit 1) and the second at 12 months (Visit 2). Between these visits, surveillance for febrile illness was performed regularly, preferably once a week by telephone or in person, including a home visit at least every other week. In the event of fever, information was collected by telephone; 3 call attempts were made by the study nurse at 2 different times during the first day and another call was made on the next day. Unscheduled visits to the study site were required as soon as possible in case of suspected dengue case (SDC); preferably within 5 days following the onset of symptoms . Visit procedures are outlined in Figure 1.The follow-up was originally planned to last 2 years. However, the study was prematurely terminated by the sponsor at 12 months of follow-up because in December 2017 the sponsor had decided to deprioritize the development of the dengue purified inactivated vaccine candidate to which the study was related. Therefore, only 2 scheduled visits were made . Blood samples were collected in serum-separation tubes  or 367986 (5.0 mL)). At scheduled visits for patients aged from 2 months to 2 years, 3.5 mL of blood was collected and for those aged from 2 years to 50 years, 2 × 5 mL of blood was collected. Blood was centrifuged at room temperature to obtain serum, which was allocated as follows: 0.2 mL  for a dengue IgG enzyme-linked immunosorbent assay  [Blood samples for laboratory assays were collected at Visit 1 and at all unscheduled visits ( Panbio)  and 1 mLSimplexa Dengue Kit, Focus Diagnostics) [SD Bioline Dengue Duo, Abbott) [Platelia Dengue NS1 Ag, Bio-Rad) for the qualitative or semiquantitative detection of DENV NS1 [At SDC visits, for early presenters, blood was similarly collected and serum similarly prepared and allocated for the assays as follows: 0.75 mL  stored at −70 °C and sent on dry ice to another laboratory for RT-qPCR (nostics) . This asnostics) . A secon Abbott)  used to  Abbott)  and a onDENV NS1 . The rapvirologically confirmed dengue case was a SDC confirmed by either RT-qPCR or NS1. A probable SDC was negative or RT-qPCR or NS1 assay not performed and was (a) DENV antibody IgM positive, or (b) was DENV antibody IgG positive.A study participant was considered to have a SDC in the presence of body temperature ≥38.0 °C lasting from 36–48 h to 7 days within the past 8 days, accompanied by other signs and symptoms that the study investigator felt could be related to dengue. These other dengue symptoms or signs might not have had a defined focus or an obvious reason unrelated to dengue (based on the judgment of the physician) and include upper respiratory tract and/or gastrointestinal symptoms, headache, joint ache, and others. A The sample size calculation was performed at the multicenter level of the study, and with a target population of 500 for the Philippine site. The target population was estimated at 1750 overall participants (approximately 300 to 500 per site). A dropout rate of 5% per year was estimated, which would lead to approximately 1662 participants completing the first year of follow-up and approximately 1579 completing 2 years of follow-up, accumulating 3327 patient years.The incidence of dengue was likely to vary by site and by age group. The study population should thus include between 30% and 50% of adults, but the distribution was not further specified as it would make operational feasibility more complex.Supposing that the study population was composed of 70% of children with an expected incidence of 8 RT-qPCR-confirmed dengue cases per 1000 person–years and 30% of adults with an expected incidence of 5 RT-qPCR-confirmed dengue cases per 1000 person–years, the study would detect about 19 cases in children and 5 cases in adults (12 cases in Year 1 with a cohort of 1750 participants and 12 cases in Year 2 with a cohort of about 1662 participants). The overall incidence rate would be 7.1 per 1000 person–years with an exact Poisson 95% confidence intervals (CI) of  and with a CI based on the normal approximation and accounting for the design effect of .The enrollment was conducted by household. Each household could be considered as a cluster and this induced a design effect to account for the between-cluster variability when estimating CI of the incidence rates. The design effect measures the increase in the standard error of the incidence rate estimate due to the sampling design used and is given by D = 1 + (b–1)ρ, where ρ is the intracluster correlation (a measure of the rate of homogeneity within clusters) and b is the average number of individuals sampled per household. Here, b was assumed to be 3. Although in theory “ρ” can have a value up to 1, in practice values >0.4 are uncommon. A conservative estimate of >0.4 was used for this study. The design effect was then estimated at 1.8.Statistical analysis was performed on the per-protocol cohort that included all evaluable eligible participants. Demographic characteristics  were summarized using descriptive statistics. The incidence proportion of cases confirmed virologically, cases confirmed only with RT-qPCR, and probable SDCs during the study period were calculated. As the correlation of the observations among individuals from the same household  was anticipated, these proportions were also estimated using a marginal logistic model using generalized estimating equations (GEE) accounting for correlated data. The 95% CI accounting for correlated data was computed for all estimated incidence proportions. GEE is the extension of the generalized linear models  for the analysis of correlated data such as clustered data or repeated measurements. However, given the small number of participants per cluster (household) in this study, the GEE method may downwardly bias standard error estimates. Therefore, if the estimated design effect was <1, then the classical logistic regression model not accounting for correlated data was used instead of GEE to estimate the incidence proportion and the 95% CI.The clinical symptoms reported during SDCs were tabulated. The proportion (with 95% CI) of participants with a DENV antibody IgG positive result (ELISA) at Visit 1 was estimated by age group using the same methodology as for the incidence proportions. All statistical analyses were performed using Statistical Analysis Systems (SAS Institute) software (version 9.4).Table 1). Of the 500 individuals tested for anti-DENV IgG seropositivity at enrollment, 494 yielded available results and were included in the statistical analysis. Almost all study participants older than 9 years were anti-DENV IgG-positive  (Table 2).Of the 500 individuals enrolled, 498 completed the study follow-up through Month 12 (Visit 2). One left the study area, and another withdrew consent. Almost one-third of the participants were adolescents and another one-third were adults aged up to 50 years . The overall incidence proportion of virologically and only RT-qPCR confirmed dengue cases over the 16-month study period was 0.8 (95% CI 0.3–2.1).Figure 2). These confirmed cases of dengue were in children aged 4, 5, 11, and 14 years. No probable dengue cases were recorded.Nine SDCs were reported, 4 were reported from January to April 2018, and 5 from July to September 2018 and none required hospitalization. Four SDCs were confirmed virologically as being cases of dengue; the other 5 SDCs did not meet the criteria for virological confirmation. Two of the 4 dengue cases confirmed virologically were correctly diagnosed  visit for dengue suspicion. The next most common symptoms were cough and headache (Table 3). No serious adverse events (SAEs) related to study procedures were reported.As shown in The present study was conducted to determine the incidence of dengue among household members aged 6 months to 50 years in a dengue-endemic municipality. Active surveillance was implemented to detect SDCs, in parallel with routine passive surveillance. All reported SDCs were detected by the active-surveillance process and all dengue cases confirmed virologically occurred from July to September, during the rainy summer monsoon season for the Philippines , a perioDengue was common in the study community, as demonstrated by the high IgG prevalence at enrollment, with nearly 4 of 5 study participants tested positive for DENV IgG. Consistent with previously published epidemiological data, almost all adults were seropositive for DENV IgG antibody at study enrollment, suggesting that most of the adult population in this area had been infected previously by dengue . A studyDespite active (weekly) surveillance in our study, the incidence of virologically confirmed dengue was low. The detection of SDCs requires a sensitive definition. Our surveillance strategy was focused on the occurrence of an acute febrile illness lasting at least 36–48 h. This criterion to define a SDC requiring clinical and laboratory evaluation may have been too stringent in the context of the common use of antipyretics. In addition, atypical presentations of dengue have been described and deserve more attention ,29,30.Dengue infections may be asymptomatic in 75% overall . These iThe Philippine dengue surveillance system depends mostly on cases in hospitalized patients with severe symptoms. For example, in 2010–2014, 93% of reported cases were in hospitalized patients . TherefoThe findings presented here bring a modest contribution to our current knowledge of the dengue epidemiology in the Philippines, which, although a highly endemic area, has only limited dengue seroepidemiology data . ExistinThe premature discontinuation of the study is the major limitation of the present data because the short follow-up did not allow us to (a) observe community trends, (b) characterize dengue incidence in an additional season, and (c) characterize the spatiotemporal distribution of the cases, among households and in the broader community. Moreover, the sample size calculation was not made at the individual country level, but on an overall international, multicenter study level. The present data might not be generalizable outside the study’s population.GEE is an extension of the generalized linear model and is used for the analysis of correlated data, such as clustered data and is applicable because the participants in the present study were within households  and correlation of the observations between individuals from the same household  was anticipated. The model as applied to the study has no covariates. Statistical methodologies developed for simple random samples were not selected as the primary method as they assume independence of the observations . Ignoring the clustering of observations could result in underestimating the variance of the estimated proportion. A limitation of GEE is mainly due to the number of individuals per household (cluster). This is why we applied the rule that if the estimated design effect was <1, then the standard logistic model not accounting for correlated data was used.Another limitation is that blood samples were collected at Visit 2, but could not be analyzed due to the termination of the study. Even though it was not one of the study’s objectives, the analysis of these samples would have assisted in identifying asymptomatic cases that may have occurred after study enrollment. Furthermore, baseline measurement of antibodies against all dengue serotypes would have helped differentiate monotypic from multitypic dengue cases. Atypical and asymptomatic cases and cases in those with fever lasting <2 days have been missed due to the fever criteria used to define a SDC.Figure 3 provides a plain language summary of the study findings.Highly urbanized dengue-endemic areas, such as metropolitan Manila, in the Philippines, must acquire the organizational potential to perform adequate routine surveillance, for example through sentinel sites. It is important to characterize the entire spectrum of clinical symptoms and identify community hot-spots of transmission. Those in these endemic areas should also have the opportunity to participate in research projects to further identify the proportion of asymptomatic viremic infections and to describe how these cases may contribute to transmission. A deeper knowledge of the dengue epidemic profile and transmission in highly epidemic areas would generate information of global epidemiological interest. Improvements in the surveillance of incident cases are warranted, coupled with improvements in clinical management strategies and efficient prevention strategies. The evidence presented here will enhance efforts in shaping effective surveillance strategies and in designing future dengue vaccine trials.


Chronic traumatic encephalopathy (CTE) is a neuropathological diagnosis defined by a unique pattern of hyperphosphorylated tau (p-tau) accumulation that begins in neocortical regions of the brain. It is associated with a range of neuropsychological symptoms, but a definitive diagnosis can only be made by postmortem brain examination. In 2018, we instituted CTE screening for all autopsy brains as part of our routine departmental protocol by performing p-tau immunohistochemistry on a restricted set of 3 neocortical blocks . This strategy allowed us to identify 4 cases of low-stage CTE from 180 consecutive autopsies. Two of the 4 cases had a documented history of brain injury; for the remaining 2 cases, there was a long history of treatment-resistant tonic/clonic epilepsy suggesting that undocumented brain injuries may have occurred. Our experience indicates that 3-block CTE screening is useful in identifying CTE in routine practice. The results of this study further support the association between prior head injuries and CTE and demonstrate that, albeit uncommon, CTE does occur in the general population. Our findings suggest that p-tau screening should be routinely pursued in brain autopsy, particularly where there is a documented or likely history of traumatic brain injury. Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease strongly associated with prior exposure to repetitive head impacts (RHI) . InitialIn 2018, we identified the first case of CTE in a retired professional rugby league player . To the In the absence of a clinical history of cognitive decline or dementia, our previous screening protocols incorporated p-tau immunostaining of the hippocampus only, principally to assess for the presence of Alzheimer disease neuropathologic change (ADNC). Given that such medial temporal lobe structures are not involved until high-stage CTE, this approach means that many or most cases of low-stage CTE would not have been identified. We therefore amended our protocols in 2018 based on a prior study that found the examination of 3 neocortical blocks stained for p-tau is sufficient to detect ∼78% of known CTE cases . The ASBB, launched in March 2018, uses specialist diagnostic neuropathology, coupled with research, to understand CTE and other brain pathology that is associated with traumatic brain injury (TBI) in sport and elsewhere. We have recently published the results of the first 21 brains donated to the ASBB over a 3-year period, from March 2018 to March 2021 . These bAutopsy referrals for neuropathological examination come from a wide range of sources that include hospital autopsies, coronial cases, state-wide Creutzfeldt-Jakob disease surveillance, and brain banking for research . All departmental procedures and reporting are approved by the National Association of Testing Authorities and RPAH. Brains referred for tissue banking were obtained with informed consent (RPAH HREC #19-0010). Inclusion of coronial cases was approved by the NSW State Coroner. This study was approved by the RPAH HREC (#22-0053).A total of 192 autopsy referrals accrued over the period of March 2018 until March 2021 are included in this report. The source of individual cases is not revealed to preserve confidentiality. The majority of cases (n = 180) are representative of consecutive brain referrals to the department and included 18 cases donated via the RPAH Neuropathology Tissue and Tumour Bank (NTTB) specifically because of a clinical diagnosis of dementia. Some cases were excluded from p-tau screening. These were: (1) those under the age of 18 years, (2) those with confirmed Creutzfeldt-Jakob disease , and (3) those donated to the Multiple Sclerosis Australian Brain Bank, . The remainder of cases included in this report are derived from brain donation to the ASBB (n = 12) and all had a diagnosis of CTE, as reported previously , peri-Rolandic cortex (block B), and superior and middle temporal gyri at the level of the anterior hippocampus (block D). For ASBB cases and some NTTB dementia cases, more extensive cortical sampling was performed as per our standard procedure for ASBB donors, or when indicated for NTTB donors with neurodegeneration. p-tau IHC was performed using the AT8 monoclonal antibody  at 1:800 dilution on a Leica Bond-Max autostainer. CTE diagnosis and scoring were performed according to current NINDS/NIBIB consensus neuropathological criteria for CTE =2.433, p = 0.119. However, a history of TBI was positively associated with the presence of neurodegenerative pathology, χ2 =7.229, p = 0.007. Prior TBI was associated with a 3-fold increased chance of any neurodegenerative diagnosis, odds ratio 3.3 (95% CI 1.3–7.9). There was no difference in age between the subgroups with respect to TBI history. The average age of those with evidence of TBI history was 61 ± 21 years; the average age of those with no evidence of TBI history was 54 ± 18 years, p = 0.12, Student t-test.The routine neuropathological findings in the cohorts (with respect to neurodegeneration only) are summarized in Four individuals  in the general clinical cohort were found to harbor pathognomonic CTE lesions in one or more of the 3 blocks screened . These lClear evidence of a prior history of TBI was present in 2 of the 4 CTE cases: one was a former boxer who had sustained multiple head injuries via boxing, and another had a history of TBI via brain surgery and ensuing complications. While the other 2 had no documented evidence of prior TBI in the accompanying notes, they both had a history of treatment-resistant epilepsy with tonic-clonic seizures.We recently reported on the prevalence of CTE in the first 3 years of the Australian Sports Brain Bank, where 12 of 21 donor brains were diagnosed with CTE . HoweverIn our standard autopsy neuropathology practice, p-tau IHC was usually only performed when there was a clinical history of dementia or when initial microscopic examination indicated that it may be required and useful. Here we show that incorporating p-tau IHC on 3 routinely sampled neocortical blocks as standard procedure is useful in detecting CTE in general practice. Since establishing p-tau IHC as part of our standard autopsy protocol, we have so far identified 4 cases of CTE from 180 consecutive unselected adult brain autopsies. This equates to roughly a 2% prevalence of CTE in our diagnostic practice.We cannot say that this figure accurately reflects the prevalence of CTE in the general population of Australia but given the 4 cases found to harbor CTE had a documented or likely history of brain injury, it is consistent with CTE being a disease that is rare outside the setting of prior TBI. In our recent reporting on CTE in donors to the Australian Sports Brain Bank (ASBB) we found a greater than 50% prevalence of CTE in a cohort where all donors had a history of TBI via their involvement in sport . Here inIt is not possible to apply a meaningful statistical analysis to the relationship between known or likely TBI history and CTE prevalence with only 4 CTE cases in our cohort. However, we were able to detect a 3-fold increase in the diagnosis of any neurodegenerative pathology, including CTE, with a history of TBI. This finding is not unprecedented and is in line with multiple other studies that have linked TBI history to an increased risk of a variety of neurodegenerative conditions . AlthougOf the 4 CTE cases we report here, only one presented with CTE as the sole neurodegenerative pathology, or “pure” CTE. This individual was 1 of the 2 with epilepsy and also had a history of alcohol abuse. Substance abuse has previously been cited as a factor in CTE and CTE-like pathology encountered in routine practice ; howeverDiagnostic autopsy for CTE and its characteristic neuropathology has historically been largely restricted to individuals with a history of sports-related concussion or RHI. In this setting, CTE diagnosis involves a very broad sampling of cortical brain regions for p-tau IHC, or even whole hemisphere mounts. The 3-block restricted protocol employed in our routine practice was capable of diagnosing 75% of known CTE cases. This demonstrates that the prevalence of 2% we report here is likely to be an underestimate. However, considering the scant literature on the prevalence of CTE in the general population, the 3-block p-tau CTE screening is a practical trade-off in terms of time and expense for detecting most cases of CTE in routine practice.The data presented here are consistent with CTE being a disease caused by prior exposure to TBI. Incorporating 3 neocortical block p-tau IHC into routine neuropathology is a cost-effective means to identify CTE in the general population. At the very least, our findings should compel forensic pathologists and neuropathologists to pursue neocortical p-tau IHC in autopsy cases where there is a known or suspected history of prior TBI.


The aim was to evaluate the cost-utility of a 3-month multimodal occupational therapy intervention in addition to usual care in patients with thumb carpometacarpal osteoarthritis (CMC1 OA).A cost-utility analysis was performed alongside a multicentre randomised controlled trial including three rheumatology departments in Norway. A total of 180 patients referred to surgical consultation due to CMC1 OA were randomised to either multimodal occupational therapy including patient education, hand exercises, assistive devices and orthoses (n=90), or usual care receiving only information on OA (n=90). The outcome measure was quality-adjusted life-years (QALYs) derived from the generic questionnaire EQ-5D-5L over a 2-year period. Resource use and health-related quality of life of the patients were prospectively collected at baseline, 4, 18 and 24 months. Costs were estimated by taking a healthcare and societal perspective. The results were expressed as incremental cost-effectiveness ratios, and a probabilistic sensitivity analysis with 1000 replications following intention-to-treat principle was done to account for uncertainty in the analysis.During the 2-year follow-up period, patients receiving multimodal occupational therapy gained 0.06 more QALYs than patients receiving usual care. The mean (SD) direct costs were €3227 (3546) in the intervention group and €4378 (5487) in the usual care group, mean difference €−1151 . The intervention was the dominant treatment with a probability of 94.5% being cost-effective given the willingness-to-pay threshold of €27 500.The within-trial analysis demonstrated that the multimodal occupational therapy in addition to usual care was cost-effective at 2 years in patients with CMC1 OA.NCT01794754. A strength of the study is the involvement of a patient research partner throughout the conduct of the study, although it may be considered a limitation that only one partner was involved.Self-reported costs combined with several months between assessments may have biased the total costs of the study.We were unable to include medication costs, travel expenses or purchase of technical or medical equipment (except those provided as part of the study) in the analyses due to imprecise reporting.Hand osteoarthritis (OA) is the most common joint disease,There is a common misconception that hand OA is a normal part of ageing, and that there is ‘nothing to be done’.et al performed a trial-based economic evaluation on joint protection only, hand exercises only and a combination of joint protection and hand exercises compared with leaflet and advice over a 12-month period in patients with hand OA.et al however, providing orthoses only was found to be neither effective nor cost-effective.Oppong The aim of this study was to perform a cost-utility analysis (CUA) of a 3-month multimodal occupational therapy intervention in addition to usual care versus usual care only in patients with CMC1 OA referred to surgical consultation. The outcome measure was quality-adjusted life-years (QALYs), and the costs were estimated both as direct and indirect costs and societal costs over a 2-year period taking a healthcare and societal perspective.A CUA was performed alongside a multicentre randomised controlled trial (RCT) including rheumatology departments in three Norwegian hospitals  and is described in the protocol article.A patient research partner from the Patient Research Panel at Diakonhjemmet Hospital, who had lived with hand OA for several years, was involved throughout the project. She participated in project meetings where she gave input to the project plan, recruitment procedure and information material as well as feedback on the interpretation and dissemination of the results.Patients referred by their general practitioner for surgical consultation due to CMC1 OA at the three rheumatology departments between 2013 and 2015 were considered eligible for the study if they could speak Norwegian and had no cognitive deficits. Eligible patients received information about the study, a written consent form and a questionnaire by post. Patients who agreed to participate were scheduled for an appointment with an occupational therapist (OT) at the hospital within 2 weeks after referral. After a baseline assessment, the patients were randomised to either a 3-month multimodal occupational therapy intervention or usual care. In Norway, the waiting period between referral and the actual surgical consultation was at the time this study was conducted between 4 and 6 months, and the intervention was conducted during this waiting period. Patients were assessed again immediately before surgical consultation (approximately 4 months after baseline assessment), and 18 and 24 months after the baseline assessment . The sam10.1136/bmjopen-2022-063103.supp1Supplementary dataDuring the waiting period between referral and surgical consultation, the intervention group received a multimodal occupational therapy intervention in line with recommended first-line treatment.The control group received usual care, which generally means staying on the waiting list for consultation in specialist healthcare, that is, receiving no treatment in specialist healthcare. For the purpose of this study, the control group were assessed at the hospital at baseline and all follow-up time points by an OT. They received general written and oral information about hand OA. The control group had the same possibility as the intervention group to ask questions and be given advice.If necessary, patients in both groups could independently seek treatment in primary healthcare, such as a consultation with general practitioner or other healthcare personnel (this information was collected as part of this study). Both groups had a surgical consultation with a medical specialist approximately 4 months after the baseline assessment. All patients paid a deductible for the assessment consultations (a maximum of €259 per year).Patients were randomised using a computer-generated list with a block size of 10, stratified by hospital. Envelopes were opened by the patients after receiving information on hand OA and completing baseline assessment. The group affiliation was known to both the patients and the OTs.www.euroqol.org).The health-related quality of life (HRQoL) was measured at baseline, 4, 18 and 24 months using the generic instrument EQ-5D-5L . Although the OT assessment was done for study purposes, it was included in the cost estimates because we cannot rule out a possible positive effect of an appointment with an OT in specialist healthcare for the control group. We have not included medication costs, travelling expenses or purchase of technical or medical equipment (except assistive devices provided to patients in the intervention group) due to lack of or imprecise reporting. Many patients reported using medication when needed, making it impossible to calculate costs. Travelling expenses were not collected, and with regard to technical or medical equipment (except those provided as part of the project), it was not possible to determine if the patients had bought the equipment before or during the trial.Data on sickness absence related to surgery were collected from patient medical records. The number of hours of informal help at home due to OA  and the productivity loss  were self-reported. If not explicitly indicated by the patient, we expected that the productivity loss remained the same between assessment points, thus, if the patient reported sickness absence of 50% at 4 months and 100% at 18 months, this was calculated as 50% productivity loss between 4 and 18 months, and 100% between 18 and 24 months.The costs of specialist healthcare visits and surgery were calculated using diagnosis-related group (DRG) weights from 2019. DRG is a coding system used for administration of both clinical and financial activity in specialist healthcare. The DRG costs were estimated by multiplying the cost weighting for the specific DRG group of the patient with the unit cost of 2019.Direct costs included costs related to specialist healthcare  and primary healthcare . Indirect costs included productivity loss due to sickness absence (due to surgery or for other reasons) and costs related to help with chores at home.Differences in QALYs between the two groups were estimated using the trapezoidal method (area under the curve).Between-group differences in mean costs and mean effects were analysed using independent sample t-test. Significance level was set to 0.05. Statistical analyses were performed using Excel, SPSS V.26 and Stata V.16.A total of 180 patients were randomised to either multimodal occupational therapy in addition to usual care (n=90) or usual care only (n=90). The mean age across both groups was 63 years (SD 7.6) and 79% of the patients were women. We found no between-group differences in any of the baseline characteristics including utility score, medication use or percentage of patients working full-time or part-time .A total of 18 patients (7 in the intervention group and 11 in the control group) had missing HRQoL utility score at one or more timepoints. For these missing values we used ‘last observation carried forward’. Sensitivity analyses showed that the average HRQoL utility scores remained the same for both groups without imputation of these missing values. Missing self-reported costs were not imputed (n=16). The costs related to surgery were collected from patient medical records and were not encumbered by missing data.The total between-group difference in QALYs was 0.06  after 24 months, in favour of the intervention group. The distribution of EQ-5D-5L utility score across the different time points for the two groups are shown in 10.1136/bmjopen-2022-063103.supp2Supplementary dataThe mean cost of multimodal occupational therapy intervention  was approximately €500 per patient. Surgery accounted for the largest between-group difference related to direct healthcare costs with 22 surgical procedures in the intervention group and 33 surgical procedures in the control group, resulting in a €550 difference in surgical cost per patient between the two groups. The use of physiotherapy/occupational therapy also constituted a high cost. However, almost one-third of these costs were related to the predetermined assessment points  that were part of the study but not the actual OT intervention (€375 per patient in the control group and €307 per patient in the intervention group).Productivity loss due to sickness absence or disability benefits was the largest contributor to the total costs in both groups. However, at baseline, only 7% of the sickness absence in both groups was reported to be due to hand OA, and the percentages at 4, 18 and 24 months were 6%, 2% and 0.5%, respectively. Similar numbers of patients in the two groups were retired at baseline (34% in the control group vs 32% in the intervention group), 4 months (36% vs 34%) and 24 months (42% vs 43%). However, more patients in the intervention group (43%) retired between 4 and 18 months than in the control group (36%), indicating that fewer patients contributed to productivity loss in the intervention group during this period.The aim of this study was to evaluate the cost-utility of a 3-month multimodal occupational therapy intervention in patients with CMC1 OA in a 2-year period. Our results demonstrate that the intervention, consisting of patient education, hand exercises, orthoses and assistive devices, has the highest probability of being cost-effective, given the willingness-to-pay threshold (€27 500/QALY). The results indicate that the intervention is both less costly and more effective when provided alongside usual care. This study is one of few studies to evaluate the cost-utility of conservative treatment in hand OA, and the only study to evaluate the cost-utility of the 2018 EULAR guidelines on first-line treatment for CMC1 OA. Our findings add to the evidence base by demonstrating that multimodal occupational therapy is a cost-effective alternative for the management of CMC1 OA.et al failed to show that the combination of hand exercises and use of assistive devices (joint protection classesThe results of this study are in line with a previous study showing that hand exercises were more cost-effective than leaflet and advice in patients with hand OA.et al,In our study, we did not separately evaluate the different parts of the recommended first-line treatment. It is possible that we would have found the same results as Oppong Surgery constituted a large cost in this within-trial. Although we found a tendency towards reduction in surgical procedures in the intervention group compared with the control group, this result was not statistically significant.Productivity loss represented the largest contributor to the total costs in both groups. The between-group difference may to some extent be attributed to a higher retirement rate in the intervention group. Although most participants were of retirement age, we do not know the reason for retirement and this should therefore be reported in future studies. Additionally, to avoid bias, register data should be used for more accurate reporting of sickness absence and disability benefits.The patients reported to have OA complaints for a median of 5 years, and more than 60% showed radiographic signs of subluxation of the CMC1 joint, indicating advanced disease progression. However, only 21% of the patients had visited a physiotherapist or an OT before referral to surgical consultation.The current study is the first study assessing the cost-utility of recommended multimodal treatment in patients with CMC1 OA. It is considered a strength that the study was conducted alongside an RCT. The study also has some limitations. Almost 80% of the sample were women, which represents a higher proportion of women than the usual gender distribution in hand OA. The results may therefore not be representative for men with hand OA. The self-reported costs and the 14-month time frame between the reporting may have biased the total costs. This has most likely contributed to lower overall costs, as patients may not remember to report all consultations with healthcare personnel, especially in the time period between 4 and 18 months. On the other hand, the costs related to surgery were collected from patient medical records and will not be encumbered by the same bias. A total of 16 patients had missing data throughout the study. There may have been systematic reasons for these patients being missing, which could have affected the outcome. However, there were more missing data in the control group than the intervention group, indicating that the between-group difference could have been larger if all patients had reported direct and indirect costs. The results may have been biased by the method of imputation of missing HRQoL utility values. However, due to low number of missing values and sensitivity analyses showing no difference in mean score whether using ‘Last observation carried forward’ or not, we did not go any further with more advanced methods like multiple imputation. We were unable to include medication costs, travel expenses or purchase of technical or medical equipment, except for the assistive devices provided as part on the intervention, in the analyses. This is not in line with the most recent recommendations for cost-utility analysesThe within-trial analysis demonstrated that multimodal occupational therapy was cost-effective in addition to usual care in patients with CMC1 OA, when taking a healthcare and societal perspective. The intervention was the dominant treatment with a 94.5% probability of being cost-effective at 2 years, given the willingness-to-pay threshold of €27 500/QALY. The low cost related to the intervention supports the use of this first-line treatment in all patients with CMC1 OA.


The aim of this study was to evaluate the cost-effectiveness of ravulizumab compared with eculizumab for the treatment of adult patients with paroxysmal nocturnal hemoglobinuria (PNH) in the Netherlands.A cost-effectiveness analysis was conducted based on a Markov cohort model simulating the course of patients with PNH with clinical symptom(s) indicative of high disease activity, or who are clinically stable after having been treated with eculizumab for at least the past six months. Costs, quality of life, and the incremental cost-effectiveness ratio (ICER) were estimated over a lifetime horizon from a Dutch societal perspective. Several additional analyses were performed, including a one-way sensitivity analysis, a probabilistic sensitivity analysis, and scenario analysis.When compared with eculizumab, ravulizumab saves €266,833 and 1.57 quality adjusted life years (QALYs) are gained, resulting in a dominant ICER. Drug costs account for the majority of the total costs in both intervention groups. Cost savings were driven by the difference in total treatment costs of ravulizumab compared with eculizumab caused by the reduced administration frequency, accounting for 98% of the total cost savings. The QALY gain with ravulizumab is largely attributable to the improved quality of life associated with less frequent infusions and BTH events. At a willingness-to-pay threshold of €20,000/QALY, there is a 76.6% probability that ravulizumab would be cost-effective.The cost reduction and QALY gain associated with the lower rates of BTH and less frequent administration make ravulizumab a cost-saving and clinically beneficial substitute for eculizumab for adults with PNH in the Netherlands.The online version contains supplementary material available at 10.1007/s10198-022-01556-5. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, progressive, chronic disease, predominantly diagnosed during young adulthood (30–45 years of age). PNH is characterized by uncontrolled activation of the complement system, leading to chronic intravascular hemolysis and thrombosis. Unless the disease is managed appropriately, life-threatening complications can occur, such as thrombosis, renal insufficiency, and pulmonary hypertension –3.As reported in a 2006 study from England, the incidence of PNH is 0.13 per 100,000 persons, with an estimated 15-year prevalence of 1.59 per 100,000 ; using tPrior to the introduction in 2008 of the first complement C5 inhibitor, eculizumab, PNH treatment in the Netherlands consisted exclusively of supportive symptom management  . Unlike Despite the clinical benefits that eculizumab provides for patients with PNH, the treatment regimen has considerable practical implications for their lives. Patients require infusions of eculizumab every 2 weeks, each with an infusion duration between 25 and 45 min and a total treatment duration between two to four hours –16. FreqIn July 2019, the European Medicines Agency granted marketing authorization to ravulizumab, a new complement inhibitor , and patTwo pivotal clinical trials, ALXN1210-PNH-301 and ALXN1210-PNH-302 (hereafter referred to as Study 301 and Study 302), have shown that ravulizumab is clinically non-inferior compared with eculizumab in eculizumab-naive patients as well as eculizumab-experienced and stable patients. However, both trials showed numerically  fewer BTH events among ravulizumab-treated patients than among eculizumab-treated patients. Moreover, none of the reported BTH events in the ravulizumab arm were caused by IncC5Inhib , 22. TheThe reduced infusion frequency and lower rate of BTH events associated with ravulizumab treatment are expected to lead to a gain in QoL for patients. In addition, reduced infusion frequency may be linked to a reduction in costs compared with eculizumab in the Dutch clinical practice. The aim of this study was to evaluate the cost-effectiveness of ravulizumab compared with eculizumab in the Netherlands for the treatment of adult patients with PNH with hemolysis, with clinical symptom(s) indicative of high disease activity, or who are clinically stable after having been treated with eculizumab for at least the past six months.A Markov cohort cost-effectiveness model was developed in Microsoft Excel 2016  to determine the cost-effectiveness of ravulizumab as treatment for adult patients with PNH in the Netherlands. The study followed the ISPOR CHEERS checklist and adhered to good reporting practice for health economic evaluations .The model compared ravulizumab with eculizumab, the current standard of care . The difThe Markov model comprised the following 11 health states: nine living health states and two death health states  and adults who were clinically stable on eculizumab at the labeled dosing for at least six months . Results were calculated for the aggregate population, which was a mix of both cohorts with a distribution of 8.3% and 91.6% of Cohort 1 and 2, respectively (see Appendix 1). This distribution was calculated as the ratio of the number of new patients (Cohort 1) versus the number of prevalent patients (Cohort 2) in the Netherlands .Model patient characteristics reflect the patient populations in Studies 301 and 302  for patients without a history of IncC5Inhib BTH events , 22.Based on the subset of data identified in the previous step, a full information maximum likelihood multinomial logit model was estimated to predict the outcome state conditional on the initial state (’No BTH’).The transition equations developed in the last step were used to calculate the mean transition probabilities for each  pair. Transition probabilities were calculated for both values of the second covariate, which was a binary indicator for whether the patient received ravulizumab or eculizumab in the randomized treatment period  of the clinical study. Transition matrices for subsequent IncC5Inhib BTH events were determined in the same manner as for the initial IncC5Inhib and CAC BTH event transitions. Details on the transition probability calculations are shown in Appendix 1.Transition matrices were constructed in the following three steps:The transition probabilities were extrapolated with a time-dependent change in risk of initial IncC5Inhib BTH for Cohort 1, such that patients in Cohort 1 who have not yet experienced an initial IncC5Inhib BTH event take on the risk observed in Study 302 after 26 weeks of treatment , 22. As The transition probability to spontaneous remission was based on the study by Hillmen et al. (1995) and was equal for all living health states . BackgroThe probabilities of requiring a transfusion in the 2-week horizon of our analysis, as well as the mean number of units of pRBC required, were calculated based on 26-week patient visit-level data from Studies 301 and 302 (Appendix 1) , 22.The EORTC Quality of Life Questionnaire (30 items) (EORTC QLQ-C30) outcomes from Studies 301 and 302 were mapped onto EuroQol 5-Dimension 3-Level (EQ-5D-3L) utility values, and results were analyzed with a mixed-effects regression model per health state .The utility increments related to the reduced treatment administration of ravulizumab were calculated based on a DCE in the Netherlands.The per health state transition probabilities were corrected for the Dutch general population utility cap.The utility values were determined in multiple steps as follows:The EORTC QLQ-C30 outcomes over the period of 26 weeks were mapped onto Dutch EQ-5D-3L utility data using the direct ordinary least squares regression mapping method by Versteegh et al. (2012) . The effThe mixed-effects regression model described the associations between the utility values and BTH events, transfusions, and visit counts. A treatment indicator was omitted to avoid double counting as the utility increment of ravulizumab versus eculizumab was based on the DCE survey. Mixed-effects models were employed to better account for the heterogeneity in the panel data used from the 301 and 302 clinical trials—that is, heterogeneity between individuals in the data and natural fluctuations , 22. TheThe cost-effectiveness analysis incorporated a health-utility increment for ravulizumab versus eculizumab to reflect the patient benefit of the decreased frequency of administration , 35. TheAn age-specific utility cap for the Dutch general population was included in the modeled health state utilities . The maxBoth direct medical and societal costs were included in the model, as summarized in Table Drug costs were based on the treatment regimen and list prices for the interventions. Eculizumab treatment was administered weekly for 5 weeks , then every two weeks from week five onwards (the maintenance phase). The dose was 600 mg during the initial phase and 900 mg during the maintenance phase . PatientEculizumab was administered to patients as an intravenous infusion over 25–45 min. Ravulizumab was administered to patients as an intravenous infusion over a minimum of 102–114 min for the loading dose and a minimum of 120–140 min for the maintenance dose. The infusion costs for eculizumab were based on the 2016 Dutch Healthcare Authority (NZa) add-on tariff for eculizumab infusion . The admThe modeled medical resource utilization associated with a BTH event  were assumed to require one-time travel (back and forth) per visit. The travel costs of dialysis were based on seven sessions  to a dialysis center . It was Productivity losses resulting from both eculizumab and ravulizumab infusions and BTH events were taken into account. The costs were based on the cost of productivity per hour, corrected for the average labor participation rate. The productivity losses for eculizumab and ravulizumab administration included travel time to the clinic, infusion time for loading and maintenance doses, and recovery time. The productivity losses for transfusion and BTH events consisted of travel time to the clinic, hospital stay, and recovery time . It was A univariate sensitivity analysis was conducted to assess the impact of varying certain input variables on the ICER. In the PSA, the input values were simultaneously varied over their confidence intervals (CIs) for a total of 1,000 simulations. The parameters in the analysis were varied by their respective distributions  using the deterministic values and standard errors. We assumed the standard error to be 25% from the mean when the standard error or 95% CI was not available. For the univariate sensitivity analysis, the lower and upper bounds were set at 2.5% and 97.5%, respectively. Appendix 1 summarizes all the variables and their respective CIs and distributions. The scenario analyses conducted for the cost-effectiveness analysis comprised 25 different scenarios (Appendix 1).The aggregate population for the base case analysis comprised a total population of 84 patients as follows: 7 complement-inhibitor naïve patients and 77 eculizumab-experienced patients. The base case results show that patients treated with ravulizumab incurred €5,626,489 in total costs, whereas patients treated with eculizumab incurred €5,893,322 in total costs. Additionally, a total of 24.85 QALYs were gained in the ravulizumab cohort versus 23.28 QALYs gained in the eculizumab cohort (Table The difference in QALY gain between the two interventions is predominantly due to the lower administration frequency associated with ravulizumab as well as the absence of IncC5Inhib BTH events following ravulizumab administration. Treatment costs (drug and administration costs) are the highest contributors to the total costs in both the ravulizumab and eculizumab groups with €5,520,820 and € 5,781,406, respectively, with a difference of €260,587. This accounted for 98% of the total cost reduction. The other 2% of cost savings were due to medical costs related to the prevented BTH events (€– 1578) and societal costs (-€4699).The results of the univariate sensitivity analyses for the aggregate population are presented as tornado diagrams in Figs. The results of the PSA for the aggregate population demonstrate the outcomes for the ICER represented on a cost-effectiveness plane Fig.  and a coIn the aggregate population, the two scenarios with the most impact on the incremental costs are excluding the option of up-dosing eculizumab-treated patients and excluding discounting, resulting in a difference from the base case of €117,874 and –€766,356, respectively  and more effective (1.57 QALYs gained per patient) compared with eculizumab from a Dutch societal perspective. This means that from a healthcare as well as an economic perspective ravulizumab would be a good alternative to eculizumab as a treatment for adult patients with PNH in the Netherlands. Cost savings were driven by the difference in total treatment costs of ravulizumab compared with eculizumab, accounting for 98% of the total cost savings. The gain in QALYs is predominantly caused by the lower administration frequency associated with ravulizumab and the absence of IncC5Inhib BTH events after initiation of ravulizumab treatment.Several assumptions were required to optimally reflect everyday clinical practice while using the currently available data, and these assumptions can have a considerable impact on the overall structure of the cost-effectiveness analysis. When developing the model, for example, a lack of long-term data meant that it was necessary to extrapolate the 26-week trial data over a lifetime horizon. It was assumed that the trial results after 26 weeks would remain constant over the lifetime horizon of the model, in terms of the incidence of meeting transfusion requirements, probabilities of BTH events, and QoL outcomes. The univariate sensitivity analysis showed that the transition probability for IncC5Inhib BTH had a major effect on the outcome, which indicates that assuming that the probabilities stay equal over time may impact our outcomes. However, the 52-week results of Studies 301 and 302 support our assumption and demonstrate that the non-inferior yet higher efficacy and QoL outcomes of ravulizumab in the first 26 weeks were maintained for the following 26 weeks , 48. MorAlthough our analysis represents the Dutch clinical setting as accurately as possible, limited data availability affected our ability to comprise all aspects of complement inhibitor treatment in the Netherlands, and there are several limitations that should be taken into account when interpreting results. First, a lack of data on the number of at-home infusions and their associated costs meant that at-home administration was not implemented in our model. A Dutch study comparing at-home and in-hospital administration of trastuzumab showed that the costs are comparable overall for the two settings . AlthougIn practice, some patients may require a permanent increase in the dose of eculizumab due to an increased risk of IncC5Inhib BTH, but this was not accounted for in our base case analysis. A study in Leeds, UK, reported that around 20% of patients treated with eculizumab require a dose in excess of the standard two-weekly 900 mg maintenance dose to achieve and maintain efficacy . We condThe conservative method used to estimate mortality risks is also a potential limitation of our study resulting from a lack of data. Study 301 and Study 302 were trials with relatively small patient populations and, as a result, limited data regarding mortality risks. It was assumed that, outside BTH events, the mortality of patients with PNH was equal to the mortality of the general Dutch population and therefore health-state independent. BTH events were assumed to temporarily increase the mortality risk of patients with PNH. As only limited data were available, we based the excess mortality on a study which included only patients with PNH who were not treated with a C5-inhibititor . HoweverEconomic evaluations of healthcare interventions clinical trials often have differing recommendations for QoL data collection and analysis, and methods for converting one form of data to another can introduce uncertainty into the model. In study 301 and 302, QoL was assessed using an EORTC instrument because this questionnaire is considered the most adequate measure of QoL in PNH . HoweverAn analysis assessing patient preferences has shown that patients with PNH who had been treated with both ravulizumab and eculizumab prefer a lower frequency of infusions (once every eight-weeks rather than once every two weeks), and the improved overall QoL they experienced as a result . Study 3In addition, ravulizumab has recently been made available at a concentration ten times the standard dose , making it possible to reduce infusion duration from 120–140 min to 25–75 min . This neThis is the first study to analyze the cost-effectiveness of ravulizumab versus eculizumab for patients with PNH in the Dutch setting, following the Dutch guidelines. The model provides a reliable representation of the Dutch setting because it includes societal costs, and it is fully populated with Dutch data, Dutch costs inputs, Dutch value sets for the utility mapping, and a DCE survey specific for the Dutch population. In addition, this is the first study that estimates the cost-effectiveness of ravulizumab versus eculizumab from a societal perspective. A societal perspective is relevant for treating chronic PNH with C5-inhibitors because a reduced infusion frequency brings advantages for patient and society that are only captured when societal costs are included . The inclusion of both treatment-naïve patients and eculizumab-experienced patients is an additional strength of our model, as it provides direct comparative data for these patient populations.Our results show that ravulizumab is likely to be cost saving compared with eculizumab. However, future research is needed to determine whether the real-world long-term effects of ravulizumab support the assumption of extrapolating 26-week efficacy data to a lifetime horizon. In particular, the absence of BTH events and lack of required up-dosing among ravulizumab patients has not yet been demonstrated in the long-term and has a heavy influence on the model outcomes. Moreover, more data are needed to support the effect of the reduced administration frequency on QoL in patients with PNH because though this is currently based on a DCE, a preference-based method is preferred.In conclusion, due to fewer BTH events and the lower administration frequency, ravulizumab is cost-saving compared with eculizumab while increasing the QoL of patients. Therefore, ravulizumab is a beneficial substitute for eculizumab for newly diagnosed adult patients with PNH and patients currently treated with eculizumab.Supplementary file1 (DOCX 108 KB)Supplementary file2 (DOCX 99 KB)Below is the link to the electronic supplementary material.


This comparative effectiveness reseach study assesses the effecetiveness of adalimumab vs tofacitinib for treatment of rheumatoid arthiritis in the clinical setting. What is the effectiveness of adalimumab (ADA) compared with tofacitinib (TOF) for treatment of rheumatoid arthritis in routine clinical practice?In this comparative effectiveness study of 842 patients with rheumatoid arthritis in Australia, TOF was favored slightly at 3 months vs ADA, but there was no difference in scores between patients receiving TOF and those receiving ADA at 9 months.This study showed similar treatment effects for TOF and ADA, which is consistent with data from a randomized trial and current European Alliance of Associations for Rheumatology treatment guidelines. There is a need for observational studies to supplement evidence from clinical trials, and the target trial emulation (TTE) framework can help avoid biases that can be introduced when treatments are compared crudely using observational data by applying design principles for randomized clinical trials. Adalimumab (ADA) and tofacitinib (TOF) were shown to be equivalent in patients with rheumatoid arthritis (RA) in a randomized clinical trial, but to our knowledge, these drugs have not been compared head-to-head using routinely collected clinical data and the TTE framework.To emulate a randomized clinical trial comparing ADA vs TOF in patients with RA who were new users of a biologic or targeted synthetic disease-modifying antirheumatic drug (b/tsDMARD).This comparative effectiveness study emulating a randomized clinical trial of ADA vs TOF included Australian adults aged 18 years or older with RA in the Optimising Patient Outcomes in Australian Rheumatology (OPAL) data set. Patients were included if they initiated ADA or TOF between October 1, 2015, and April 1, 2021; were new b/tsDMARD users; and had at least 1 component of the disease activity score in 28 joints using C-reactive protein (DAS28-CRP) recorded at baseline or during follow-up.Treatment with either ADA (40 mg every 14 days) or TOF (10 mg daily).The main outcome was the estimated average treatment effect, defined as the difference in mean DAS28-CRP among patients receiving TOF compared with those receiving ADA at 3 and 9 months after initiating treatment. Missing DAS28-CRP data were multiply imputed. Stable balancing weights were used to account for nonrandomized treatment assignment.P = .02) at 3 months and −0.03  at 9 months.A total of 842 patients were identified, including 569 treated with ADA  and 273 treated with TOF . After applying stable balancing weights, mean DAS28-CRP in the ADA group was 5.3  at baseline, 2.6  at 3 months, and 2.3  at 9 months; in the TOF group, it was 5.3  at baseline, 2.4  at 3 months, and 2.3  at 9 months. The estimated average treatment effect was −0.2 (95% CI, −0.4 to −0.03; In this study, there was a modest but statistically significant reduction in DAS28-CRP at 3 months for patients receiving TOF compared with those receiving ADA and no difference between treatment groups at 9 months. Three months of treatment with either drug led to clinically relevant average reductions in mean DAS28-CRP, consistent with remission. More recently, targeted synthetic DMARDs (tsDMARDs), including janus kinase inhibitors (JAKis), have become available and are considered equivalent to bDMARDs for patients with moderate to severe disease refractory to conventional synthetic DMARD (csDMARD) therapy.2 showed that tofacitinib (TOF) combined with methotrexate was noninferior to adalimumab (ADA) combined with methotrexate. Other trials have demonstrated the efficacy of JAKi therapy in patients who have not responded to methotrexate or TNFi therapy.4 Drug retention was longer for TOF compared with TNFis in an observational study of 4023 treatment courses that combined multiple lines of therapy.5 Similar outcomes have been described for TOF and JAKi therapy compared with TNFi therapy.7 To our knowledge, there are no observational studies evaluating the effectiveness of a JAKi drug compared with a TNFi drug in a b/tsDMARD-naive patient population.Although trials directly comparing specific b/tsDMARDs head-to-head are limited, a double-blind phase 3b/4 randomized clinical trial8 However, there are significant challenges to the reliability of comparative effectiveness studies using RWDs.9 In this study, RWD refers to clinical records in patients’ EMRs that are routinely collected at the point of care. Outcomes are often recorded incompletely in registries and EMRs, patients are not randomized to treatment groups, and differences between groups need to be accounted for. There can also be differential durations of follow-up and attrition.Registries and real-world data sets (RWDs), which include routinely collected data such as electronic medical records (EMRs) and medical claims data, are a valuable source of information for understanding the effectiveness of treatments. There is increasing recognition of the complementary role for real-world evidence based on analyses of RWDs in health care and regulatory decision-making.10 In this study, we sought to emulate an RCT of ADA vs TOF in b/tsDMARD-naive patients with RA in the Optimising Patient Outcomes in Australian Rheumatology (OPAL) data set using an intention-to-treat analysis.11 To generate evidence from a large observational RWD, we developed a methodological approach to address the challenges of missing baseline and outcome data and nonrandomized treatment assignment. Our approach aimed to avoid the selection bias that could result from excluding patients with missing outcomes.Target trial emulation (TTE) is a framework for comparative effectiveness analyses whereby principles from the design of randomized clinical trials (RCTs) are applied to observational research by making explicit the design of the trial that is intended to be emulated.HC210647). Patients consented to their deidentified EMR data being used for research via an opt-out consent model. This report followed the International Society for Pharmacoeconomics and Outcomes Research (ISPOR) reporting guideline.Detailed technical methods of this comparative effectiveness study are described in eMethods 1 through 7 and the eAppendix in The prespecified protocol for the target trial to be emulated is described in eMethods 1 in Eligible patients in the emulated target trial were adults aged 18 years or older who were diagnosed with RA; whose first visit occurred between April 1, 2015, and January 1, 2021; who had no prior recorded b/tsDMARD; who had at least 6 months from their first recorded visit until baseline; and who had at least 6 months of treatment with a csDMARD immediately prior to baseline. These criteria defined (or enriched for) a cohort of new users who were b/tsDMARD naive based on their EMR and the government criteria for b/tsDMARD reimbursement. Patients were excluded if they did not have at least 1 component of the DAS28-CRP recorded at baseline, 3 months, or 9 months.Patients initiated treatment with either ADA (40 mg every 14 days) or TOF (10 mg daily) and, in the target trial, would be expected to continue treatment during follow-up unless an adverse event or contraindication occurred. The limited duration of availability of the biosimilar for ADA meant that all ADA interventions were the originator and not the biosimilar.The primary outcome was disease activity at 3 and 9 months after initiating treatment. The average treatment effect (ATE) was defined as the difference in mean DAS28-CRP among patients receiving TOF compared with those receiving ADA at 3 and 9 months.2 However, its calculation requires multiple variables that are missing in the OPAL data set. Therefore, DAS28-CRP, used in other landmark trials for RA,13 was selected as a composite outcome that represents clinician- and patient-assessed disease and an objective pathologic marker.These time points were selected because joint counts and pathologic markers are assessed for government reimbursement at 3 and 9 months. American College of Rheumatology response of at least 50% was used in the trial of ADA and TOF.This analysis used the multicenter OPAL data set, which includes the EMRs for 216 138 patients with rheumatic conditions treated by 112 rheumatologists across Australia at 43 different clinics since 2004. In Australia, government reimbursement is available for b/tsDMARDs for patients with moderate to severe disease who have not responded to at least 6 months of treatment with csDMARDs, and physicians can prescribe the b/tsDMARD that fits the patient’s clinical need. As such, there are no binding guidelines as to the order in which a b/tsDMARD class can be prescribed. Response to treatment is assessed at 3 months and at 6-month intervals thereafter. Tofacitinib is approved by the Australian Therapeutic Goods Administration for use for RA at a dosage of 5 mg twice daily.The deidentified data in the OPAL data set include demographics, disease history, disease activity measures, comorbidities, pathology, medications, patient-reported outcomes, and characteristics of the treating rheumatologist. Data were collected from April 1, 2015, to June 30, 2021. A more detailed description of the data set and a summary of the characteristics of the variables used in this analysis are given in eMethods 2 in International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10) codes C* or D00-D48 was considered to be cancer.14 A major cardiovascular event (MACE) was defined according to previously published ICD-10 codes for MACE in the context of administrative data sets and rheumatology: I21-I24 ; I63-I66 (stroke); I11, I50, and I97.1 (heart failure); and Z95 (coronary artery bypass grafting).16Treatment cessations due to an adverse reaction were described for all patients who satisfied the inclusion criteria. Treating physicians have discretion to record an adverse reaction to a medication in the EMR, and there may have been unrecorded adverse reactions. All recorded adverse reactions that were considered more serious than nonserious were described. An adverse reaction recorded using the Patients were not involved in the design of this study or consulted during selection of outcomes or the interpretation of findings. Patients will be involved in the dissemination of this research.A multistep method was developed to address the challenges of missing disease activity data and nonrandomized treatment assignment. Analysis that only uses complete cases could lead to selection bias, and thus, multiple imputation was used instead of complete-case analysis. An overview of the methods is presented in 17 Patients with no observed DAS28-CRP components at follow-up were then excluded after multiple imputation because these patients would not be informative about the treatment effect. Stable balancing weights (SBWs) were then used to account for differences in baseline characteristics between the included patients receiving ADA and the total eligible patients and between the included patients receiving TOF and the total eligible patients.18 Balance for the baseline characteristics was evaluated, including calculating standardized mean differences between the treatment groups, which express these differences in terms of the observed SDs in the sample of eligible patients. The difference in weighted DAS28-CRP was then calculated at 3 and 9 months within each imputed data set, and these estimates were pooled using Rubin rules to yield single point estimates at 3 and 9 months.19 The whole procedure was then bootstrapped using 1000 bootstrap samples to generate a 95% CI for the estimates using the percentile method.20In brief, multiple imputation by chained equations using the random forest algorithm under the missing-at-random assumption was used to impute missing data for the components of the DAS28-CRP at baseline and follow-up, generating 10 imputed data sets.P < .05. eMethods 1 through 7, eResults 1 through 4, and the eAppendix in All analyses were performed using R, version 4.0.2 . Differences in mean DAS28-CRP at follow-up were assessed using a 2-sided test and a significance level of Of the 52 338 patients with RA in the OPAL data set, 842 eligible b/tsDMARD-naive patients who were new starters of ADA  or TOF  were identified . PatientBefore SBWs were applied, there were small differences between the treatment groups . For exaAfter SBWs were applied, the standardized mean differences in baseline characteristics were between −0.03 and 0.03, within the conventional threshold of 0.1 for propensity score matching . This in21After weighting, mean DAS28-CRP decreased from 5.3  at baseline to 2.6  at 3 months and 2.3  at 9 months in the ADA group (eResults 5 in P = .02) at 3 months and −0.03  at 9 months  for MACE . Additionally, there was 1 case of pulmonary embolism in each of the ADA (0.1%) and TOF (0.2%) treatment groups, the former of which was life-threatening but considered unrelated to treatment. There was 1 case of deep vein thrombosis in the TOF group.There were no adverse reactions recorded using 2 Our findings are also consistent with an observational study that combined multiple lines of therapy and showed slightly longer drug retention for TOF therapy compared with TNFi therapy.5 Our study benefitted from the large number of patients with RA in the OPAL data set, which enabled the analysis to focus on the effectiveness of TOF vs ADA received as the first b/tsDMARD. Our findings support the 2019 European Alliance of Associations for Rheumatology (EULAR) recommendations, which treat JAKis and bDMARDs as equivalent when used as first-line therapy.1In this comparative effectiveness study using the TTE framework, we found a modest but statistically significant reduction in disease activity associated with TOF compared with ADA at 3 months and no difference between drugs at 9 months in patients with RA who were b/tsDMARD naive. These results may be generalizable to patients with RA who have not responded to csDMARD therapy and are eligible to initiate their first b/tsDMARD, subject to the limitations of the study. Although the outcomes, time points, and superiority design differ, the small effect size in our analysis is consistent with the previous findings of noninferiority in the American College of Rheumatology response of at least 50% at 6 months for these drugs in a clinical trial.23 Given the well-known limitations of observational research, the need for quality in comparative effectiveness studies has been recognized.9 Although there have been few applications of the TTE framework in rheumatology,26 there have been numerous studies using propensity score methods, including propensity score matching and inverse probability of treatment weighting,34 and some of these are limited by time-related biases, such as comparing drugs or classes received as different lines of therapy.35 Covariate balancing is an alternative approach to propensity score methods and can lead to better balance while avoiding some of the known pitfalls of propensity score methods, such as model misspecification and dispersed weights.37 Additionally, this study dealt with the challenge of missing outcomes data, which is a source of bias not often addressed in observational studies.Observational studies are an important supplement to RCTs for assessing whether trial findings can be reproduced in everyday practice in a less restricted patient population and for guiding treatment decisions that occur outside the idealized setting of a trial.22 Although the rates of MACE and other cardiovascular events in our study are consistent with those previously reported, this study was not set up to address safety, and we only described recorded adverse reactions in patients who received ADA or TOF as first-line b/tsDMARD therapy. Follow-up was limited, and physicians’ discretion to record adverse reactions limit the conclusions that can be drawn from these data. As more data on these risks become available, the safety of these drugs will be better understood.38Safety signals, although typically rare, are important in connection with ADA and TOF, especially in light of the relatively higher risks of MACE and cancer associated with JAKi therapy compared with TNFi therapy.As an analysis of observational data, the challenges of missing outcomes data and nonrandomized treatment assignment were possible limitations to this study that may impact interpretation of the generalizability of the results. Our analysis relied on some assumptions, and violation of these would limit the reliability of the results. We assumed the joint counts, CRP levels, and patient global scores were missing at random . Under this assumption, the missing data on joint counts, CRP levels, and patient global scores could be accounted for in the imputation model by the characteristics of the patients, the treating rheumatologists, and the clinics, which were fully observed, and accounting for these variables could produce unbiased results in the analysis. Availability of nursing staff time and other characteristics of the clinics and individual rheumatologists are plausible variables to explain whether a rheumatologist in a busy clinic would be able to prioritize recording complete data on all outcomes during a consultation with a patient. Nevertheless, we acknowledge that there may have been bias that could not be addressed if there were further unmeasured sources of missing data that were not accounted for in the imputation model.40 It is possible that assignment to ADA or TOF was confounded by unmeasured characteristics that were not accounted for in this analysis. E-values for the ATE suggest that these results may be sensitive to unmeasured confounding  the probability of initiating treatment with ADA or TOF may have depended on the measured baseline characteristics but did not depend on future disease activity at follow-up time points, (2) the probability of treatment assignment did not equal 0, and (3) a patient's observed DAS28-CRP at follow-up was the same as that patient's potential DAS28-CRP at follow-up after they followed the treatment that they were observed to be assigned to.2 and support the current EULAR treatment guidelines. The analysis serves as an exemplar of the TTE framework applied to an incomplete RWD.In this comparative effectiveness study, DAS28-CRP was significantly lower at 3 months for patients treated with TOF compared with ADA. However, 3 months of treatment with either drug led to substantive reductions in mean DAS28-CRP, consistent with remission. There was no difference in DAS28-CRP between patients receiving TOF or ADA at 9 months. The results of this observational study are consistent with clinical trial data


DNA methylation marks that are inherited from parents to offspring are known to play a role in cancer risk and could explain part of the familial risk for cancer. We therefore conducted a genome-wide search for heritable methylation marks associated with prostate cancer risk. Peripheral blood DNA methylation was measured for 133 of the 469 members of 25 multiple-case prostate cancer families, using the EPIC array. We used these families to systematically search the genome for methylation marks with Mendelian patterns of inheritance, then we tested the 1,000 most heritable marks for association with prostate cancer risk. After correcting for multiple testing, 41 heritable methylation marks were associated with prostate cancer risk. Separate analyses, based on 869 incident cases and 869 controls from a prospective cohort study, showed that 9 of these marks near the metastable epiallele VTRNA2-1 were also nominally associated with aggressive prostate cancer risk in the population.The online version contains supplementary material available at 10.1007/s10689-022-00325-w. MLH1 and MSH2 [Most DNA methylation marks are erased before or soon after conception, however methylation marks are known to be effectively inherited from parents to offspring in rare cases, e.g. near Some epimutations, and most methylation marks that are strongly associated with an mQTL, are systemic  , which mOne of the most important risk factors for prostate cancer is having a family history of the disease but less than half of the familial risk for prostate cancer is explained by the currently identified genetic risk factors. This is despite modern genomic studies of prostate cancer risk being based on tens of thousands of cases, suggesting that many heritable risk factors for prostate cancer might exist that are not genetic. We therefore conducted a systematic, genome-wide search for heritable methylation marks associated with prostate cancer risk, and assessed the extent to which their variation was explained by common genetic variants.Our study was based on previously reported methods , 5, 6 anThe family-based phase was based on 25 multiple-case prostate cancer families drawn from the Australian Prostate Cancer Family Study (APCFS) . Periphefamilies . MethylaBecause we were interested in causes of familial prostate cancer, we selected only the 1,000 most heritable methylation marks. For each of these, we calculated the probability that each family member carries a hypothetical genetic variant causing aberrant methylation at the mark, based on M-values and family structure but not ages or affected statuses. We tested these carrier probabilities for association with prostate cancer using Cox proportional hazards survival models. We accounted for multiple testing  using the Bonferroni p-value threshold of 0.05/1000. Risk estimates from the family-based phase are biased by ascertainment so are not presented, though p-values are valid because the test statistic is not affected by ascertainment under the null hypothesis.VTRNA2-1 have bimodal distributions so, as a sensitivity analysis, we also dichotomised the methylation values of these sites and estimated their associations with prostate cancer using conditional logistic regression, as in the main analyses, above.The population-based phase was based on unrelated individuals recruited irrespective of family history to the Melbourne Collaborative Cohort Study (MCCS) , 9. PeriThe 1,000 most heritable methylation marks from the family-based phase are listed in Supplementary Table 1. Of these 1,000 methylation marks, 41 were associated with prostate cancer risk at the Bonferroni-corrected significance level  with aggressive prostate cancer were found for all 9 marks near VTRNA2-1) also have nominally significant associations with aggressive prostate cancer risk in the general population. Note that we would not expect all 41 methylation marks to be associated with risk in the population, e.g. BRCA1 is usually not detected by genome-wide association studies. Also, the magnitude of risk in the population-based and familial settings could differ greatly, due to rare mQTLs or epimutations causing changes in methylation that are many times the population standard deviation.Our study has identified 41 methylation marks associated with familial prostate cancer, and 9 of these marks . Down regulation of VTRNA2-1 in a variety of tumours and cancer cell lines has been well documented and associated with promoter CpG hypermethylation. As we found previously using the same datasets, VTRNA2-1 methylation marks are associated with aggressive prostate cancer in the population [VTRNA2-1 locus as a metastable epiallele because the loss of imprinting in this region occurs systemically, can be modulated by the periconceptional environment and persists through adulthood [Nine of the 41 heritable methylation marks associated with prostate cancer risk are in the imprinted see also . Imprintpulation  and are pulation . We and dulthood .PM20D1), a known methylation and expression quantitative trait locus associated with risk of Alzheimer’s disease. These and the other annotated and unannotated CpGs identified in this study require further research to understand the biological explanation for their association with heritable prostate cancer risk. Non-genetic causes of heritability could not be investigated in the current study, but it is possible that familial environmental and lifestyle factors play a role in determining DNA methylation at these loci.Seven of the heritable methylation marks associated with prostate cancer risk are located at peptidase M20 domain containing 1 (Despite having a modest sample size, we were able to identify many heritable, cancer-associated methylation marks. Excluding non-heritable methylation marks before testing for association with cancer excludes many marks that cannot cause familial cancer, while retaining any that can. Our method of excluding non-heritable marks before testing for association with disease is therefore a very powerful way of enriching the candidate set of methylation marks for those that could cause familial disease. Further discussion of the methodology can be found in Joo et al., .Heritable methylation marks can mimic the effects of genetic variants, so identifying them is similar in many ways to finding genetic loci that are associated with cancer. As for genetic loci, these marks can implicate new biological mechanisms and therefore shed light on the processes of prostate cancer initiation and progression. These methylation marks could also be used in risk-prediction algorithms to give more precise estimates of a person’s risk of prostate cancer, and so provide more tailored screening.Our study has several strengths, including its use of an innovative method to identify heritable methylation marks, its method of enriching the candidate set of methylation marks for those that could cause familial disease, and its use of a cohort study to further investigate the findings from the family-based phase. The main weakness of the study was that some of the marks from the family-based phase could not be investigated in the population-based phase, due to the use of different arrays in the two phases. Our modest sample size is also a weakness, though this is offset by the enrichment step described above.VTRNA2-1 likely to be associated with aggressive prostate cancer in the general population.In summary, our study has identified 41 heritable methylation marks that are associated with prostate cancer risk in the context of multiple-case families, with 9 of these marks near Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3